WorldWideScience

Sample records for human fumarylacetoacetate hydrolase

  1. THE HUMAN FUMARYLACETOACETATE GENE : CHARACTERIZATION OF RESTRICTION-FRAGMENT-LENGTH-POLYMORPHISMS AND IDENTIFICATION OF HAPLOTYPES IN TYROSINEMIA TYPE-1 AND PSEUDODEFICIENCY

    NARCIS (Netherlands)

    ROOTWELT, H; KVITTINGEN, EA; HOIE, K; AGSTERIBBE, E; HARTOG, M; BERGER, R

    Deficiency of human fumarylacetoacetase (FAH) activity results in hereditary tyrosinemia type I. Using the restriction enzymes BglII, KpnI and StuI and a 1.3-kb cDNA probe for the FAH gene, we have found 6 restriction fragment length polymorphisms (RFLPs). These RFLPs were utilised in 3 tyrosinemia

  2. Epoxide hydrolase affects estrogen production in the human ovary.

    Science.gov (United States)

    Hattori, N; Fujiwara, H; Maeda, M; Fujii, S; Ueda, M

    2000-09-01

    To investigate the mechanisms of ovarian cell differentiation, we raised a new monoclonal antibody, HCL-3, which reacted with human luteal cells. It also reacted with human and porcine hepatocytes. The immunoaffinity-purified HCL-3 antigen from human corpora lutea (CL) was shown to be a 46-kDa protein. The N-terminal 22 amino acids of the 46-kDa protein from porcine liver exhibited high homology (82%) to human microsomal epoxide hydrolase (mEH). The purified HCL-3 antigen from human CL or porcine liver showed EH enzyme activity, confirming that HCL-3 antigen is identical to mEH, which is reported to detoxify the toxic substrates in the liver. In human follicles, mEH was immunohistochemically detected on granulosa and theca interna cells. In the menstrual and pregnant CL, mEH was also expressed on large and small luteal cells. A competitive inhibitor of EH, 1,2-epoxy-3,3,3-trichloropropane, inhibited the conversion of estradiol from testosterone by granulosa cells cultured in vitro, indicating the involvement of mEH in ovarian estrogen production. Because anticonvulsant sodium valproate and its analogues were reported to inhibit EH enzyme activity, these findings provide a new insight into the etiology of endocrine disorders that are frequently observed among epileptic patients taking anticonvulsant drugs.

  3. Hepatic cholesterol ester hydrolase in human liver disease.

    Science.gov (United States)

    Simon, J B; Poon, R W

    1978-09-01

    Human liver contains an acid cholesterol ester hydrolase (CEH) of presumed lysosomal origin, but its significance is unknown. We developed a modified CEH radioassay suitable for needle biopsy specimens and measured hepatic activity of this enzyme in 69 patients undergoing percutaneous liver biopsy. Histologically normal livers hydrolyzed 5.80 +/- 0.78 SEM mumoles of cholesterol ester per hr per g of liver protein (n, 10). Values were similar in alcoholic liver disease (n, 17), obstructive jaundice (n, 9), and miscellaneous hepatic disorders (n, 21). In contrast, mean hepatic CEH activity was more than 3-fold elevated in 12 patients with acute hepatitis, 21.05 +/- 2.45 SEM mumoles per hr per g of protein (P less than 0.01). In 2 patients studied serially, CEH returned to normal as hepatitis resolved. CEH activity in all patients paralleled SGOT levels (r, 0.84; P less than 0.01). There was no correlation with serum levels of free or esterified cholesterol nor with serum activity of lecithin-cholesterol acyltransferase, the enzyme responsible for cholesterol esterification in plasma. These studies confirm the presence of CEH activity in human liver and show markedly increased activity in acute hepatitis. The pathogenesis and clinical significance of altered hepatic CEH activity in liver disease require further study.

  4. Fumarylacetoacetate hydrolase deficient pigs are a novel large animal model of metabolic liver disease

    Directory of Open Access Journals (Sweden)

    Raymond D. Hickey

    2014-07-01

    FAH-deficiency produced a lethal defect in utero that was corrected by administration of 2-(2-nitro-4-trifluoromethylbenzoyl-1,3 cyclohexanedione (NTBC throughout pregnancy. Animals on NTBC were phenotypically normal at birth; however, the animals were euthanized approximately four weeks after withdrawal of NTBC due to clinical decline and physical examination findings of severe liver injury and encephalopathy consistent with acute liver failure. Biochemical and histological analyses, characterized by diffuse and severe hepatocellular damage, confirmed the diagnosis of severe liver injury. FAH−/− pigs provide the first genetically engineered large animal model of a metabolic liver disorder. Future applications of FAH−/− pigs include discovery research as a large animal model of HT1 and spontaneous acute liver failure, and preclinical testing of the efficacy of liver cell therapies, including transplantation of hepatocytes, liver stem cells, and pluripotent stem cell-derived hepatocytes.

  5. Development of organophosphate hydrolase activity in a bacterial homolog of human cholinesterase

    Science.gov (United States)

    Legler, Patricia; Boisvert, Susanne; Compton, Jaimee; Millard, Charles

    2014-07-01

    We applied a combination of rational design and directed evolution (DE) to Bacillus subtilis p-nitrobenzyl esterase (pNBE) with the goal of enhancing organophosphorus acid anhydride hydrolase (OPAAH) activity. DE started with a designed variant, pNBE A107H, carrying a histidine homologous with human butyrylcholinesterase G117H to find complementary mutations that further enhance its OPAAH activity. Five sites were selected (G105, G106, A107, A190, and A400) within a 6.7 Å radius of the nucleophilic serine O?. All 95 variants were screened for esterase activity with a set of five substrates: pNP-acetate, pNP-butyrate, acetylthiocholine, butyrylthiocholine, or benzoylthiocholine. A microscale assay for OPAAH activity was developed for screening DE libraries. Reductions in esterase activity were generally concomitant with enhancements in OPAAH activity. One variant, A107K, showed an unexpected 7-fold increase in its kcat/Km for benzoylthiocholine, demonstrating that it is also possible to enhance the cholinesterase activity of pNBE. Moreover, DE resulted in at least three variants with modestly enhanced OPAAH activity compared to wild type pNBE. A107H/A190C showed a 50-fold increase in paraoxonase activity and underwent a slow time- and temperature-dependent change affecting the hydrolysis of OPAA and ester substrates. Structural analysis suggests that pNBE may represent a precursor leading to human cholinesterase and carboxylesterase 1 through extension of two vestigial specificity loops; a preliminary attempt to transfer the Ω-loop of BChE into pNBE is described. pNBE was tested as a surrogate scaffold for mammalian esterases. Unlike butyrylcholinesterase and pNBE, introducing a G143H mutation (equivalent to G117H) did not confer detectable OP hydrolase activity on human carboxylesterase 1. We discuss the importance of the oxyanion-hole residues for enhancing the OPAAH activity of selected serine hydrolases.

  6. Molecular Basis of Prodrug Activation by Human Valacyclovirase, an [alpha]-Amino Acid Ester Hydrolase

    Energy Technology Data Exchange (ETDEWEB)

    Lai, Longsheng; Xu, Zhaohui; Zhou, Jiahai; Lee, Kyung-Dall; Amidon, Gordon L. (Michigan)

    2008-07-08

    Chemical modification to improve biopharmaceutical properties, especially oral absorption and bioavailability, is a common strategy employed by pharmaceutical chemists. The approach often employs a simple structural modification and utilizes ubiquitous endogenous esterases as activation enzymes, although such enzymes are often unidentified. This report describes the crystal structure and specificity of a novel activating enzyme for valacyclovir and valganciclovir. Our structural insights show that human valacyclovirase has a unique binding mode and specificity for amino acid esters. Biochemical data demonstrate that the enzyme hydrolyzes esters of {alpha}-amino acids exclusively and displays a broad specificity spectrum for the aminoacyl moiety similar to tricorn-interacting aminopeptidase F1. Crystal structures of the enzyme, two mechanistic mutants, and a complex with a product analogue, when combined with biochemical analysis, reveal the key determinants for substrate recognition; that is, a flexible and mostly hydrophobic acyl pocket, a localized negative electrostatic potential, a large open leaving group-accommodating groove, and a pivotal acidic residue, Asp-123, after the nucleophile Ser-122. This is the first time that a residue immediately after the nucleophile has been found to have its side chain directed into the substrate binding pocket and play an essential role in substrate discrimination in serine hydrolases. These results as well as a phylogenetic analysis establish that the enzyme functions as a specific {alpha}-amino acid ester hydrolase. Valacyclovirase is a valuable target for amino acid ester prodrug-based oral drug delivery enhancement strategies.

  7. Mice lacking lipid droplet-associated hydrolase, a gene linked to human prostate cancer, have normal cholesterol ester metabolism

    DEFF Research Database (Denmark)

    Kory, Nora; Grond, Susanne; Kamat, Siddhesh S

    2017-01-01

    Variations in the gene LDAH (C2ORF43), which encodes lipid droplet-associated hydrolase (LDAH), are among few loci associated with human prostate cancer. Homologs of LDAH have been identified as proteins of lipid droplets (LDs). LDs are cellular organelles that store neutral lipids...

  8. Discovery of α-L-arabinopyranosidases from human gut microbiome expands the diversity within glycoside hydrolase family 42

    DEFF Research Database (Denmark)

    Viborg, Alexander Holm; Katayama, Takane; Arakawa, Takatoshi

    2017-01-01

    Enzymes of the glycoside hydrolase family 42 (GH42) are widespread in bacteria of the human gut microbiome and play fundamental roles in the decomposition of both milk and plant oligosaccharides. All GH42 enzymes characterized so far have β-galactosidase activity. Here, we report the existence...

  9. Tissue Expressions of Soluble Human Epoxide Hydrolase-2 Enzyme in Patients with Temporal Lobe Epilepsy.

    Science.gov (United States)

    Ahmedov, Merdin Lyutviev; Kemerdere, Rahsan; Baran, Oguz; Inal, Berrin Bercik; Gumus, Alper; Coskun, Cihan; Yeni, Seher Naz; Eren, Bulent; Uzan, Mustafa; Tanriverdi, Taner

    2017-10-01

    We sought to simply demonstrate how levels of soluble human epoxide hydrolase-2 show changes in both temporal the cortex and hippocampal complex in patients with temporal lobe epilepsy. A total of 20 patients underwent anterior temporal lobe resection due to temporal lobe epilepsy. The control group comprised 15 people who died in traffic accidents or by falling from a height, and their autopsy findings were included. Adequately sized temporal cortex and hippocampal samples were removed from each patient during surgery, and the same anatomic structures were removed from the control subjects during the autopsy procedures. Each sample was stored at -80°C as rapidly as possible until the enzyme assay. The temporal cortex in the epilepsy patients had a significantly higher enzyme level than did the temporal cortex of the control group (P = 0.03). Correlation analysis showed that as the enzyme level increases in the temporal cortex, it also increases in the hippocampal complex (r 2  = 0.06, P = 0.00001). More important, enzyme tissue levels showed positive correlations with seizure frequency in both the temporal cortex and hippocampal complex in patients (r 2  = 0.7, P = 0.00001 and r 2  = 0.4, P = 0.003, respectively). The duration of epilepsy was also positively correlated with the hippocampal enzyme level (r 2  = 0.06, P = 0.00001). Soluble human epoxy hydrolase enzyme-2 is increased in both lateral and medial temporal tissues in temporal lobe epilepsy. Further studies should be conducted as inhibition of this enzyme has resulted in a significant decrease in or stopping of seizures and attenuated neuroinflammation in experimental epilepsy models in the current literature. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Ubiquitin Carboxy-Terminal HydrolaseL3 Correlates with Human Sperm Count, Motility and Fertilization.

    Science.gov (United States)

    Wang, Meijiao; Yu, Tinghe; Hu, Lina; Cheng, Zhi; Li, Min

    2016-01-01

    Ubiquitin C-terminal hydrolase L3 (UCHL3) belongs to the group of deubiquitinating enzymes and plays a part in apoptosis of germ cells and the differentiation of spermatocytes into spermatids. However, the exact role of UCHL3 in human spermatogenesis and sperm function remains unknown. Here we examined the level and activity of UCHL3 in spermatozoa from men with asthenozoospermia (A), oligoasthenozoospermia (OA) or normozoospermia (N). Immunofluorescence indicated that UCHL3 was mainly localized in the acrosome and throughout the flagella, and western blotting revealed a lower level in A or OA compared with N (p sperm count, concentration and motility. The UCHL3 level was positively correlated with the normal fertilization rate (FR) and percentage of embryos suitable for transfer/cryopreservation of in vitro fertilization (IVF). The UCHL3 activity was also positively correlated with FR, the percentage of embryos suitable for transfer/cryopreservation and high-quality embryos rate of IVF. Aforementioned correlations were not manifested in intra-cytoplasmic sperm injection (ICSI). These findings suggest that UCHL3 may play a role in male infertility.

  11. Lysophosphatidylcholine hydrolases of human erythrocytes, lymphocytes, and brain: Sensitive targets of conserved specificity for organophosphorus delayed neurotoxicants

    International Nuclear Information System (INIS)

    Vose, Sarah C.; Holland, Nina T.; Eskenazi, Brenda; Casida, John E.

    2007-01-01

    Brain neuropathy target esterase (NTE), associated with organophosphorus (OP)-induced delayed neuropathy, has the same OP inhibitor sensitivity and specificity profiles assayed in the classical way (paraoxon-resistant, mipafox-sensitive hydrolysis of phenyl valerate) or with lysophosphatidylcholine (LysoPC) as the substrate. Extending our earlier observation with mice, we now examine human erythrocyte, lymphocyte, and brain LysoPC hydrolases as possible sensitive targets for OP delayed neurotoxicants and insecticides. Inhibitor profiling of human erythrocytes and lymphocytes gave the surprising result of essentially the same pattern as with brain. Human erythrocyte LysoPC hydrolases are highly sensitive to OP delayed neurotoxicants, with in vitro IC 50 values of 0.13-85 nM for longer alkyl analogs, and poorly sensitive to the current OP insecticides. In agricultural workers, erythrocyte LysoPC hydrolyzing activities are similar for newborn children and their mothers and do not vary with paraoxonase status but have high intersample variation that limits their use as a biomarker. Mouse erythrocyte LysoPC hydrolase activity is also of low sensitivity in vitro and in vivo to the OP insecticides whereas the delayed neurotoxicant ethyl n-octylphosphonyl fluoride inhibits activity in vivo at 1-3 mg/kg. Overall, inhibition of blood LysoPC hydrolases is as good as inhibition of brain NTE as a predictor of OP inducers of delayed neuropathy. NTE and lysophospholipases (LysoPLAs) both hydrolyze LysoPC, yet they are in distinct enzyme families with no sequence homology and very different catalytic sites. The relative contributions of NTE and LysoPLAs to LysoPC hydrolysis and clearance from erythrocytes, lymphocytes, and brain remain to be defined

  12. Structure-activity relationships of amide-phosphonate derivatives as inhibitors of the human soluble epoxide hydrolase.

    Science.gov (United States)

    Kim, In-Hae; Park, Yong-Kyu; Nishiwaki, Hisashi; Hammock, Bruce D; Nishi, Kosuke

    2015-11-15

    Structure-activity relationships of amide-phosphonate derivatives as inhibitors of the human soluble epoxide hydrolase (sEH) were investigated. First, a series of alkyl or aryl groups were substituted on the carbon alpha to the phosphonate function in amide compounds to see whether substituted phosphonates can act as a secondary pharmacophore. A tert-butyl group (16) on the alpha carbon was found to yield most potent inhibition on the target enzyme. A 4-50-fold drop in inhibition was induced by other substituents such as aryls, substituted aryls, cycloalkyls, and alkyls. Then, the modification of the O-substituents on the phosphonate function revealed that diethyl groups (16 and 23) were preferable for inhibition to other longer alkyls or substituted alkyls. In amide compounds with the optimized diethylphosphonate moiety and an alkyl substitution such as adamantane (16), tetrahydronaphthalene (31), or adamantanemethane (36), highly potent inhibitions were gained. In addition, the resulting potent amide-phosphonate compounds had reasonable water solubility, suggesting that substituted phosphonates in amide inhibitors are effective for both inhibition potency on the human sEH and water solubility as a secondary pharmacophore. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Glycoside hydrolases having multiple hydrolase activities

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Zhiwei; Friedland, Gregory D.; Chhabra, Swapnil R.; Chivian, Dylan C.; Simmons, Blake A

    2017-08-08

    Glycoside hydrolases having at least two different hydrolytic activities are provided. In one embodiment, an isolated recombinant hydrolase having at least two activities selected from a group including asparagine derivatives, glutamine derivatives, and histidine derivatives is provided. Further, a method of generating free sugars from a mixture comprising asparagine derivatives, glutamine derivatives, and histidine derivatives is provided.

  14. Monoclonal antibodies reveal multiple forms of expression of human microsomal epoxide hydrolase

    Energy Technology Data Exchange (ETDEWEB)

    Duan, Hongying; Takagi, Akira [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Kayano, Hidekazu [Department of Pathology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Koyama, Isamu [Department of Digestive and General Surgery, Saitama International Medical Center, Faculty of Medicine, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1298 (Japan); Morisseau, Christophe; Hammock, Bruce D. [Department of Entomology and Cancer Center, University of California, Davis, One Shields Avenue, Davis, CA 95616-8584 (United States); Akatsuka, Toshitaka, E-mail: akatsuka@saitama-med.ac.jp [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan)

    2012-04-01

    In a previous study, we developed five kinds of monoclonal antibodies against different portions of human mEH: three, anti-N-terminal; one, anti-C-terminal; one, anti-conformational epitope. Using them, we stained the intact and the permeabilized human cells of various kinds and performed flow cytometric analysis. Primary hepatocytes and peripheral blood mononuclear cells (PBMC) showed remarkable differences. On the surface, hepatocytes exhibited 4 out of 5 epitopes whereas PBMC did not show any of the epitopes. mEH was detected inside both cell types, but the most prominent expression was observed for the conformational epitope in the hepatocytes and the two N-terminal epitopes in PBMC. These differences were also observed between hepatocyte-derived cell lines and mononuclear cell-derived cell lines. In addition, among each group, there were several differences which may be related to the cultivation, the degree of differentiation, or the original cell subsets. We also noted that two glioblastoma cell lines reveal marked expression of the conformational epitope on the surface which seemed to correlate with the brain tumor-associated antigen reported elsewhere. Several cell lines also underwent selective permeabilization before flow cytometric analysis, and we noticed that the topological orientation of mEH on the ER membrane in those cells was in accordance with the previous report. However, the orientation on the cell surface was inconsistent with the report and had a great variation between the cells. These findings show the multiple mode of expression of mEH which may be possibly related to the multiple roles that mEH plays in different cells. -- Highlights: ► We examine expression of five mEH epitopes in human cells. ► Remarkable differences exist between hepatocytes and PBMC. ► mEH expression in cell lines differs depending on several factors. ► Some glioblastoma cell lines reveal marked surface expression of mEH. ► Topology of mEH on the cell

  15. Development of monoclonal antibodies to human microsomal epoxide hydrolase and analysis of “preneoplastic antigen”-like molecules

    Energy Technology Data Exchange (ETDEWEB)

    Duan, Hongying [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Yoshimura, Kazunori [Department of Physiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Kobayashi, Nobuharu; Sugiyama, Kazuo [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Sawada, Jun-ichi; Saito, Yoshiro [Division of Biochemistry and Immunochemistry, National Institute of Health Sciences, Kamiyoga 1-18-1, Setagaya-ku, Tokyo 158-8501 (Japan); Morisseau, Christophe; Hammock, Bruce D. [Department of Entomology and Cancer Center, University of California, Davis, One Shields Avenue, Davis, CA 95616-8584 (United States); Akatsuka, Toshitaka, E-mail: akatsuka@saitama-med.ac.jp [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan)

    2012-04-01

    Microsomal epoxide hydrolase (mEH) is a drug metabolizing enzyme which resides on the endoplasmic reticulum (ER) membrane and catalyzes the hydration of reactive epoxide intermediates that are formed by cytochrome P450s. mEH is also thought to have a role in bile acid transport on the plasma membrane of hepatocytes. It is speculated that efficient execution of such multiple functions is secured by its orientation and association with cytochrome P450 enzymes on the ER membrane and formation of a multiple transport system on the plasma membrane. In certain disease status, mEH loses its association with the membrane and can be detected as distinct antigens in the cytosol of preneoplastic foci of liver (preneoplastic antigen), in the serum in association with hepatitis C virus infection (AN antigen), or in some brain tumors. To analyze the antigenic structures of mEH in physiological and pathological conditions, we developed monoclonal antibodies against different portions of mEH. Five different kinds of antibodies were obtained: three, anti-N-terminal portions; one anti-C-terminal; and one, anti-conformational epitope. By combining these antibodies, we developed antigen detection methods which are specific to either the membrane-bound form or the linearized form of mEH. These methods detected mEH in the culture medium released from a hepatocellular carcinoma cell line and a glioblastoma cell line, which was found to be a multimolecular complex with a unique antigenic structure different from that of the membrane-bound form of mEH. These antibodies and antigen detection methods may be useful to study pathological changes of mEH in various human diseases. -- Highlights: ► Monoclonal antibodies against different portions of mEH were developed. ► They discriminate between the membrane-bound and the linearized forms of mEH. ► We analyze the antigenic structure of the altered form of mEH in tumor cells. ► Preneoplastic antigen is a multimolecular complex of mEH with

  16. Nanobody Based Immunoassay for Human Soluble Epoxide Hydrolase Detection Using Polymeric Horseradish Peroxidase (PolyHRP) for Signal Enhancement: The Rediscovery of PolyHRP?

    Science.gov (United States)

    Li, Dongyang; Cui, Yongliang; Morisseau, Christophe; Gee, Shirley J; Bever, Candace S; Liu, Xiangjiang; Wu, Jian; Hammock, Bruce D; Ying, Yibin

    2017-06-06

    Soluble epoxide hydrolase (sEH) is a potential pharmacological target for treating hypertension, vascular inflammation, cancer, pain, and multiple cardiovascular related diseases. A variable domain of the heavy chain antibody (termed single domain antibody (sdAb), nanobody, or VHH) possesses the advantages of small size, high stability, ease of genetic manipulation, and ability for continuous manufacture, making such nanobody a superior choice as an immunoreagent. In this work, we developed an ultrasensitive nanobody based immunoassay for human sEH detection using polymeric horseradish peroxidase (PolyHRP) for signal enhancement. Llama nanobodies against human sEH were used as the detection antibody in sandwich enzyme linked immunosorbent assays (ELISA) with polyclonal anti-sEH as the capture antibody. A conventional sandwich ELISA using a horseradish peroxidase (HRP) labeled anti-hemeagglutinin (HA) tag as the tracer showed a marginal sensitivity (0.0015 optical density (OD)·mL/ng) and limit of detection (LOD) of 3.02 ng/mL. However, the introduction of the PolyHRP as the tracer demonstrated a 141-fold increase in the sensitivity (0.21 OD·mL/ng) and 57-fold decrease in LOD (0.05 ng/mL). Systematic comparison of three different tracers in four ELISA formats demonstrated the overwhelming advantage of PolyHRP as a label for nanobody based immunoassay. This enhanced sEH immunoassay was further evaluated in terms of selectivity against other epoxide hydrolases and detection of the target protein in human tissue homogenate samples. Comparison with an enzyme activity based assay and a Western blot for sEH detection reveals good correlation with the immunoassay. This work demonstrates increased competiveness of nanobodies for practical sEH protein detection utilizing PolyHRP. It is worthwhile to rediscover the promising potential of PolyHRP in nanobody and other affinity based methods after its low-profile existence for decades.

  17. Variants of glycoside hydrolases

    Science.gov (United States)

    Teter, Sarah [Davis, CA; Ward, Connie [Hamilton, MT; Cherry, Joel [Davis, CA; Jones, Aubrey [Davis, CA; Harris, Paul [Carnation, WA; Yi, Jung [Sacramento, CA

    2011-04-26

    The present invention relates to variants of a parent glycoside hydrolase, comprising a substitution at one or more positions corresponding to positions 21, 94, 157, 205, 206, 247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, and optionally further comprising a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2, wherein the variants have glycoside hydrolase activity. The present invention also relates to nucleotide sequences encoding the variant glycoside hydrolases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.

  18. The activity of the endocannabinoid metabolising enzyme fatty acid amide hydrolase in subcutaneous adipocytes correlates with BMI in metabolically healthy humans

    Directory of Open Access Journals (Sweden)

    Alexander Stephen PH

    2011-08-01

    Full Text Available Abstract Background The endocannabinoid system (ECS is a ubiquitously expressed signalling system, with involvement in lipid metabolism and obesity. There are reported changes in obesity of blood concentrations of the endocannabinoids anandamide (AEA and 2-arachidonoylglcyerol (2-AG, and of adipose tissue expression levels of the two key catabolic enzymes of the ECS, fatty acid amide hydrolase (FAAH and monoacylglycerol lipase (MGL. Surprisingly, however, the activities of these enzymes have not been assayed in conditions of increasing adiposity. The aim of the current study was to investigate whether FAAH and MGL activities in human subcutaneous adipocytes are affected by body mass index (BMI, or other markers of adiposity and metabolism. Methods Subcutaneous abdominal mature adipocytes, fasting blood samples and anthropometric measurements were obtained from 28 metabolically healthy subjects representing a range of BMIs. FAAH and MGL activities were assayed in mature adipocytes using radiolabelled substrates. Serum glucose, insulin and adipokines were determined using ELISAs. Results MGL activity showed no relationship with BMI or other adiposity indices, metabolic markers (fasting serum insulin or glucose or serum adipokine levels (adiponectin, leptin or resistin. In contrast, FAAH activity in subcutaneous adipocytes correlated positively with BMI and waist circumference, but not with skinfold thickness, metabolic markers or serum adipokine levels. Conclusions In this study, novel evidence is provided that FAAH activity in subcutaneous mature adipocytes increases with BMI, whereas MGL activity does not. These findings support the hypothesis that some components of the ECS are upregulated with increasing adiposity in humans, and that AEA and 2-AG may be regulated differently.

  19. DZNep, inhibitor of S-adenosylhomocysteine hydrolase, down-regulates expression of SETDB1 H3K9me3 HMTase in human lung cancer cells.

    Science.gov (United States)

    Lee, Ju-Kyung; Kim, Keun-Cheol

    2013-09-06

    3-Deazaneplanocin A (DZNep), an epigenetic anticancer drug, leads to the indirect suppression of S-adenosyl methionine-dependent cellular methylations by inhibiting S-adenosyl homocystein (AdoHcy) hydrolase. Although it is well known that DZNep targets the degradation of EZH2 protein, H3K27me3 HMTase, there are still uncertainties about the regulation of other types of HMTases during cell death. In this study, we describe that SETDB1 gene expression was regulated by DZNep treatment in human lung cancer cells. We confirm that DZNep induced growth inhibition and increased the dead cell population of lung cancer cells. DZNep treatment affected histone methylations, including H3K27me3 and H3K9me3, but not H3K4me3. Reduced levels of H3K27me3 and H3K9me3 were related with the decreased EZH2 and SETDB1 proteins. Real time PCR analysis showed that SETDB1 gene expression was decreased by DZNep treatment, but no effect was observed for EZH2 gene expression. We cloned the promoter region of SETDB1 and SUV39H1 genes, and performed luciferase assays. The promoter activity of SETDB1 gene was down regulated by DZNep treatment, whereas no effect on SUV39H1 promoter activity was observed. In conclusion, we suggest that DZNep regulates not only on H3K27me3 HMTase EZH2, but also H3K9 HMTase SETDB1 gene expression at the transcription level, implicating that the mechanism of action of DZNep targets multiple HMTases during the death of lung cancer cells. Copyright © 2013 Elsevier Inc. All rights reserved.

  20. Nanobody based immunoassay for human soluble epoxide hydrolase detection using polyHRP for signal enhancement—the rediscovery of polyHRP

    Science.gov (United States)

    Soluble epoxide hydrolase (sEH) is a potential pharmacological target for treating hypertension, vascular inflammation, cancer, pain and multiple cardiovascular related diseases. A variable domain of a heavy chain only antibody (termed sdAb, nanobody or VHH) possesses advantages of small size, high ...

  1. Peptidoglycan Hydrolases of Escherichia coli

    Science.gov (United States)

    van Heijenoort, Jean

    2011-01-01

    Summary: The review summarizes the abundant information on the 35 identified peptidoglycan (PG) hydrolases of Escherichia coli classified into 12 distinct families, including mainly glycosidases, peptidases, and amidases. An attempt is also made to critically assess their functions in PG maturation, turnover, elongation, septation, and recycling as well as in cell autolysis. There is at least one hydrolytic activity for each bond linking PG components, and most hydrolase genes were identified. Few hydrolases appear to be individually essential. The crystal structures and reaction mechanisms of certain hydrolases having defined functions were investigated. However, our knowledge of the biochemical properties of most hydrolases still remains fragmentary, and that of their cellular functions remains elusive. Owing to redundancy, PG hydrolases far outnumber the enzymes of PG biosynthesis. The presence of the two sets of enzymes acting on the PG bonds raises the question of their functional correlations. It is difficult to understand why E. coli keeps such a large set of PG hydrolases. The subtle differences in substrate specificities between the isoenzymes of each family certainly reflect a variety of as-yet-unidentified physiological functions. Their study will be a far more difficult challenge than that of the steps of the PG biosynthesis pathway. PMID:22126997

  2. Further characterization of intestinal lactase/phlorizin hydrolase

    DEFF Research Database (Denmark)

    Skovbjerg, H; Norén, O; Sjöström, H

    1982-01-01

    Pig intestinal lactase/phlorizin hydrolase (EC 3.2.1.23/62) was purified in its amphiphilic form by immunoadsorbent chromatography. The purified enzyme was free of other known brush border enzymes and appeared homogeneous in immunoelectrophoresis and polyacrylamide gel electrophoresis in the pres......Pig intestinal lactase/phlorizin hydrolase (EC 3.2.1.23/62) was purified in its amphiphilic form by immunoadsorbent chromatography. The purified enzyme was free of other known brush border enzymes and appeared homogeneous in immunoelectrophoresis and polyacrylamide gel electrophoresis...... in the presence of SDS. Pig lactase/phlorizin hydrolase was shown to have the same quaternary structure as the human enzyme, i.e., built up of two polypeptides of the same molecular weight (160000). In addition to hydrolyzing lactose, phlorizin and a number of synthetic substrates, both the human and the pig...... membranes (basolateral and intracellular membranes) exhibited in SDS-polyacrylamide gel electrophoresis the same size of constituent polypeptides and the same catalytic and immunological properties as a normal brush border lactase/phlorizin hydrolase....

  3. Fluorometric determination of paraoxon in human serum using a gold nanoparticle-immobilized organophosphorus hydrolase and coumarin 1 as a competitive inhibitor

    International Nuclear Information System (INIS)

    Kamelipour, Nahid; Mohsenifar, Afshin; Rahmani-Cherati, Tavoos; Tabatabaei, Meisam; Khoshnevisan, Kamyar; Allameh, Abdolamir; Milani, Majid M.; Etemadikia, Batool; Najavand, Saeid

    2014-01-01

    A dimeric organophosphorus hydrolase (OPH; EC 3.1.8.1; 72 kDa) was isolated from wild-type bacteria, analyzed for its 16s rRNA sequence, purified, and immobilized on gold nanoparticles (AuNPs) to form the transducer part of a biosensor. The isolated strain was identified as Pseudomonas aeruginosa. The AuNPs were characterized by transmission electron microscopy and localized surface plasmon resonance. Covalent binding of OPH to the AuNPs was confirmed by spectrophotometry, enzymatic activity assays, and FTIR spectroscopy. Coumarin 1, a competitive inhibitor of OPH, was used as a fluorogenic probe. The bioconjugates quench the emission of coumarin 1 upon binding, but the addition of paraoxon results in an enhancement of fluorescence that is directly proportional to the concentration of paraoxon. The gold-OPH conjugates were then used to determine paraoxon in serum samples spiked with varying levels of paraoxon. The method works in the 50 to 1,050 nM concentration range, has a low standard deviation (with a CV of 5.7–11 %), and a detection limit as low as 5 × 10 −11 M. (author)

  4. Evaluation of fish models of soluble epoxide hydrolase inhibition.

    OpenAIRE

    Newman, J W; Denton, D L; Morisseau, C; Koger, C S; Wheelock, C E; Hinton, D E; Hammock, B D

    2001-01-01

    Substituted ureas and carbamates are mechanistic inhibitors of the soluble epoxide hydrolase (sEH). We screened a set of chemicals containing these functionalities in larval fathead minnow (Pimphales promelas) and embryo/larval golden medaka (Oryzias latipes) models to evaluate the utility of these systems for investigating sEH inhibition in vivo. Both fathead minnow and medaka sEHs were functionally similar to the tested mammalian orthologs (murine and human) with respect to substrate hydrol...

  5. Lysophosphatidic acids are new substrates for the phosphatase domain of soluble epoxide hydrolase[S

    OpenAIRE

    Oguro, Ami; Imaoka, Susumu

    2012-01-01

    Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that has a C-terminus epoxide hydrolase domain and an N-terminus phosphatase domain. The endogenous substrates of epoxide hydrolase are known to be epoxyeicosatrienoic acids, but the endogenous substrates of the phosphatase activity are not well understood. In this study, to explore the substrates of sEH, we investigated the inhibition of the phosphatase activity of sEH toward 4-methylumbelliferyl phosphate by using lecithin and its hyd...

  6. A new insight into the physiological role of bile salt hydrolase among intestinal bacteria from the genus Bifidobacterium.

    Science.gov (United States)

    Jarocki, Piotr; Podleśny, Marcin; Glibowski, Paweł; Targoński, Zdzisław

    2014-01-01

    This study analyzes the occurrence of bile salt hydrolase in fourteen strains belonging to the genus Bifidobacterium. Deconjugation activity was detected using a plate test, two-step enzymatic reaction and activity staining on a native polyacrylamide gel. Subsequently, bile salt hydrolases from B. pseudocatenulatum and B. longum subsp. suis were purified using a two-step chromatographic procedure. Biochemical characterization of the bile salt hydrolases showed that the purified enzymes hydrolyzed all of the six major human bile salts under the pH and temperature conditions commonly found in the human gastrointestinal tract. Next, the dynamic rheometry was applied to monitor the gelation process of deoxycholic acid under different conditions. The results showed that bile acids displayed aqueous media gelating properties. Finally, gel-forming abilities of bifidobacteria exhibiting bile salt hydrolase activity were analyzed. Our investigations have demonstrated that the release of deconjugated bile acids led to the gelation phenomenon of the enzymatic reaction solution containing purified BSH. The presented results suggest that bile salt hydrolase activity commonly found among intestinal microbiota increases hydrogel-forming abilities of certain bile salts. To our knowledge, this is the first report showing that bile salt hydrolase activity among Bifidobacterium is directly connected with the gelation process of bile salts. In our opinion, if such a phenomenon occurs in physiological conditions of human gut, it may improve bacterial ability to colonize the gastrointestinal tract and their survival in this specific ecological niche.

  7. Cytosolic cholesterol ester hydrolase in adrenal cortex

    OpenAIRE

    Tocher, Douglas R.

    1983-01-01

    Cholesterol ester hydrolase (CEH) in adrenocortical cytosol was known to be phosphorylated and activated, in response to ACTH in a cAMPdependent protein kinase mediated process. The purification of CEH from bovine adrenocortical cytosol was attempted. The use of detergents to solubilise the enzyme from lipid-rich aggregates was investigated and sodium cholate was found to be effective. A purification procedure using cholate solubilised enzyme was developed. The detergent int...

  8. In Silico Investigation of Flavonoids as Potential Trypanosomal Nucleoside Hydrolase Inhibitors

    Directory of Open Access Journals (Sweden)

    Christina Hung Hung Ha

    2015-01-01

    Full Text Available Human African Trypanosomiasis is endemic to 37 countries of sub-Saharan Africa. It is caused by two related species of Trypanosoma brucei. Current therapies suffer from resistance and public accessibility of expensive medicines. Finding safer and effective therapies of natural origin is being extensively explored worldwide. Pentamidine is the only available therapy for inhibiting the P2 adenosine transporter involved in the purine salvage pathway of the trypanosomatids. The objective of the present study is to use computational studies for the investigation of the probable trypanocidal mechanism of flavonoids. Docking experiments were carried out on eight flavonoids of varying level of hydroxylation, namely, flavone, 5-hydroxyflavone, 7-hydroxyflavone, chrysin, apigenin, kaempferol, fisetin, and quercetin. Using AutoDock 4.2, these compounds were tested for their affinity towards inosine-adenosine-guanosine nucleoside hydrolase and the inosine-guanosine nucleoside hydrolase, the major enzymes of the purine salvage pathway. Our results showed that all of the eight tested flavonoids showed high affinities for both hydrolases (lowest free binding energy ranging from −10.23 to −7.14 kcal/mol. These compounds, especially the hydroxylated derivatives, could be further studied as potential inhibitors of the nucleoside hydrolases.

  9. Crystal structure of bile salt hydrolase from Lactobacillus salivarius.

    Science.gov (United States)

    Xu, Fuzhou; Guo, Fangfang; Hu, Xiao Jian; Lin, Jun

    2016-05-01

    Bile salt hydrolase (BSH) is a gut-bacterial enzyme that negatively influences host fat digestion and energy harvesting. The BSH enzyme activity functions as a gateway reaction in the small intestine by the deconjugation of glycine-conjugated or taurine-conjugated bile acids. Extensive gut-microbiota studies have suggested that BSH is a key mechanistic microbiome target for the development of novel non-antibiotic food additives to improve animal feed production and for the design of new measures to control obesity in humans. However, research on BSH is still in its infancy, particularly in terms of the structural basis of BSH function, which has hampered the development of BSH-based strategies for improving human and animal health. As an initial step towards the structure-function analysis of BSH, C-terminally His-tagged BSH from Lactobacillus salivarius NRRL B-30514 was crystallized in this study. The 1.90 Å resolution crystal structure of L. salivarius BSH was determined by molecular replacement using the structure of Clostridium perfringens BSH as a starting model. It revealed this BSH to be a member of the N-terminal nucleophile hydrolase superfamily. Crystals of apo BSH belonged to space group P21212, with unit-cell parameters a = 90.79, b = 87.35, c = 86.76 Å (PDB entry 5hke). Two BSH molecules packed perfectly as a dimer in one asymmetric unit. Comparative structural analysis of L. salivarius BSH also identified potential residues that contribute to catalysis and substrate specificity.

  10. Hydrolase activity in Jerusalem artichoke and chicory

    Energy Technology Data Exchange (ETDEWEB)

    Klaushofer, H.; Abraham, B.; Leichtfried, G.

    1988-03-01

    Post-harvest storage of chicory and Jerusalem artichoke and overwintering of Jerusalem artichoke in the soil cause a more or less pronounced shortening of the fructan chain, depending on the variety. The proportion of fructose in the total fructan thus shifts towards glucose. This reduction on the fructose/glucose ratio is undesirable if the intention is to obtain a sweetener of high fructose content. In this work an attempt was made, via the quantity of fructose formed after a 4(3)-hour reaction of a tuber (root) extract with inulin, to assign a characteristic value to the depolymerization tendency of the material in question. However, since the plant extract not only contains enzymes (hydrolase A and B) that shorten the fructan chains but the activity of fructosyltransferase (SST, FFT) and enzymes of microbial origin (inulinase II, invertase) must also be considered, the concept of 'hydrolase activity' used by the authors is essentially an expression of 'total activity'. The activity unit (EU) is defined as the ability to split of 1 ..mu..mol of fructose from (chicory) inulin per minute under experimental conditions. Values of 0.25 to 0.77 EU/g dry solids were found in Jerusalem artichoke. Considerable differences may occur between varieties from the same cultivated area and the same harvest period. With one and the same variety, the activity appears to be subject to marked yearly fluctuations, so that at present, because of hydrolase activity, nothing certain can be said about the depolymerization tendency of a variety.

  11. Lysophosphatidic acids are new substrates for the phosphatase domain of soluble epoxide hydrolase[S

    Science.gov (United States)

    Oguro, Ami; Imaoka, Susumu

    2012-01-01

    Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that has a C-terminus epoxide hydrolase domain and an N-terminus phosphatase domain. The endogenous substrates of epoxide hydrolase are known to be epoxyeicosatrienoic acids, but the endogenous substrates of the phosphatase activity are not well understood. In this study, to explore the substrates of sEH, we investigated the inhibition of the phosphatase activity of sEH toward 4-methylumbelliferyl phosphate by using lecithin and its hydrolyzed products. Although lecithin itself did not inhibit the phosphatase activity, the hydrolyzed lecithin significantly inhibited it, suggesting that lysophospholipid or fatty acid can inhibit it. Next, we investigated the inhibition of phosphatase activity by lysophosphatidyl choline, palmitoyl lysophosphatidic acid, monopalmitoyl glycerol, and palmitic acid. Palmitoyl lysophosphatidic acid and fatty acid efficiently inhibited phosphatase activity, suggesting that lysophosphatidic acids (LPAs) are substrates for the phosphatase activity of sEH. As expected, palmitoyl, stearoyl, oleoyl, and arachidonoyl LPAs were efficiently dephosphorylated by sEH (Km, 3–7 μM; Vmax, 150–193 nmol/min/mg). These results suggest that LPAs are substrates of sEH, which may regulate physiological functions of cells via their metabolism. PMID:22217705

  12. Glycoside Hydrolases across Environmental Microbial Communities.

    Directory of Open Access Journals (Sweden)

    Renaud Berlemont

    2016-12-01

    Full Text Available Across many environments microbial glycoside hydrolases support the enzymatic processing of carbohydrates, a critical function in many ecosystems. Little is known about how the microbial composition of a community and the potential for carbohydrate processing relate to each other. Here, using 1,934 metagenomic datasets, we linked changes in community composition to variation of potential for carbohydrate processing across environments. We were able to show that each ecosystem-type displays a specific potential for carbohydrate utilization. Most of this potential was associated with just 77 bacterial genera. The GH content in bacterial genera is best described by their taxonomic affiliation. Across metagenomes, fluctuations of the microbial community structure and GH potential for carbohydrate utilization were correlated. Our analysis reveals that both deterministic and stochastic processes contribute to the assembly of complex microbial communities.

  13. Novel microbial epoxide hydrolases for biohydrolysis of glycidyl derivatives

    Czech Academy of Sciences Publication Activity Database

    Kotík, Michael; Břicháč, Jiří; Kyslík, Pavel

    2005-01-01

    Roč. 120, - (2005), s. 364-375 ISSN 0168-1656 Institutional research plan: CEZ:AV0Z5020903 Keywords : screening * epoxide hydrolase * biotransformation Subject RIV: EE - Microbiology, Virology Impact factor: 2.687, year: 2005

  14. Use of full recovery hydrolasing equipment for facility decommissioning - 16325

    International Nuclear Information System (INIS)

    Martin, Scott A.; Adams, Scott R.

    2009-01-01

    The removal of surface contamination is a major challenge for nearly all nuclear facilities undergoing, or awaiting, decommissioning. Conventional means of surface decontamination can expose workers to unnecessary hazards, and are often not fit-for-purpose due to size constraints or weight restrictions. Additionally, conventional methods are not always easily deployed remotely due to their complexity or required services. The use of ultra high pressure water for surface decontamination, known as hydrolasing, is recognized as a technology which can be used in various applications requiring surface removal. Hydrolasing is an advantageous technology for many reasons including its versatility, overall simplicity and relative ease of remote deployment. For the nuclear industry, one of the largest challenges with regards to the use of hydrolasing is the requirement for the full recovery of the injected water and removed solids. For nonnuclear applications, there is often no requirement for recovery of the liquid and solid waste, which has led to few system designs which will recover the waste in full. S.A. Robotics' experience with the deployment of ultra high pressure water systems for nuclear applications has shown that full recovery of injected water and removed solids is achievable in both underwater and in-air applications. Innovative equipment and system design have allowed S.A. Robotics' hydrolasing systems to achieve near 100% solid and liquid recovery during concrete hydrolasing. This technology has been deployed for Fluor Hanford at Hanford's K-Basins, as well as for UKAEA as part of the Windscale Piles decommissioning project. The purpose of this paper is to provide a short description of the hydrolasing process and the associated waste issues, describe the unique design features of S.A. Robotics' hydrolasing systems which combat these issues, and provide an overview of two of the hydrolasing projects that S.A. Robotics has completed. (authors)

  15. Structure of HsaD, a steroid-degrading hydrolase, from Mycobacterium tuberculosis

    International Nuclear Information System (INIS)

    Lack, Nathan; Lowe, Edward D.; Liu, Jie; Eltis, Lindsay D.; Noble, Martin E. M.; Sim, Edith; Westwood, Isaac M.

    2007-01-01

    The structure of HsaD, a carbon–carbon bond serine hydrolase involved in steroid catabolism that is critical for the survival of M. tuberculosis inside human macrophages, has been solved by X-ray crystallography. Data were collected at the Diamond Light Source in Oxfordshire, England: this paper describes one of the first structures determined at the new synchrotron. Tuberculosis is a major cause of death worldwide. Understanding of the pathogenicity of Mycobacterium tuberculosis has been advanced by gene analysis and has led to the identification of genes that are important for intracellular survival in macrophages. One of these genes encodes HsaD, a meta-cleavage product (MCP) hydrolase that catalyzes the hydrolytic cleavage of a carbon–carbon bond in cholesterol metabolism. This paper describes the production of HsaD as a recombinant protein and, following crystallization, the determination of its three-dimensional structure to 2.35 Å resolution by X-ray crystallography at the Diamond Light Source in Oxfordshire, England. To the authors’ knowledge, this study constitutes the first report of a structure determined at the new synchrotron facility. The volume of the active-site cleft of the HsaD enzyme is more than double the corresponding active-site volumes of related MCP hydrolases involved in the catabolism of aromatic compounds, consistent with the specificity of HsaD for steroids such as cholesterol. Knowledge of the structure of the enzyme facilitates the design of inhibitors

  16. Brucella abortus choloylglycine hydrolase affects cell envelope composition and host cell internalization.

    Directory of Open Access Journals (Sweden)

    María Inés Marchesini

    Full Text Available Choloylglycine hydrolase (CGH, E.C. 3.5.1.24 is a conjugated bile salt hydrolase that catalyses the hydrolysis of the amide bond in conjugated bile acids. Bile salt hydrolases are expressed by gastrointestinal bacteria, and they presumably decrease the toxicity of host's conjugated bile salts. Brucella species are the causative agents of brucellosis, a disease affecting livestock and humans. CGH confers Brucella the ability to deconjugate and resist the antimicrobial action of bile salts, contributing to the establishment of a successful infection through the oral route in mice. Additionally, cgh-deletion mutant was also attenuated in intraperitoneally inoculated mice, which suggests that CGH may play a role during systemic infection other than hydrolyzing conjugated bile acids. To understand the role CGH plays in B. abortus virulence, we infected phagocytic and epithelial cells with a cgh-deletion mutant (Δcgh and found that it is defective in the internalization process. This defect along with the increased resistance of Δcgh to the antimicrobial action of polymyxin B, prompted an analysis of the cell envelope of this mutant. Two-dimensional electrophoretic profiles of Δcgh cell envelope-associated proteins showed an altered expression of Omp2b and different members of the Omp25/31 family. These results were confirmed by Western blot analysis with monoclonal antibodies. Altogether, the results indicate that Brucella CGH not only participates in deconjugation of bile salts but also affects overall membrane composition and host cell internalization.

  17. Ubiquitin C-Terminal Hydrolase L1 in Tumorigenesis

    Directory of Open Access Journals (Sweden)

    Jennifer Hurst-Kennedy

    2012-01-01

    Full Text Available Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1, aka PGP9.5 is an abundant, neuronal deubiquitinating enzyme that has also been suggested to possess E3 ubiquitin-protein ligase activity and/or stabilize ubiquitin monomers in vivo. Recent evidence implicates dysregulation of UCH-L1 in the pathogenesis and progression of human cancers. Although typically only expressed in neurons, high levels of UCH-L1 have been found in many nonneuronal tumors, including breast, colorectal, and pancreatic carcinomas. UCH-L1 has also been implicated in the regulation of metastasis and cell growth during the progression of nonsmall cell lung carcinoma, colorectal cancer, and lymphoma. Together these studies suggest UCH-L1 has a potent oncogenic role and drives tumor development. Conversely, others have observed promoter methylation-mediated silencing of UCH-L1 in certain tumor subtypes, suggesting a potential tumor suppressor role for UCH-L1. In this paper, we provide an overview of the evidence supporting the involvement of UCH-L1 in tumor development and discuss the potential mechanisms of action of UCH-L1 in oncogenesis.

  18. Inhibiting Inosine Hydrolase and Alanine Racemase to Enhance the Germination of Bacillus anthracis Sterne Spores: Potential Spore Decontamination Strategies

    Science.gov (United States)

    2015-06-19

    decontamination strategies>> Maryline DEFEZ 1𔃼, Melissa HUNTER3J Susan WELKOS :~J Christopher COTE3 1 University Grenoble-Alpes, Grenoble, France. 1...inosine hydrolase and alanine racemase to enhance the germination of Bacillus anthracis Sterne spores potential spore decontamination strategies 5a...8217 • Accidentally in Humans • Natural reservoir is soil • Anthrax Disease Cycle: - animals infected by soilborne spores in food and water or bites from certain

  19. Evaluation of fish models of soluble epoxide hydrolase inhibition.

    Science.gov (United States)

    Newman, J W; Denton, D L; Morisseau, C; Koger, C S; Wheelock, C E; Hinton, D E; Hammock, B D

    2001-01-01

    Substituted ureas and carbamates are mechanistic inhibitors of the soluble epoxide hydrolase (sEH). We screened a set of chemicals containing these functionalities in larval fathead minnow (Pimphales promelas) and embryo/larval golden medaka (Oryzias latipes) models to evaluate the utility of these systems for investigating sEH inhibition in vivo. Both fathead minnow and medaka sEHs were functionally similar to the tested mammalian orthologs (murine and human) with respect to substrate hydrolysis and inhibitor susceptibility. Low lethality was observed in either larval or embryonic fish exposed to diuron [N-(3,4-dichlorophenyl), N'-dimethyl urea], desmethyl diuron [N-(3,4-dichlorophenyl), N'-methyl urea], or siduron [N-(1-methylcyclohexyl), N'-phenyl urea]. Dose-dependent inhibition of sEH was a sublethal effect of substituted urea exposure with the potency of siduron diuron = diuron, differing from the observed in vitro sEH inhibition potency of siduron > desmethyl diuron > diuron. Further, siduron exposure synergized the toxicity of trans-stilbene oxide in fathead minnows. Medaka embryos exposed to diuron, desmethyl diuron, or siduron displayed dose-dependent delays in hatch, and elevated concentrations of diuron and desmethyl diuron produced developmental toxicity. The dose-dependent toxicity and in vivo sEH inhibition correlated, suggesting a potential, albeit undefined, relationship between these factors. Additionally, the observed inversion of in vitro to in vivo potency suggests that these fish models may provide tools for investigating the in vivo stability of in vitro inhibitors while screening for untoward effects. PMID:11171526

  20. Enantioselectivity of a recombinant epoxide hydrolase from Agrobacterium radiobacter

    NARCIS (Netherlands)

    Lutje Spelberg, Jeffrey H.; Rink, Rick; Kellogg, Richard M.; Janssen, Dick B.

    1998-01-01

    The recombinant epoxide hydrolase from Agrobacterium radiobacter AD1 was used to obtain enantiomerically pure epoxides by means of a kinetic resolution. Epoxides such as styrene oxide and various derivatives thereof and phenyl glycidyl ether were obtained in high enantiomeric excess and in

  1. Properties of epoxide hydrolase from the yeast Rhodotorula glutinis

    NARCIS (Netherlands)

    Ariës-Kronenburg, N.A.E.

    2002-01-01

    Epoxide hydrolases are ubiquitous enzymes that can be found in nearly all living organisms. Some of the enzymes play an important role in detoxifying xenobiotic and metabolic compounds. Others are important in the growth of organisms like

  2. Identification and characterization of some Aspergillus pectinolytic glycoside hydrolases

    NARCIS (Netherlands)

    Zandleven, J.S.

    2006-01-01

    Keywords: Aspergillusniger , Arabidopsis thaliana , homogalacturonan, rhamnogalacturonan, xylogalacturonan, xylogalacturonan hydrolase, exo-polygalacturonasePectinases are used for many food

  3. Method for enhancing amidohydrolase activity of fatty acid amide hydrolase

    Science.gov (United States)

    John, George; Nagarajan, Subbiah; Chapman, Kent; Faure, Lionel; Koulen, Peter

    2017-12-26

    A method for enhancing amidohydrolase activity of Fatty Acid Amide Hydrolase (FAAH) is disclosed. The method comprising administering a phenoxyacyl-ethanolamide that causes the enhanced activity. The enhanced activity can have numerous effects on biological organisms including, for example, enhancing the growth of certain seedlings.

  4. Method for enhancing amidohydrolase activity of fatty acid amide hydrolase

    Science.gov (United States)

    John, George; Nagarajan, Subbiah; Chapman, Kent; Faure, Lionel; Koulen, Peter

    2016-10-25

    A method for enhancing amidohydrolase activity of Fatty Acid Amide Hydrolase (FAAH) is disclosed. The method comprising administering a phenoxyacylethanolamide that causes the enhanced activity. The enhanced activity can have numerous effects on biological organisms including, for example, enhancing the growth of certain seedlings. The subject matter disclosed herein relates to enhancers of amidohydrolase activity.

  5. A remote but significant sequence homology between glycoside hydrolase clan GH-H and glycoside hydrolase family GH 31

    DEFF Research Database (Denmark)

    Janecek, S.; Svensson, Birte; MacGregor, E.A.

    2007-01-01

    Although both the α-amylase super-family, i.e. the glycoside hydrolase (GH) clan GH-H (the GH families 13, 70 and 77), and family GH31 share some characteristics, their different catalytic machinery prevents classification of GH31 in clan GH-H. A significant but remote evolutionary relatedness is...

  6. Poly(aspartic acid) (PAA) hydrolases and PAA biodegradation: current knowledge and impact on applications.

    Science.gov (United States)

    Hiraishi, Tomohiro

    2016-02-01

    Thermally synthesized poly(aspartic acid) (tPAA) is a bio-based, biocompatible, biodegradable, and water-soluble polymer that has a high proportion of β-Asp units and equivalent moles of D- and L-Asp units. Poly(aspartic acid) (PAA) hydrolase-1 and hydrolase-2 are tPAA biodegradation enzymes purified from Gram-negative bacteria. PAA hydrolase-1 selectively cleaves amide bonds between β-Asp units via an endo-type process, whereas PAA hydrolase-2 catalyzes the exo-type hydrolysis of the products of tPAA hydrolysis by PAA hydrolase-1. The novel reactivity of PAA hydrolase-1 makes it a good candidate for a biocatalyst in β-peptide synthesis. This mini-review gives an overview of PAA hydrolases with emphasis on their biochemical and functional properties, in particular, PAA hydrolase-1. Functionally related enzymes, such as poly(R-3-hydroxybutyrate) depolymerases and β-aminopeptidases, are compared to PAA hydrolases. This mini-review also provides findings that offer an insight into the catalytic mechanisms of PAA hydrolase-1 from Pedobacter sp. KP-2.

  7. In-silico gene co-expression network analysis in Paracoccidioides brasiliensis with reference to haloacid dehalogenase superfamily hydrolase gene

    Directory of Open Access Journals (Sweden)

    Raghunath Satpathy

    2015-01-01

    Full Text Available Context: Paracoccidioides brasiliensis, a dimorphic fungus is the causative agent of paracoccidioidomycosis, a disease globally affecting millions of people. The haloacid dehalogenase (HAD superfamily hydrolases enzyme in the fungi, in particular, is known to be responsible in the pathogenesis by adhering to the tissue. Hence, identification of novel drug targets is essential. Aims: In-silico based identification of co-expressed genes along with HAD superfamily hydrolase in P. brasiliensis during the morphogenesis from mycelium to yeast to identify possible genes as drug targets. Materials and Methods: In total, four datasets were retrieved from the NCBI-gene expression omnibus (GEO database, each containing 4340 genes, followed by gene filtration expression of the data set. Further co-expression (CE study was performed individually and then a combination these genes were visualized in the Cytoscape 2. 8.3. Statistical Analysis Used: Mean and standard deviation value of the HAD superfamily hydrolase gene was obtained from the expression data and this value was subsequently used for the CE calculation purpose by selecting specific correlation power and filtering threshold. Results: The 23 genes that were thus obtained are common with respect to the HAD superfamily hydrolase gene. A significant network was selected from the Cytoscape network visualization that contains total 7 genes out of which 5 genes, which do not have significant protein hits, obtained from gene annotation of the expressed sequence tags by BLAST X. For all the protein PSI-BLAST was performed against human genome to find the homology. Conclusions: The gene co-expression network was obtained with respect to HAD superfamily dehalogenase gene in P. Brasiliensis.

  8. Crystallization of mouse S-adenosyl-l-homocysteine hydrolase

    International Nuclear Information System (INIS)

    Ishihara, Masaaki; Kusakabe, Yoshio; Ohsumichi, Tsuyoshi; Tanaka, Nobutada; Nakanishi, Masayuki; Kitade, Yukio; Nakamura, Kazuo T.

    2010-01-01

    Mouse S-adenosyl-l-homocysteine hydrolase has been crystallized in the presence of the reaction product adenosine. Diffraction data to 1.55 Å resolution were collected using synchrotron radiation. S-Adenosyl-l-homocysteine hydrolase (SAHH; EC 3.3.1.1) catalyzes the reversible hydrolysis of S-adenosyl-l-homocysteine to adenosine and l-homocysteine. For crystallographic investigations, mouse SAHH (MmSAHH) was overexpressed in bacterial cells and crystallized using the hanging-drop vapour-diffusion method in the presence of the reaction product adenosine. X-ray diffraction data to 1.55 Å resolution were collected from an orthorhombic crystal form belonging to space group I222 with unit-cell parameters a = 100.64, b = 104.44, c = 177.31 Å. Structural analysis by molecular replacement is in progress

  9. Structural insight into catalytic mechanism of PET hydrolase

    OpenAIRE

    Han, Xu; Liu, Weidong; Huang, Jian-Wen; Ma, Jiantao; Zheng, Yingying; Ko, Tzu-Ping; Xu, Limin; Cheng, Ya-Shan; Chen, Chun-Chi; Guo, Rey-Ting

    2017-01-01

    PET hydrolase (PETase), which hydrolyzes polyethylene terephthalate (PET) into soluble building blocks, provides an attractive avenue for the bioconversion of plastics. Here we present the structures of a novel PETase from the PET-consuming microbe Ideonella sakaiensis in complex with substrate and product analogs. Through structural analyses, mutagenesis, and activity measurements, a substrate-binding mode is proposed, and several features critical for catalysis are elucidated.

  10. Structural insight into catalytic mechanism of PET hydrolase.

    Science.gov (United States)

    Han, Xu; Liu, Weidong; Huang, Jian-Wen; Ma, Jiantao; Zheng, Yingying; Ko, Tzu-Ping; Xu, Limin; Cheng, Ya-Shan; Chen, Chun-Chi; Guo, Rey-Ting

    2017-12-13

    PET hydrolase (PETase), which hydrolyzes polyethylene terephthalate (PET) into soluble building blocks, provides an attractive avenue for the bioconversion of plastics. Here we present the structures of a novel PETase from the PET-consuming microbe Ideonella sakaiensis in complex with substrate and product analogs. Through structural analyses, mutagenesis, and activity measurements, a substrate-binding mode is proposed, and several features critical for catalysis are elucidated.

  11. Inhibition of Xenobiotic-Degrading Hydrolases by Organophosphinates

    Science.gov (United States)

    1986-07-01

    M 4 Q r 000 44 Table 11. Purification of arylester hydrolase Specific Total Total Activity Volume Activity Proteina (Umoles/ Purifi- Fraction (mL...did get re-adjusted after the sample was applied. After the sample was applied the column was washed with the above MES buffer an.+eluted with 100 ml...Lieske (94) and compared them to the reversed phase HPLC retention times we have previously reported (16). We get an excellent linear correlation

  12. IMMOBILIZATION OF TANNIN ACYL HYDROLASE FROM ASPERGILLUS NIGER

    OpenAIRE

    B. Lenin Kumar*, N. Lokeswari and D. Sriramireddy

    2013-01-01

    ABSTRACT: Tannin acyl hydrolase, commonly referred to as tannase (E.C. 3.1.1.20), an inducible extra-cellular enzyme produced by a number of animals, plants and microbes. In this investigation, tannase production under solid-state fermentation by using Aspergillus niger and the waste residue of cashew husk was used as substrate for obtaining the desired fermented product. Microbial tannase is more stable than tannase from other sources like plants or animals. Tannase from fungal sources are r...

  13. Annotation and comparative analysis of the glycoside hydrolase genes in Brachypodium distachyon

    Directory of Open Access Journals (Sweden)

    Wu Jiajie

    2010-10-01

    Full Text Available Abstract Background Glycoside hydrolases cleave the bond between a carbohydrate and another carbohydrate, a protein, lipid or other moiety. Genes encoding glycoside hydrolases are found in a wide range of organisms, from archea to animals, and are relatively abundant in plant genomes. In plants, these enzymes are involved in diverse processes, including starch metabolism, defense, and cell-wall remodeling. Glycoside hydrolase genes have been previously cataloged for Oryza sativa (rice, the model dicotyledonous plant Arabidopsis thaliana, and the fast-growing tree Populus trichocarpa (poplar. To improve our understanding of glycoside hydrolases in plants generally and in grasses specifically, we annotated the glycoside hydrolase genes in the grasses Brachypodium distachyon (an emerging monocotyledonous model and Sorghum bicolor (sorghum. We then compared the glycoside hydrolases across species, at the levels of the whole genome and individual glycoside hydrolase families. Results We identified 356 glycoside hydrolase genes in Brachypodium and 404 in sorghum. The corresponding proteins fell into the same 34 families that are represented in rice, Arabidopsis, and poplar, helping to define a glycoside hydrolase family profile which may be common to flowering plants. For several glycoside hydrolase familes (GH5, GH13, GH18, GH19, GH28, and GH51, we present a detailed literature review together with an examination of the family structures. This analysis of individual families revealed both similarities and distinctions between monocots and eudicots, as well as between species. Shared evolutionary histories appear to be modified by lineage-specific expansions or deletions. Within GH families, the Brachypodium and sorghum proteins generally cluster with those from other monocots. Conclusions This work provides the foundation for further comparative and functional analyses of plant glycoside hydrolases. Defining the Brachypodium glycoside hydrolases sets

  14. Insights into Substrate Specificity of NlpC/P60 Cell Wall Hydrolases Containing Bacterial SH3 Domains

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.; Patin, Delphine; Farr, Carol L.; Grant, Joanna C.; Chiu, Hsiu-Ju; Jaroszewski, Lukasz; Knuth, Mark W.; Godzik, Adam; Lesley, Scott A.; Elsliger, Marc-André; Deacon, Ashley M.; Wilson, Ian A.

    2015-09-15

    analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.

  15. Autolysis of dairy leuconostocs and detection of peptidoglycan hydrolases by renaturing SDS-PAGE.

    Science.gov (United States)

    Cibik, R; Chapot-Chartier, M P

    2000-11-01

    The autolysis of lactic acid bacteria plays a major role during cheese ripening. The aim of this study was to evaluate the autolytic properties and peptidoglycan hydrolase content of dairy leuconostocs. Autolysis of 59 strains of dairy Leuconostoc was examined under starvation conditions in potassium phosphate buffer. The ability of dairy leuconostocs to lyse is strain dependant and not related to the species. The peptidoglycan hydrolase profile of Leuc. mesenteroides subsp. mesenteroides 10L was analysed by renaturing gel electrophoresis. Two major activity bands migrating at 41 and 52 kDa were observed. According to the specificity analysis, strain 10L seems to contain a glycosidase and an N-acetyl-muramyl-L-alanine amidase, or an endopeptidase. The peptidoglycan hydrolase profiles of various Leuconostoc species were also compared. Several peptidoglycan hydrolase activities could be detected in the different Leuconostoc species. Further characterization of the peptidoglycan hydrolases will help to control autolysis of leuconostocs in cheese.

  16. Degradation of Polyester Polyurethane by Bacterial Polyester Hydrolases

    Directory of Open Access Journals (Sweden)

    Juliane Schmidt

    2017-02-01

    Full Text Available Polyurethanes (PU are widely used synthetic polymers. The growing amount of PU used industrially has resulted in a worldwide increase of plastic wastes. The related environmental pollution as well as the limited availability of the raw materials based on petrochemicals requires novel solutions for their efficient degradation and recycling. The degradation of the polyester PU Impranil DLN by the polyester hydrolases LC cutinase (LCC, TfCut2, Tcur1278 and Tcur0390 was analyzed using a turbidimetric assay. The highest hydrolysis rates were obtained with TfCut2 and Tcur0390. TfCut2 also showed a significantly higher substrate affinity for Impranil DLN than the other three enzymes, indicated by a higher adsorption constant K. Significant weight losses of the solid thermoplastic polyester PU (TPU Elastollan B85A-10 and C85A-10 were detected as a result of the enzymatic degradation by all four polyester hydrolases. Within a reaction time of 200 h at 70 °C, LCC caused weight losses of up to 4.9% and 4.1% of Elastollan B85A-10 and C85A-10, respectively. Gel permeation chromatography confirmed a preferential degradation of the larger polymer chains. Scanning electron microscopy revealed cracks at the surface of the TPU cubes as a result of enzymatic surface erosion. Analysis by Fourier transform infrared spectroscopy indicated that the observed weight losses were a result of the cleavage of ester bonds of the polyester TPU.

  17. Compositional profile of α / β-hydrolase fold proteins in mangrove soil metagenomes: prevalence of epoxide hydrolases and haloalkane dehalogenases in oil-contaminated sites.

    Science.gov (United States)

    Jiménez, Diego Javier; Dini-Andreote, Francisco; Ottoni, Júlia Ronzella; de Oliveira, Valéria Maia; van Elsas, Jan Dirk; Andreote, Fernando Dini

    2015-05-01

    The occurrence of genes encoding biotechnologically relevant α/β-hydrolases in mangrove soil microbial communities was assessed using data obtained by whole-metagenome sequencing of four mangroves areas, denoted BrMgv01 to BrMgv04, in São Paulo, Brazil. The sequences (215 Mb in total) were filtered based on local amino acid alignments against the Lipase Engineering Database. In total, 5923 unassembled sequences were affiliated with 30 different α/β-hydrolase fold superfamilies. The most abundant predicted proteins encompassed cytosolic hydrolases (abH08; ∼ 23%), microsomal hydrolases (abH09; ∼ 12%) and Moraxella lipase-like proteins (abH04 and abH01; mangroves BrMgv01-02-03. This suggested selection and putative involvement in local degradation/detoxification of the pollutants. Seven sequences that were annotated as genes for putative epoxide hydrolases and five for putative haloalkane dehalogenases were found in a fosmid library generated from BrMgv02 DNA. The latter enzymes were predicted to belong to Actinobacteria, Deinococcus-Thermus, Planctomycetes and Proteobacteria. Our integrated approach thus identified 12 genes (complete and/or partial) that may encode hitherto undescribed enzymes. The low amino acid identity (< 60%) with already-described genes opens perspectives for both production in an expression host and genetic screening of metagenomes. © 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  18. Compositional profile of α/β-hydrolase fold proteins in mangrove soil metagenomes: prevalence of epoxide hydrolases and haloalkane dehalogenases in oil-contaminated sites

    Science.gov (United States)

    Jiménez, Diego Javier; Dini-Andreote, Francisco; Ottoni, Júlia Ronzella; de Oliveira, Valéria Maia; van Elsas, Jan Dirk; Andreote, Fernando Dini

    2015-01-01

    The occurrence of genes encoding biotechnologically relevant α/β-hydrolases in mangrove soil microbial communities was assessed using data obtained by whole-metagenome sequencing of four mangroves areas, denoted BrMgv01 to BrMgv04, in São Paulo, Brazil. The sequences (215 Mb in total) were filtered based on local amino acid alignments against the Lipase Engineering Database. In total, 5923 unassembled sequences were affiliated with 30 different α/β-hydrolase fold superfamilies. The most abundant predicted proteins encompassed cytosolic hydrolases (abH08; ∼ 23%), microsomal hydrolases (abH09; ∼ 12%) and Moraxella lipase-like proteins (abH04 and abH01; mangroves BrMgv01-02-03. This suggested selection and putative involvement in local degradation/detoxification of the pollutants. Seven sequences that were annotated as genes for putative epoxide hydrolases and five for putative haloalkane dehalogenases were found in a fosmid library generated from BrMgv02 DNA. The latter enzymes were predicted to belong to Actinobacteria, Deinococcus-Thermus, Planctomycetes and Proteobacteria. Our integrated approach thus identified 12 genes (complete and/or partial) that may encode hitherto undescribed enzymes. The low amino acid identity (< 60%) with already-described genes opens perspectives for both production in an expression host and genetic screening of metagenomes. PMID:25171437

  19. Cloning, recombinant production, crystallization and preliminary X-ray diffraction analysis of a family 101 glycoside hydrolase from Streptococcus pneumoniae

    International Nuclear Information System (INIS)

    Gregg, Katie J.; Boraston, Alisdair B.

    2009-01-01

    The catalytic module of a family 101 glycoside hydrolase from S. pneumoniae was cloned, recombinantly produced and crystallized. Streptococcus pneumoniae is a serious human pathogen that is responsible for a wide range of diseases including pneumonia, meningitis, septicaemia and otitis media. The full virulence of this bacterium is reliant on carbohydrate processing and metabolism, as revealed by biochemical and genetic studies. One carbohydrate-processing enzyme is a family 101 glycoside hydrolase (SpGH101) that is responsible for catalyzing the liberation of galactosyl β1,3-N-acetyl-d-galactosamine (Galβ1,3GalNAc) α-linked to serine or threonine residues of mucin-type glycoproteins. The 124 kDa catalytic module of this enzyme (SpGH101CM) was cloned and overproduced in Escherichia coli and purified. Crystals were obtained in space group P2 1 and diffracted to 2.0 Å resolution, with unit-cell parameters a = 81.86, b = 88.91, c = 88.77 Å, β = 112.46°. SpGH101CM also qualitatively displayed good activity towards the synthetic substrate p-nitrophenyl-2-acetamido-2-deoxy-3-O-(β-d-galactopyranosyl) -α-d-galactopyranoside, which is consistent with the classification of this enzyme as an endo-α-N-acetylgalactosaminidase

  20. Les lipases sont des hydrolases atypiques : principales caractéristiques et applications

    Directory of Open Access Journals (Sweden)

    Fickers P.

    2008-01-01

    Full Text Available ipases are atypical hydrolases: principal characteristics and applications. Due to their kinetic and substrate specificities, triacylglycerol acyl-hydrolases or lipases are atypical enzymes. In function of their microenvironment, lipases are able to act as hydrolases in aqueous solution or as biocatalysts in organic synthesis. As hydrolases, they are responsible of the triglycerids catabolism into fatty acids and glycerol. In many organisms, this reaction plays a major role in the fat and lipid metabolism. In addition, lipases are also able to hydrolyse phospholipids and cholesterol esters. In organic solvent, lipases could catalyse reactions such as esterifications, acidolysis or alcoolysis with enantio-, regio- and chimioselectivity. Lipases form a mixed class of enzyme due to their animal, vegetal or microbial origins. All those properties led to the development of many applications in the food and chemical industries but also in the medical and therapeutic field.

  1. Dysregulation of soluble epoxide hydrolase and lipidomic profiles in anorexia nervosa

    KAUST Repository

    Shih, P. B.; Yang, J.; Morisseau, C.; German, J. B.; Scott-Van Zeeland, A. A.; Armando, A. M.; Quehenberger, O.; Bergen, A. W.; Magistretti, Pierre J.; Berrettini, W.; Halmi, K. A.; Schork, N.; Hammock, B. D.; Kaye, W.

    2015-01-01

    Individuals with anorexia nervosa (AN) restrict eating and become emaciated. They tend to have an aversion to foods rich in fat. Because epoxide hydrolase 2 (EPHX2) was identified as a novel AN susceptibility gene, and because its protein product

  2. Purification and characterisation of a novel enantioselective epoxide hydrolase from Aspergillus niger M200

    Czech Academy of Sciences Publication Activity Database

    Kotík, Michael; Kyslík, Pavel

    2006-01-01

    Roč. 1760, - (2006), s. 245-252 ISSN 0006-3002 Institutional research plan: CEZ:AV0Z50200510 Keywords : epoxide hydrolase * enantioselectivity * aspergillus niger Subject RIV: EE - Microbiology, Virology

  3. α-Amylase: an enzyme specificity found in various families of glycoside hydrolases

    DEFF Research Database (Denmark)

    Janeček, Štefan; Svensson, Birte; MacGregor, E. Ann

    2014-01-01

    of all carbohydrate-active enzymes, it is one of the most frequently occurring glycoside hydrolases (GH). α-Amylase is the main representative of family GH13, but it is probably also present in the families GH57 and GH119, and possibly even in GH126. Family GH13, known generally as the main α...... investigation because of an obvious, but unexpected, homology with inverting β-glucan-active hydrolases....

  4. HYDROLASING OF CONTAMINATED UNDERWATER BASIN SURFACES AT THE HANFORD K AREA

    International Nuclear Information System (INIS)

    CHRONISTER, G.B.

    2005-01-01

    This paper discusses selecting and implementing hydrolasing technology to reduce radioactive contamination in preparing to dispose of the K Basins; two highly contaminated concrete basins at the Hanford Site. A large collection of spent nuclear fuel stored for many years underwater at the K Basins has been removed to stable, dry, safe storage. Remediation activities have begun for the remaining highly contaminated water. sludge, and concrete basin structures. Hydrolasing will be used to decontaminate and prepare the basin structures for disposal

  5. Regulation of calcium release from the endoplasmic reticulum by the serine hydrolase ABHD2.

    Science.gov (United States)

    Yun, Bogeon; Lee, HeeJung; Powell, Roger; Reisdorph, Nichole; Ewing, Heather; Gelb, Michael H; Hsu, Ku-Lung; Cravatt, Benjamin F; Leslie, Christina C

    2017-09-02

    The serine hydrolase inhibitors pyrrophenone and KT195 inhibit cell death induced by A23187 and H 2 O 2 by blocking the release of calcium from the endoplasmic reticulum and mitochondrial calcium uptake. The effect of pyrrophenone and KT195 on these processes is not due to inhibition of their known targets, cytosolic phospholipase A 2 and α/β-hydrolase domain-containing (ABHD) 6, respectively, but represent off-target effects. To identify targets of KT195, fibroblasts were treated with KT195-alkyne to covalently label protein targets followed by click chemistry with biotin azide, enrichment on streptavidin beads and tryptic peptide analysis by mass spectrometry. Although several serine hydrolases were identified, α/β-hydrolase domain-containing 2 (ABHD2) was the only target in which both KT195 and pyrrophenone competed for binding to KT195-alkyne. ABHD2 is a serine hydrolase with a predicted transmembrane domain consistent with its pull-down from the membrane proteome. Subcellular fractionation showed localization of ABHD2 to the endoplasmic reticulum but not to mitochondria or mitochondrial-associated membranes. Knockdown of ABHD2 with shRNA attenuated calcium release from the endoplasmic reticulum, mitochondrial calcium uptake and cell death in fibroblasts stimulated with A23187. The results describe a novel mechanism for regulating calcium transfer from the endoplasmic reticulum to mitochondria that involves the serine hydrolase ABHD2. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Expression of Nudix hydrolase genes in barley under UV irradiation

    Science.gov (United States)

    Tanaka, Sayuri; Sugimoto, Manabu; Kihara, Makoto

    Seed storage and cultivation should be necessary to self-supply foods when astronauts would stay and investigate during long-term space travel and habitation in the bases on the Moon and Mars. Thought the sunlight is the most importance to plants, both as the ultimate energy source and as an environmental signal regulating growth and development, UV presenting the sunlight can damage many aspects of plant processes at the physiological and DNA level. Especially UV-C, which is eliminated by the stratospheric ozone layer, is suspected to be extremely harmful and give a deadly injury to plants in space. However, the defense mechanism against UV-C irradiation damage in plant cells has not been clear. In this study, we investigated the expression of Nudix hydrolases, which defense plants from biotic / abiotic stress, in barley under UV irradiation. The genes encoding the amino acid sequences, which show homology to those of 28 kinds of Nudix hydrolases in Arabidopsis thaliana, were identified in the barley full-length cDNA library. BLAST analysis showed 14 kinds of barley genes (HvNUDX1-14), which encode the Nudix motif sequence. A phylogenetic tree showed that HvNUDX1, HvNUDX7, HvNUDX9 and HvNUDX11 belonged to the ADP-ribose pyrophosphohydrolase, ADP-sugar pyrophosphohydrolase, NAD(P)H pyrophosphohydrolase and FAD pyrophosphohydrolase subfamilies, respectively, HvNUDX3, HvNUDX6, and HvNUDX8 belonged to the Ap _{n}A pyrophosphohydrolase subfamilies, HvNUDX5 and HvNUDX14 belonged to the coenzyme A pyrophosphohydrolase subfamilies, HvNUDX12 and HvNUDX13 belonged to the Ap _{4}A pyrophosphohydrolase subfamilies. Induction of HvNUDX genes by UV-A (340nm), UV-B (312nm), and UV-C (260nm) were analyzed by quantitative RT-PCR. The results showed that HvNUDX4 was induced by UV-A and UV-B, HvNUDX6 was induced by UV-B and UV-C, and HvNUDX7 and HvNUDX14 were induced by UV-C, significantly. Our results suggest that the response of HvNUDXs to UV irradiation is different by UV

  7. Crystal Structure of Homo Sapiens PTD012 Reveals a Zinc-Containing Hydrolase Fold

    Energy Technology Data Exchange (ETDEWEB)

    Manjasetty,B.; Bussow, K.; Fieber-ErdMan, M.; Roske, Y.; Gobam, J.; Scheich, C.; Gotz, F.; Niesen, F.; Heinemann, U.

    2006-01-01

    The human protein PTD012 is the longer product of an alternatively spliced gene and was described to be localized in the nucleus. The X-ray structure analysis at 1.7 Angstroms resolution of PTD012 through SAD phasing reveals a monomeric protein and a novel fold. The shorter splice form was also studied and appears to be unfolded and non-functional. The structure of PTD012 displays an {alpha}{beta}{beta}{alpha} four-layer topology. A metal ion residing between the central {beta}-sheets is partially coordinated by three histidine residues. X-ray absorption near-edge structure (XANES) analysis identifies the PTD012-bound ion as Zn{sup 2+}. Tetrahedral coordination of the ion is completed by the carboxylate oxygen atom of an acetate molecule taken up from the crystallization buffer. The binding of Zn{sup 2+} to PTD012 is reminiscent of zinc-containing enzymes such as carboxypeptidase, carbonic anhydrase, and {beta}-lactamase. Biochemical assays failed to demonstrate any of these enzyme activities in PTD012. However, PTD012 exhibits ester hydrolase activity on the substrate p-nitrophenyl acetate.

  8. Occurrence of urea-based soluble epoxide hydrolase inhibitors from the plants in the order Brassicales.

    Directory of Open Access Journals (Sweden)

    Seiya Kitamura

    Full Text Available Recently, dibenzylurea-based potent soluble epoxide hydrolase (sEH inhibitors were identified in Pentadiplandra brazzeana, a plant in the order Brassicales. In an effort to generalize the concept, we hypothesized that plants that produce benzyl glucosinolates and corresponding isothiocyanates also produce these dibenzylurea derivatives. Our overall aim here was to examine the occurrence of urea derivatives in Brassicales, hoping to find biologically active urea derivatives from plants. First, plants in the order Brassicales were analyzed for the presence of 1, 3-dibenzylurea (compound 1, showing that three additional plants in the order Brassicales produce the urea derivatives. Based on the hypothesis, three dibenzylurea derivatives with sEH inhibitory activity were isolated from maca (Lepidium meyenii roots. Topical application of one of the identified compounds (compound 3, human sEH IC50 = 222 nM effectively reduced pain in rat inflammatory pain model, and this compound was bioavailable after oral administration in mice. The biosynthetic pathway of these urea derivatives was investigated using papaya (Carica papaya seed as a model system. Finally, a small collection of plants from the Brassicales order was grown, collected, extracted and screened for sEH inhibitory activity. Results show that several plants of the Brassicales order could be potential sources of urea-based sEH inhibitors.

  9. Resveratrol, a Red Wine Polyphenol, Suppresses Pancreatic Cancer by Inhibiting Leukotriene A4 Hydrolase

    Science.gov (United States)

    Oi, Naomi; Jeong, Chul-Ho; Nadas, Janos; Cho, Yong-Yeon; Pugliese, Angelo; Bode, Ann M.; Dong, Zigang

    2016-01-01

    The anticancer effects of red wine have attracted considerable attention. Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a well-known polyphenolic compound of red wine with cancer chemopreventive activity. However, the basis for this activity is unclear. We studied leukotriene A4 hydrolase (LTA4H) as a relevant target in pancreatic cancer. LTA4H knockdown limited the formation of leukotriene B4 (LTB4), the enzymatic product of LTA4H, and suppressed anchorage-independent growth of pancreatic cancer cells. An in silico shape similarity algorithm predicted that LTA4H might be a potential target of resveratrol. In support of this idea, we found that resveratrol directly bound to LTA4H in vitro and in cells and suppressed proliferation and anchorage-independent growth of pancreatic cancer by inhibiting LTB4 production and expression of the LTB4 receptor 1 (BLT1). Notably, resveratrol exerted relatively stronger inhibitory effects than bestatin, an established inhibitor of LTA4H activity, and the inhibitory effects of resveratrol were reduced in cells where LTA4H was suppressed by shRNA-mediated knockdown. Importantly, resveratrol inhibited tumor formation in a xenograft mouse model of human pancreatic cancer by inhibiting LTA4H activity. Our findings identify LTA4H as a functionally important target for mediating the anticancer properties of resveratrol. PMID:20952510

  10. Structure-Based Optimization of Arylamides as Inhibitors of Soluble Epoxide Hydrolase

    Energy Technology Data Exchange (ETDEWEB)

    Eldrup, Anne B.; Soleymanzadeh, Fariba; Taylor, Steven J.; Muegge, Ingo; Farrow, Neil A.; Joseph, David; McKellop, Keith; Man, Chuk C.; Kukulka, Alison; De Lombaert, Stephane; (Boehringer)

    2009-11-04

    Inhibition of soluble epoxide hydrolase (sEH) is hypothesized to lead to an increase in circulating levels of epoxyeicosatrienoic acids, resulting in the potentiation of their in vivo pharmacological properties. As part of an effort to identify inhibitors of sEH with high and sustained plasma exposure, we recently performed a high throughput screen of our compound collection. The screen identified N-(3,3-diphenyl-propyl)-nicotinamide as a potent inhibitor of sEH. Further profiling of this lead revealed short metabolic half-lives in microsomes and rapid clearance in the rat. Consistent with these observations, the determination of the in vitro metabolic profile of N-(3,3-diphenyl-propyl)-nicotinamide in rat liver microsomes revealed extensive oxidative metabolism and a propensity for metabolite switching. Lead optimization, guided by the analysis of the solid-state costructure of N-(3,3-diphenyl-propyl)-nicotinamide bound to human sEH, led to the identification of a class of potent and selective inhibitors. An inhibitor from this class displayed an attractive in vitro metabolic profile and high and sustained plasma exposure in the rat after oral administration.

  11. Lipid-lowering effect of bergamot polyphenolic fraction: role of pancreatic cholesterol ester hydrolase.

    Science.gov (United States)

    Musolino, V; Gliozzi, M; Carresi, C; Maiuolo, J; Mollace, R; Bosco, F; Scarano, F; Scicchitano, M; Maretta, A; Palma, E; Iannone, M; Morittu, V M; Gratteri, S; Muscoli, C; Fini, M; Mollace, V

    2017-01-01

    Bergamot polyphenolic fraction (BPF) has been shown to positively modulate several mechanisms involved in metabolic syndrome, suggesting its use in therapy. In particular, it is able to induce a significant amelioration of serum lipid profile in hyperlipemic patients at different levels. The purpose of our study was to investigate the effect of BPF on cholesterol absorption physiologically mediated by pancreatic cholesterol ester hydrolase (pCEH). An in vitro activity assay was performed to study the effect of BPF on pCEH, whereas the rate of cholesterol absorption was evaluated through in vivo studies. In particular, male, Sprague-Dawley rats (200–225 g) were fed either normal chow or chow supplemented with 0.5% cholic acid, 5.5% peanut oil, and varying amounts of cholesterol (0 to 1.5%). BPF (10 mg/Kg) was daily administrated by means of a gastric gavage to animals fed with lipid supplemented diet for 4 weeks and, at the end of the study, plasma lipids and liver cholesteryl esters were measured in all experimental groups. Our results show that BPF was able to inhibit pCEH activity and this effect was confirmed, in vivo, via detection of lymphatic cholesteryl ester in rats fed with a cholesterol-rich diet. This evidence clarifies a further mechanism responsible for the hypolipemic properties of BPF previously observed in humans, confirming its beneficial effect in the therapy of hypercholesterolemia and in the treatment of metabolic syndrome.

  12. A sensitive and specific radiochromatographic assay of fatty acid amide hydrolase activity.

    Science.gov (United States)

    Maccarrone, M; Bari, M; Agrò, A F

    1999-02-15

    A radiochromatographic method has been set up in order to determine fatty acid amide hydrolase (FAAH) activity, based on reversed-phase high-performance liquid chromatography and on-line scintillation counting. The reaction products were separated using a C18 column eluted with methanol-water-acetic acid and quantitated with an external standard. Baseline separation of the acid product from the substrate was completed in less than 4 min, with a detection limit of 2.5 fmol arachidonic acid at a signal to noise ratio of 4:1. The method enabled to determine the kinetic constants (i.e., apparent Km of 2.0 +/- 0.2 microM and Vmax of 800 +/- 75 pmol. min-1. mg protein-1 toward anandamide) and the substrate specificity of human brain FAAH, as well as the extent of enzyme inhibition by some anandamide congeners. The femtomole sensitivity and the accuracy of the method allow detection and characterization of the activity of FAAH in very minute tissue samples or in samples where the enzymatic activity is very low. Copyright 1999 Academic Press.

  13. Microfluidic glycosyl hydrolase screening for biomass-to-biofuel conversion.

    Science.gov (United States)

    Bharadwaj, Rajiv; Chen, Zhiwei; Datta, Supratim; Holmes, Bradley M; Sapra, Rajat; Simmons, Blake A; Adams, Paul D; Singh, Anup K

    2010-11-15

    The hydrolysis of biomass to fermentable sugars using glycosyl hydrolases such as cellulases and hemicellulases is a limiting and costly step in the conversion of biomass to biofuels. Enhancement in hydrolysis efficiency is necessary and requires improvement in both enzymes and processing strategies. Advances in both areas in turn strongly depend on the progress in developing high-throughput assays to rapidly and quantitatively screen a large number of enzymes and processing conditions. For example, the characterization of various cellodextrins and xylooligomers produced during the time course of saccharification is important in the design of suitable reactors, enzyme cocktail compositions, and biomass pretreatment schemes. We have developed a microfluidic-chip-based assay for rapid and precise characterization of glycans and xylans resulting from biomass hydrolysis. The technique enables multiplexed separation of soluble cellodextrins and xylose oligomers in around 1 min (10-fold faster than HPLC). The microfluidic device was used to elucidate the mode of action of Tm_Cel5A, a novel cellulase from hyperthermophile Thermotoga maritima . The results demonstrate that the cellulase is active at 80 °C and effectively hydrolyzes cellodextrins and ionic-liquid-pretreated switchgrass and Avicel to glucose, cellobiose, and cellotriose. The proposed microscale approach is ideal for quantitative large-scale screening of enzyme libraries for biomass hydrolysis, for development of energy feedstocks, and for polysaccharide sequencing.

  14. Heterologous expression of the methyl carbamate-degrading hydrolase MCD.

    Science.gov (United States)

    Naqvi, Tatheer; Cheesman, Matthew J; Williams, Michelle R; Campbell, Peter M; Ahmed, Safia; Russell, Robyn J; Scott, Colin; Oakeshott, John G

    2009-10-26

    The methyl carbamate-degrading hydrolase (MCD) of Achromobacter WM111 has considerable potential as a pesticide bioremediation agent. However this potential has been unrealisable until now because of an inability to express MCD in heterologous hosts such as Escherichia coli. Herein, we describe the first successful attempt to express appreciable quantities of MCD in active form in E. coli, and the subsequent characterisation of the heterologously expressed material. We find that the properties of this material closely match the previously reported properties of MCD produced from Achromobacter WM111. This includes the presence of two distinct forms of the enzyme that we show are most likely due to the presence of two functional translational start sites. The purified enzyme catalyses the hydrolysis of a carbamate (carbaryl), a carboxyl ester (alpha-naphthyl acetate) and a phophotriester (dimethyl umbelliferyl phosphate) and it is relatively resistant to thermal and solvent-mediated denaturation. The robust nature and catalytic promiscuity of MCD suggest that it could be exploited for various biotechnological applications.

  15. Fractionation and Characterization of Tannin Acyl Hydrolase from Aspergillus niger

    Directory of Open Access Journals (Sweden)

    YUNITA ARIAN SANI ANWAR

    2009-09-01

    Full Text Available We previously produced tannin acyl hydrolase (tannase from Aspergillus niger isolated from cacao pod. In the present study the enzyme was subjected to fractionation by ammonium sulphate followed by dialysis process. The saturation level of ammonium sulphate used was 30-80% where the best enzyme activity was obtained at the saturation level of 60%. Compared to that of crude enzyme, specific activity of tannase after dialysis was four folds. Characterization results showed that optimum activity was at 35-50 oC and pH 6. Tannase was activated by K+ and Na+ at concentration of 0.01 and 0.05 M respectively. Mg2+ was found activate tannase only at 0.01 M. Addition of metal ions like Zn2+, Cu2+, Ca2+, Mn2+ and Fe2+ inhibited the enzyme activity. Kinetics analysis of various substrates tested showed that the Km value of tannic acid and gallotannin was 0.401 and 6.611 mM respectively. Vmax value of tannic acid was 10.804 U/ml and of gallotannin was 12.406 U/ml. Based on Michaelis-Menten constant (Km, the tannase obtained in the present study was more active in hydrolysing depside bonds rather than ester bonds.

  16. Fractionation and Characterization of Tannin Acyl Hydrolase from Aspergillus niger

    Directory of Open Access Journals (Sweden)

    YUNITA ARIAN SANI ANWAR

    2009-09-01

    Full Text Available We previously produced tannin acyl hydrolase (tannase from Aspergillus niger isolated from cacao pod. In the present study the enzyme was subjected to fractionation by ammonium sulphate followed by dialysis process. The saturation level of ammonium sulphate used was 30–80% where the best enzyme activity was obtained at the saturation level of 60%. Compared to that of crude enzyme, specific activity of tannase after dialysis was four folds. Characterization results showed that optimum activity was at 35–50 °C and pH 6. Tannase was activated by K+ and Na+ at concentration of 0.01 and 0.05 M respectively. Mg2+ was found activate tannase only at 0.01 M. Addition of metal ions like Zn2+, Cu2+, Ca2+, Mn2+ and Fe2+ inhibited the enzyme activity. Kinetics analysis of various substrates tested showed that the Km value of tannic acid and gallotannin was 0.401 and 6.611 mM respectively. Vmax value of tannic acid was 10.804 U/ml and of gallotannin was 12.406 U/ml. Based on Michaelis-Menten constant (Km, the tannase obtained in the present study was more active in hydrolysing depside bonds rather than ester bonds.

  17. Soluble epoxide hydrolase inhibitory activity of anthraquinone components from Aloe.

    Science.gov (United States)

    Sun, Ya Nan; Kim, Jang Hoon; Li, Wei; Jo, A Reum; Yan, Xi Tao; Yang, Seo Young; Kim, Young Ho

    2015-10-15

    Aloe is a short-stemmed succulent herb widely used in traditional medicine to treat various diseases and as raw material in cosmetics and heath foods. In this study, we isolated and identified two new anthraquinone derivatives, aloinoside C (6) and aloinoside D (7), together with six known compounds from an aqueous dissolved Aloe exudate. Their structures were identified by spectroscopic analysis. The inhibitory effects of the isolated compounds on soluble epoxide hydrolase (sEH) were evaluated. Compounds 1-8 inhibited sEH activity potently, with IC50 values ranging from 4.1±0.6 to 41.1±4.2 μM. A kinetic analysis of compounds 1-8 revealed that the inhibitory actions of compounds 1, 6 and 8 were non-competitive, whereas those of compounds 2-5 and 7 were the mixed-type. Molecular docking increases our understanding of receptor-ligand binding of all compounds. These results demonstrate that compounds 1-8 from Aloe are potential sEH inhibitors. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Compositional profile of α / β-hydrolase fold proteins in mangrove soil metagenomes : Prevalence of epoxide hydrolases and haloalkane dehalogenases in oil-contaminated sites

    NARCIS (Netherlands)

    Jiménez Avella, Diego; Dini Andreote, Francisco; Ottoni, Júlia Ronzella; de Oliveira, Valéria Maia; van Elsas, Jan Dirk; Andreote, Fernando Dini

    The occurrence of genes encoding biotechnologically relevant α/β-hydrolases in mangrove soil microbial communities was assessed using data obtained by whole-metagenome sequencing of four mangroves areas, denoted BrMgv01 to BrMgv04, in São Paulo, Brazil. The sequences (215 Mb in total) were filtered

  19. The apo structure of sucrose hydrolase from Xanthomonas campestris pv. campestris shows an open active-site groove

    DEFF Research Database (Denmark)

    Champion, Elise; Remaud-Simeon, Magali; Skov, Lars Kobberøe

    2009-01-01

    Glycoside hydrolase family 13 (GH-13) mainly contains starch-degrading or starch-modifying enzymes. Sucrose hydrolases utilize sucrose instead of amylose as the primary glucosyl donor. Here, the catalytic properties and X-ray structure of sucrose hydrolase from Xanthomonas campestris pv. campestris...... of GH-13. Comparisons with structures of the highly similar sucrose hydrolase from X. axonopodis pv. glycines most notably showed that residues Arg516 and Asp138, which form a salt bridge in the X. axonopodis sucrose complex and define part of the subsite -1 glucosyl-binding determinants...

  20. Bioprospecting metagenomics of decaying wood: mining for new glycoside hydrolases

    Directory of Open Access Journals (Sweden)

    Li Luen-Luen

    2011-08-01

    Full Text Available Abstract Background To efficiently deconstruct recalcitrant plant biomass to fermentable sugars in industrial processes, biocatalysts of higher performance and lower cost are required. The genetic diversity found in the metagenomes of natural microbial biomass decay communities may harbor such enzymes. Our goal was to discover and characterize new glycoside hydrolases (GHases from microbial biomass decay communities, especially those from unknown or never previously cultivated microorganisms. Results From the metagenome sequences of an anaerobic microbial community actively decaying poplar biomass, we identified approximately 4,000 GHase homologs. Based on homology to GHase families/activities of interest and the quality of the sequences, candidates were selected for full-length cloning and subsequent expression. As an alternative strategy, a metagenome expression library was constructed and screened for GHase activities. These combined efforts resulted in the cloning of four novel GHases that could be successfully expressed in Escherichia coli. Further characterization showed that two enzymes showed significant activity on p-nitrophenyl-α-L-arabinofuranoside, one enzyme had significant activity against p-nitrophenyl-β-D-glucopyranoside, and one enzyme showed significant activity against p-nitrophenyl-β-D-xylopyranoside. Enzymes were also tested in the presence of ionic liquids. Conclusions Metagenomics provides a good resource for mining novel biomass degrading enzymes and for screening of cellulolytic enzyme activities. The four GHases that were cloned may have potential application for deconstruction of biomass pretreated with ionic liquids, as they remain active in the presence of up to 20% ionic liquid (except for 1-ethyl-3-methylimidazolium diethyl phosphate. Alternatively, ionic liquids might be used to immobilize or stabilize these enzymes for minimal solvent processing of biomass.

  1. Glycoside hydrolase gene transcription by Alicyclobacillus acidocaldarius during growth on wheat arabinoxylan and monosaccharides: a proposed xylan hydrolysis mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Brady D.; Apel, William A.; Sheridan, Peter P.; DeVeaux, Linda C.

    2018-04-16

    Background Metabolism of carbon bound in wheat arabinoxylan (WAX) polysaccharides by bacteria requires a number of glycoside hydrolases active toward different bonds between sugars and other molecules. Alicyclobacillus acidocaldarius is a Gram-positive thermoacidophilic bacterium capable of growth on a variety of mono-, di-, oligo-, and polysaccharides. Nineteen proposed glycoside hydrolases have been annotated in the A. acidocaldarius Type Strain ATCC27009/DSM 446 genome. Results Molecular analysis using high-density oligonucleotide microarrays was performed on A. acidocaldarius strain ATCC27009 when growing on WAX. When a culture growing exponentially at the expense of arabinoxylan saccharides was challenged with glucose or xylose, most glycoside hydrolases were down-regulated. Interestingly, regulation was more intense when xylose was added to the culture than when glucose was added, a clear departure from classical carbon catabolite repression demonstrated by many Gram-positive bacteria. In silico analyses of the regulated glycoside hydrolases, along with the results from the microarray analyses, yielded a potential mechanism for arabinoxylan metabolism by A. acidocaldarius. Glycoside hydrolases expressed by this strain may have broad substrate specificity, and initial hydrolysis is catalyzed by an extracellular xylanase, while subsequent steps are likely performed inside the growing cell. Conclusions Glycoside hydrolases, for the most part, appear to be found in clusters, throughout the A. acidocaldarius genome. Not all of the glycoside hydrolase genes found at loci within these clusters were regulated during the experiment, indicating that a specific subset of the 19 glycoside hydrolase genes found in A. acidocaldarius were used during metabolism of WAX. While specific functions of the glycoside hydrolases was not tested as part of the research discussed, many of the glycoside hydrolases found in the A. acidocaldarius Type Strain appear to have a broader

  2. 1 kb of the lactase-phlorizin hydrolase promoter directs post-weaning decline and small intestinal-specific expression in transgenic mice

    DEFF Research Database (Denmark)

    Troelsen, J T; Mehlum, A; Spodsberg, N

    1994-01-01

    Adult-type hypolactasia is a genetic condition making approximately one half of the human population intolerant to milk because of abdominal symptoms. The cause is a post-weaning down-regulation of the intestinal-specific enzyme lactase-phlorizin hydrolase (LPH) reducing the intestinal capacity...... to hydrolyze lactose. We here demonstrate that the stretch -17 to -994 in the pig LPH-promoter carries cis-elements which direct a small intestinal-specific expression and a post-weaning decline of a linked rabbit beta-globin gene. These data demonstrate that the post-weaning decline of LPH is mainly due...

  3. Peptidoglycan Hydrolases of Local Lactic Acid Bacteria from Kazakh Traditional Food

    Directory of Open Access Journals (Sweden)

    Serik Shaikhin

    2014-01-01

    Full Text Available Introduction: Peptidoglycan (PG is a major component of the cell wall of Gram-positive bacteria and is essential for maintaining the integrity of the bacterial cell and its shape. The bacteria synthesize PG hydrolases, which are capable of cleaving the covalent bonds of PG. They also play an important role in modeling PG, which is required for bacterial growth and division. In an era of increasing antibiotic-resistant pathogens, PG hydrolases that destroy these important structures of the cell wall act as a potential source of new antimicrobials. The aim of this study is to identify the main PG hydrolases of local lactic acid bacteria isolated from traditional foods that enhance probiotic activity of a biological preparation. Methods. Lactococcus lactis 17А and Lactococcus garvieae 19А were isolated from the traditional sausage-like meat product called kazy. They were isolated according to standards methods of microbiology. Genetic identification of the isolates were tested by determining the nucleotide sequences of 16S rDNA. The Republican collection of microorganisms took strains of Lactobacillus casei subsp. Rhamnosus 13-P, L. delbrueckii subsp. lactis CG-1 B-RKM 0044 from cheese, Lactobacillus casei subsp. casei B-RKM 0202 from homemade butter. They used the standard technique of renaturating polyacrylamide gel electrophoresis to detect PG hydrolases activity. Results. According to the profiles of PG hydrolase activity on zymograms, the enzymes of Lactococci 17A and 19A in kazy are similar in electrophoretic mobility to major autolysin AcmA, while the lactobacilli of industrial and home-made dairy products have enzymes similar to extracellular proteins p40 and p75, which have probiotic activity. Conclusions. Use of peptidoglycan hydrolases seems to be an interesting approach in the fight against multi-drug resistant strains of bacteria and could be a valuable tool for the treatment of diseases caused by these microorganisms in Kazakhstan.

  4. Epoxide hydrolase-lasalocid a structure provides mechanistic insight into polyether natural product biosynthesis.

    Science.gov (United States)

    Wong, Fong T; Hotta, Kinya; Chen, Xi; Fang, Minyi; Watanabe, Kenji; Kim, Chu-Young

    2015-01-14

    Biosynthesis of some polyether natural products involves a kinetically disfavored epoxide-opening cyclic ether formation, a reaction termed anti-Baldwin cyclization. One such example is the biosynthesis of lasalocid A, an ionophore antibiotic polyether. During lasalocid A biosynthesis, an epoxide hydrolase, Lsd19, converts the bisepoxy polyketide intermediate into the tetrahydrofuranyl-tetrahydropyran product. We report the crystal structure of Lsd19 in complex with lasalocid A. The structure unambiguously shows that the C-terminal domain of Lsd19 catalyzes the intriguing anti-Baldwin cyclization. We propose a general mechanism for epoxide selection by ionophore polyether epoxide hydrolases.

  5. High-throughput analysis of endogenous fruit glycosyl hydrolases using a novel chromogenic hydrogel substrate assay

    DEFF Research Database (Denmark)

    Schückel, Julia; Kracun, Stjepan Kresimir; Lausen, Thomas Frederik

    2017-01-01

    A broad range of enzyme activities can be found in a wide range of different fruits and fruiting bodies but there is a lack of methods where many samples can be handled in a high-throughput and efficient manner. In particular, plant polysaccharide degrading enzymes – glycosyl hydrolases (GHs) play...... led to a more profound understanding of the importance of GH activity and regulation, current methods for determining glycosyl hydrolase activity are lacking in throughput and fail to keep up with data output from transcriptome research. Here we present the use of a versatile, easy...

  6. α/β-hydrolase domain containing protein 15 (ABHD15--an adipogenic protein protecting from apoptosis.

    Directory of Open Access Journals (Sweden)

    Evelyn Walenta

    Full Text Available Our knowledge about adipocyte metabolism and development is steadily growing, yet many players are still undefined. Here, we show that α/β-hydrolase domain containing protein 15 (Abhd15 is a direct and functional target gene of peroxisome proliferator-activated receptor gamma (PPARγ, the master regulator of adipogenesis. In line, Abhd15 is mainly expressed in brown and white adipose tissue and strongly upregulated during adipogenesis in various murine and human cell lines. Stable knockdown of Abhd15 in 3T3-L1 cells evokes a striking differentiation defect, as evidenced by low lipid accumulation and decreased expression of adipocyte marker genes. In preconfluent cells, knockdown of Abhd15 leads to impaired proliferation, which is caused by apoptosis, as we see an increased SubG1 peak, caspase 3/7 activity, and BAX protein expression as well as a reduction in anti-apoptotic BCL-2 protein. Furthermore, apoptosis-inducing amounts of palmitic acid evoke a massive increase of Abhd15 expression, proposing an apoptosis-protecting role for ABHD15. On the other hand, in mature adipocytes physiological (i.e. non-apoptotic concentrations of palmitic acid down-regulate Abhd15 expression. Accordingly, we found that the expression of Abhd15 in adipose tissue is reduced in physiological situations with high free fatty acid levels, like high-fat diet, fasting, and aging as well as in genetically obese mice. Collectively, our results position ABHD15 as an essential component in the development of adipocytes as well as in apoptosis, thereby connecting two substantial factors in the regulation of adipocyte number and size. Together with its intricate regulation by free fatty acids, ABHD15 might be an intriguing new target in obesity and diabetes research.

  7. Characterization of fatty acid amide hydrolase activity by a fluorescence-based assay.

    Science.gov (United States)

    Dato, Florian M; Maaßen, Andreas; Goldfuß, Bernd; Pietsch, Markus

    2018-04-01

    Fatty acid amide hydrolase (FAAH) is involved in many human diseases, particularly cancer, pain and inflammation as well as neurological, metabolic and cardiovascular disorders. Therefore, FAAH is an attractive target for the development of low-molecular-weight inhibitors as therapeutics, which requires robust assays that can be used for high-throughput screening (HTS) of compound libraries. Here, we report the development of a fluorometric assay based on FAAH's ability to effectively hydrolyze medium-chain fatty acid amides, introducing N-decanoyl-substituted 5-amino-2-methoxypyridine (D-MAP) as new amide substrate. D-MAP is cleaved by FAAH with an 8-fold larger specificity constant than the previously reported octanoyl-analog Oc-MAP (V max /K m of 1.09 and 0.134 mL min -1 mg -1 , respectively), with both MAP derivatives possessing superior substrate properties and much increased aqueous solubility compared to the respective p-nitroaniline compounds D-pNA and Oc-pNA. The new assay with D-MAP as substrate is highly sensitive using a lower enzyme concentration (1 μg mL -1 ) than literature-reported fluorimetric FAAH assays. In addition, D-MAP was validated in comparison to the substrate Oc-MAP for the characterization of FAAH inhibitors by means of the reference compounds URB597 and TC-F2 and was shown to be highly suitable for HTS in both kinetic and endpoint assays (Z' factors of 0.81 and 0.78, respectively). Copyright © 2018 Elsevier Inc. All rights reserved.

  8. Bile Salt Hydrolase Activities: A Novel Target to Screen Anti-Giardia Lactobacilli?

    Directory of Open Access Journals (Sweden)

    Thibault Allain

    2018-02-01

    Full Text Available Giardia duodenalis is a protozoan parasite responsible for giardiasis, a disease characterized by intestinal malabsorption, diarrhea and abdominal pain in a large number of mammal species. Giardiasis is one of the most common intestinal parasitic diseases in the world and thus a high veterinary, and public health concern. It is well-established that some probiotic bacteria may confer protection against this parasite in vitro and in vivo and we recently documented the implication of bile-salt hydrolase (BSH-like activities from strain La1 of Lactobacillus johnsonii as mediators of these effects in vitro. We showed that these activities were able to generate deconjugated bile salts that were toxic to the parasite. In the present study, a wide collection of lactobacilli strains from different ecological origins was screened to assay their anti-giardial effects. Our results revealed that the anti-parasitic effects of some of the strains tested were well-correlated with the expression of BSH-like activities. The two most active strains in vitro, La1 and Lactobacillus gasseri CNCM I-4884, were then tested for their capacity to influence G. duodenalis infection in a suckling mice model. Strikingly, only L. gasseri CNCM I-4884 strain was able to significantly antagonize parasite growth with a dramatic reduction of the trophozoites load in the small intestine. Moreover, this strain also significantly reduced the fecal excretion of Giardia cysts after 5 days of treatment, which could contribute to blocking the transmission of the parasite, in contrast of La1 where no effect was observed. This study represents a step toward the development of new prophylactic strategies to combat G. duodenalis infection in both humans and animals.

  9. How to find soluble proteins: a comprehensive analysis of alpha/beta hydrolases for recombinant expression in E. coli

    Directory of Open Access Journals (Sweden)

    Barth Sandra

    2005-04-01

    Full Text Available Abstract Background In screening of libraries derived by expression cloning, expression of active proteins in E. coli can be limited by formation of inclusion bodies. In these cases it would be desirable to enrich gene libraries for coding sequences with soluble gene products in E. coli and thus to improve the efficiency of screening. Previously Wilkinson and Harrison showed that solubility can be predicted from amino acid composition (Biotechnology 1991, 9(5:443–448. We have applied this analysis to members of the alpha/beta hydrolase fold family to predict their solubility in E. coli. alpha/beta hydrolases are a highly diverse family with more than 1800 proteins which have been grouped into homologous families and superfamilies. Results The predicted solubility in E. coli depends on hydrolase size, phylogenetic origin of the host organism, the homologous family and the superfamily, to which the hydrolase belongs. In general small hydrolases are predicted to be more soluble than large hydrolases, and eukaryotic hydrolases are predicted to be less soluble in E. coli than prokaryotic ones. However, combining phylogenetic origin and size leads to more complex conclusions. Hydrolases from prokaryotic, fungal and metazoan origin are predicted to be most soluble if they are of small, medium and large size, respectively. We observed large variations of predicted solubility between hydrolases from different homologous families and from different taxa. Conclusion A comprehensive analysis of all alpha/beta hydrolase sequences allows more efficient screenings for new soluble alpha/beta hydrolases by the use of libraries which contain more soluble gene products. Screening of hydrolases from families whose members are hard to express as soluble proteins in E. coli should first be done in coding sequences of organisms from phylogenetic groups with the highest average of predicted solubility for proteins of this family. The tools developed here can be used

  10. Soluble epoxide hydrolase in the generation and maintenance of high blood pressure in spontaneously hypertensive rats

    NARCIS (Netherlands)

    Koeners, Maarten P.; Wesseling, Sebastiaan; Ulu, Arzu; Lopez Sepulveda, Rocio; Morisseau, Christophe; Braam, Branko; Hammock, Bruce D.; Joles, Jaap A.

    Koeners MP, Wesseling S, Ulu A, Sepulveda RL, Morisseau C, Braam B, Hammock BD, Joles JA. Soluble epoxide hydrolase in the generation and maintenance of high blood pressure in spontaneously hypertensive rats. Am J Physiol Endocrinol Metab 300: E691-E698, 2011. First published January 25, 2011; doi:

  11. Regulatory regions in the rat lactase-phlorizin hydrolase gene that control cell-specific expression

    NARCIS (Netherlands)

    Verhave, Menno; Krasinski, Stephen D.; Christian, Sara I.; van Schaik, Sandrijn; van den Brink, Gijs R.; Doting, Edwina M. H.; Maas, Saskia M.; Wolthers, Katja C.; Grand, Richard J.; Montgomery, Robert K.

    2004-01-01

    OBJECTIVES: Lactase-phlorizin hydrolase (LPH) is an enterocyte-specific gene whose expression has been well-characterized, not only developmentally but also along the crypt-villus axis and along the length of the small bowel. Previous studies from the authors' laboratory have demonstrated that 2 kb

  12. High-throughput screening for gene libraries expressing carbohydrate hydrolase activity

    NARCIS (Netherlands)

    Leemhuis, Hans; Euverink, Gert-Jan W.; Dijkhuizen, Lubbert

    2003-01-01

    A simple and fast method is described allowing screening of large number of Escherichia coli clones (4000 per day) for the presence of functional or improved carbohydrate hydrolase enzymes. The procedure is relatively cheap and has the advantage that carbohydrate degrading activity can be directly

  13. Steady state kinetic analysis of substrate specificity of glycoside hydrolases from families 13 and 38

    DEFF Research Database (Denmark)

    Nielsen, Jonas Willum

    Glycosidases are widespread in nature, where they perform a diverse range of functions. The glycoside hydrolase (GH) family 38, α-mannosidase II enzymes play a crucial role in mammalian cells, in the maturation of N-glycosylated proteins in the Golgi apparatus and in catabolism in cytosol...

  14. Improvement of enantioselectivity by immobilized imprinting of epoxide hydrolase from Rhodotorula glutinis

    NARCIS (Netherlands)

    Kronenburg, N.A.E.; Bont, de J.A.M.; Fischer, L.

    2001-01-01

    The yeast Rhodotorula glutinis contains an enantioselective, membrane-associated epoxide hydrolase (EH). Partially purified EH was immobilized in a two-step procedure. In the first step, the proteins were derivatized with itaconic anhydride. In the second step, the derivatized proteins were

  15. Cloning and characterization of an epoxide hydrolase-encoding gene from Rhodotorula glutinis

    NARCIS (Netherlands)

    Visser, H.; Vreugdenhil, S.; Bont, de J.A.M.; Verdoes, J.C.

    2000-01-01

    We cloned and characterized the epoxide hydrolase gene, EPH1, from Rhodotorula glutinis. The EPH1 open reading frame of 1230 bp was interrupted by nine introns and encoded a polypeptide of 409 amino acids with a calculated molecular mass of 46.3 kDa. The amino acid sequence was similar to that of

  16. Mode of action of xylogalacturonan hydrolase towards xylogalacturonan and xylogalacturonan oligosaccharides

    NARCIS (Netherlands)

    Zandleven, J.S.; Beldman, G.; Bosveld, M.; Benen, J.A.E.; Voragen, A.G.J.

    2005-01-01

    XGH (xylogalacturonan hydrolase; GH 28) is an enzyme that is capable of degrading XGA (xylogalacturonan), which is a polymer of ¿-D-galacturonic acid, highly substituted with ß-D-xylose. XGA is present in cell walls of various plants and exudates, such as gum tragacanth. XGA oligosaccharides were

  17. Enzymatic degradation studies of xylogalacturonans from apple and potato, using xylogalacturonan hydrolase

    NARCIS (Netherlands)

    Zandleven, J.S.; Beldman, G.; Bosveld, M.; Schols, H.A.; Voragen, A.G.J.

    2006-01-01

    Action of xylogalacturonan hydrolase (XGH) towards xylogalacturonan (XGA) present in the alkali saponified ¿modified hairy regions¿ from potato and apple pectin was studied. Analysis of enzymatic degradation products from XGA in these complex pectins demonstrated that the degradable

  18. The role of epoxide hydrolase Y113H gene variant in pancreatic diseases.

    NARCIS (Netherlands)

    Ockenga, J.; Strunck, S.; Post, C.; Schulz, H.U.; Halangk, J.; Pfutzer, R.H.; Lohr, M.; Oettle, H.; Kage, A.; Rosendahl, J.; Keim, V.; Drenth, J.P.H.; Jansen, J.B.M.J.; Lochs, H.; Witt, H.

    2009-01-01

    OBJECTIVES: Chronic pancreatitis (CP) and pancreatic adenocarcinoma (pCA) are associated with risk factors such as alcohol intake and tobacco smoking. Microsomal epoxide hydrolase (EPHX1) is a phase II detoxifying enzyme capable of tobacco-borne toxicant inactivation. We studied the role of the

  19. Oxidoreductive Cellulose Depolymerization by the Enzymes Cellobiose Dehydrogenase and Glycoside Hydrolase 61▿†

    Science.gov (United States)

    Langston, James A.; Shaghasi, Tarana; Abbate, Eric; Xu, Feng; Vlasenko, Elena; Sweeney, Matt D.

    2011-01-01

    Several members of the glycoside hydrolase 61 (GH61) family of proteins have recently been shown to dramatically increase the breakdown of lignocellulosic biomass by microbial hydrolytic cellulases. However, purified GH61 proteins have neither demonstrable direct hydrolase activity on various polysaccharide or lignacious components of biomass nor an apparent hydrolase active site. Cellobiose dehydrogenase (CDH) is a secreted flavocytochrome produced by many cellulose-degrading fungi with no well-understood biological function. Here we demonstrate that the binary combination of Thermoascus aurantiacus GH61A (TaGH61A) and Humicola insolens CDH (HiCDH) cleaves cellulose into soluble, oxidized oligosaccharides. TaGH61A-HiCDH activity on cellulose is shown to be nonredundant with the activities of canonical endocellulase and exocellulase enzymes in microcrystalline cellulose cleavage, and while the combination of TaGH61A and HiCDH cleaves highly crystalline bacterial cellulose, it does not cleave soluble cellodextrins. GH61 and CDH proteins are coexpressed and secreted by the thermophilic ascomycete Thielavia terrestris in response to environmental cellulose, and the combined activities of T. terrestris GH61 and T. terrestris CDH are shown to synergize with T. terrestris cellulose hydrolases in the breakdown of cellulose. The action of GH61 and CDH on cellulose may constitute an important, but overlooked, biological oxidoreductive system that functions in microbial lignocellulose degradation and has applications in industrial biomass utilization. PMID:21821740

  20. Fungal lytic polysaccharide monooxygenases bind starch and β-cyclodextrin similarly to amylolytic hydrolases

    DEFF Research Database (Denmark)

    Nekiunaite, Laura; Isaksen, Trine; Vaaje-Kolstad, Gustav

    2016-01-01

    , the clustering of CBM20s from starch-targeting LPMOs and hydrolases was in accord with taxonomy and did not correlate to appended catalytic activity. Altogether, these results demonstrate that the CBM20-binding scaffold is retained in the evolution of hydrolytic and oxidative starch-degrading activities....

  1. Supplementing with non-glycoside hydrolase proteins enhances enzymatic deconstruction of plant biomass.

    Science.gov (United States)

    Su, Xiaoyun; Zhang, Jing; Mackie, Roderick I; Cann, Isaac K O

    2012-01-01

    The glycoside hydrolases (GH) of Caldicellulosiruptor bescii are thermophilic enzymes, and therefore they can hydrolyze plant cell wall polysaccharides at high temperatures. Analyses of two C. bescii glycoside hydrolases, CbCelA-TM1 and CbXyn10A with cellulase and endoxylanase activity, respectively, demonstrated that each enzyme is highly thermostable under static incubation at 70°C. Both enzymes, however, rapidly lost their enzymatic activities when incubated at 70°C with end-over-end shaking. Since crowding conditions, even at low protein concentrations, seem to influence enzymatic properties, three non-glycoside hydrolase proteins were tested for their capacity to stabilize the thermophilic proteins at high temperatures. The three proteins investigated were a small heat shock protein CbHsp18 from C. bescii, a histone MkHistone1 from Methanopyrus kandleri, and bovine RNase A, from a commercial source. Fascinatingly, each of these proteins increased the thermostability of the glycoside hydrolases at 70°C during end-over-end shaking incubation, and this property translated into increases in hydrolysis of several substrates including the bioenergy feedstock Miscanthus. Furthermore, MkHistone1 and RNase A also altered the initial products released from the cello-oligosaccharide cellopentaose during hydrolysis with the cellodextrinase CbCdx1A, which further demonstrated the capacity of the three non-GH proteins to influence hydrolysis of substrates by the thermophilic glycoside hydrolases. The non-GH proteins used in the present report were small proteins derived from each of the three lineages of life, and therefore expand the space from which different polypeptides can be tested for their influence on plant cell wall hydrolysis, a critical step in the emerging biofuel industry.

  2. Supplementing with non-glycoside hydrolase proteins enhances enzymatic deconstruction of plant biomass.

    Directory of Open Access Journals (Sweden)

    Xiaoyun Su

    Full Text Available The glycoside hydrolases (GH of Caldicellulosiruptor bescii are thermophilic enzymes, and therefore they can hydrolyze plant cell wall polysaccharides at high temperatures. Analyses of two C. bescii glycoside hydrolases, CbCelA-TM1 and CbXyn10A with cellulase and endoxylanase activity, respectively, demonstrated that each enzyme is highly thermostable under static incubation at 70°C. Both enzymes, however, rapidly lost their enzymatic activities when incubated at 70°C with end-over-end shaking. Since crowding conditions, even at low protein concentrations, seem to influence enzymatic properties, three non-glycoside hydrolase proteins were tested for their capacity to stabilize the thermophilic proteins at high temperatures. The three proteins investigated were a small heat shock protein CbHsp18 from C. bescii, a histone MkHistone1 from Methanopyrus kandleri, and bovine RNase A, from a commercial source. Fascinatingly, each of these proteins increased the thermostability of the glycoside hydrolases at 70°C during end-over-end shaking incubation, and this property translated into increases in hydrolysis of several substrates including the bioenergy feedstock Miscanthus. Furthermore, MkHistone1 and RNase A also altered the initial products released from the cello-oligosaccharide cellopentaose during hydrolysis with the cellodextrinase CbCdx1A, which further demonstrated the capacity of the three non-GH proteins to influence hydrolysis of substrates by the thermophilic glycoside hydrolases. The non-GH proteins used in the present report were small proteins derived from each of the three lineages of life, and therefore expand the space from which different polypeptides can be tested for their influence on plant cell wall hydrolysis, a critical step in the emerging biofuel industry.

  3. Structural analysis of Clostridium acetobutylicum ATCC 824 glycoside hydrolase from CAZy family GH105

    International Nuclear Information System (INIS)

    Germane, Katherine L.; Servinsky, Matthew D.; Gerlach, Elliot S.; Sund, Christian J.; Hurley, Margaret M.

    2015-01-01

    The crystal structure of the protein product of the C. acetobutylicum ATCC 824 gene CA-C0359 is structurally similar to YteR, an unsaturated rhamnogalacturonyl hydrolase from B. subtilis strain 168. Substrate modeling and electrostatic studies of the active site of the structure of CA-C0359 suggests that the protein can now be considered to be part of CAZy glycoside hydrolase family 105. Clostridium acetobutylicum ATCC 824 gene CA-C0359 encodes a putative unsaturated rhamnogalacturonyl hydrolase (URH) with distant amino-acid sequence homology to YteR of Bacillus subtilis strain 168. YteR, like other URHs, has core structural homology to unsaturated glucuronyl hydrolases, but hydrolyzes the unsaturated disaccharide derivative of rhamnogalacturonan I. The crystal structure of the recombinant CA-C0359 protein was solved to 1.6 Å resolution by molecular replacement using the phase information of the previously reported structure of YteR (PDB entry (http://scripts.iucr.org/cgi-bin/cr.cgi?rm)) from Bacillus subtilis strain 168. The YteR-like protein is a six-α-hairpin barrel with two β-sheet strands and a small helix overlaying the end of the hairpins next to the active site. The protein has low primary protein sequence identity to YteR but is structurally similar. The two tertiary structures align with a root-mean-square deviation of 1.4 Å and contain a highly conserved active pocket. There is a conserved aspartic acid residue in both structures, which has been shown to be important for hydration of the C=C bond during the release of unsaturated galacturonic acid by YteR. A surface electrostatic potential comparison of CA-C0359 and proteins from CAZy families GH88 and GH105 reveals the make-up of the active site to be a combination of the unsaturated rhamnogalacturonyl hydrolase and the unsaturated glucuronyl hydrolase from Bacillus subtilis strain 168. Structural and electrostatic comparisons suggests that the protein may have a slightly different substrate

  4. Structural analysis of Clostridium acetobutylicum ATCC 824 glycoside hydrolase from CAZy family GH105

    Energy Technology Data Exchange (ETDEWEB)

    Germane, Katherine L., E-mail: katherine.germane.civ@mail.mil [Oak Ridge Associated Universities, 4692 Millennium Drive, Suite 101, Belcamp, MD 21017 (United States); Servinsky, Matthew D. [US Army Research Laboratory, 2800 Powder Mill Road, Adelphi, MD 20783 (United States); Gerlach, Elliot S. [Federal Staffing Resources, 2200 Somerville Road, Annapolis, MD 21401 (United States); Sund, Christian J. [US Army Research Laboratory, 2800 Powder Mill Road, Adelphi, MD 20783 (United States); Hurley, Margaret M., E-mail: katherine.germane.civ@mail.mil [US Army Research Laboratory, 4600 Deer Creek Loop, Aberdeen Proving Ground, MD 21005 (United States); Oak Ridge Associated Universities, 4692 Millennium Drive, Suite 101, Belcamp, MD 21017 (United States)

    2015-07-29

    The crystal structure of the protein product of the C. acetobutylicum ATCC 824 gene CA-C0359 is structurally similar to YteR, an unsaturated rhamnogalacturonyl hydrolase from B. subtilis strain 168. Substrate modeling and electrostatic studies of the active site of the structure of CA-C0359 suggests that the protein can now be considered to be part of CAZy glycoside hydrolase family 105. Clostridium acetobutylicum ATCC 824 gene CA-C0359 encodes a putative unsaturated rhamnogalacturonyl hydrolase (URH) with distant amino-acid sequence homology to YteR of Bacillus subtilis strain 168. YteR, like other URHs, has core structural homology to unsaturated glucuronyl hydrolases, but hydrolyzes the unsaturated disaccharide derivative of rhamnogalacturonan I. The crystal structure of the recombinant CA-C0359 protein was solved to 1.6 Å resolution by molecular replacement using the phase information of the previously reported structure of YteR (PDB entry (http://scripts.iucr.org/cgi-bin/cr.cgi?rm)) from Bacillus subtilis strain 168. The YteR-like protein is a six-α-hairpin barrel with two β-sheet strands and a small helix overlaying the end of the hairpins next to the active site. The protein has low primary protein sequence identity to YteR but is structurally similar. The two tertiary structures align with a root-mean-square deviation of 1.4 Å and contain a highly conserved active pocket. There is a conserved aspartic acid residue in both structures, which has been shown to be important for hydration of the C=C bond during the release of unsaturated galacturonic acid by YteR. A surface electrostatic potential comparison of CA-C0359 and proteins from CAZy families GH88 and GH105 reveals the make-up of the active site to be a combination of the unsaturated rhamnogalacturonyl hydrolase and the unsaturated glucuronyl hydrolase from Bacillus subtilis strain 168. Structural and electrostatic comparisons suggests that the protein may have a slightly different substrate

  5. Cloning, expression and characterization of a mammalian Nudix hydrolase-like enzyme that cleaves the pyrophosphate bond of UDP-glucose.

    Science.gov (United States)

    Yagi, Toshihiro; Baroja-Fernández, Edurne; Yamamoto, Ryuji; Muñoz, Francisco José; Akazawa, Takashi; Hong, Kyoung Su; Pozueta-Romero, Javier

    2003-03-01

    A distinct UDP-glucose (UDPG) pyrophosphatase (UGPPase, EC 3.6.1.45) has been characterized using pig kidney ( Sus scrofa ). This enzyme hydrolyses UDPG, the precursor molecule of numerous glycosylation reactions in animals, to produce glucose 1-phosphate (G1P) and UMP. Sequence analyses of the purified enzyme revealed that, similar to the case of a nucleotide-sugar hydrolase controlling the intracellular levels of ADP-glucose linked to glycogen biosynthesis in Escherichia coli [Moreno-Bruna, Baroja-Fernández, Muñoz, Bastarrica-Berasategui, Zandueta-Criado, Rodri;guez-López, Lasa, Akazawa and Pozueta-Romero (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 8128-8132], UGPPase appears to be a member of the ubiquitously distributed group of nucleotide pyrophosphatases designated Nudix hydrolases. A complete cDNA of the UGPPase-encoding gene, designated UGPP, was isolated from a human thyroid cDNA library and expressed in E. coli. The resulting cells accumulated a protein that showed kinetic properties identical to those of pig UGPPase.

  6. Variation in bleomycin hydrolase gene is associated with reduced survival after chemotherapy for testicular germ cell cancer

    NARCIS (Netherlands)

    de Haas, Esther C.; Zwart, Nynke; Meijer, Coby; Nuver, Janine; Boezen, H. Marike; Suurmeijer, Albert J. H.; Hoekstra, Harald J.; van der Steege, Gerrit; Sleijfer, Dirk Th.; Gietema, Jourik A.

    2008-01-01

    Purpose Response to chemotherapy may be determined by gene polymorphisms involved in metabolism of cytotoxic drugs. A plausible candidate is the gene for bleomycin hydrolase (BLMH), an enzyme that inactivates bleomycin, an essential component of chemotherapy regimens for disseminated testicular

  7. Lipoxin Generation Is Related to Soluble Epoxide Hydrolase Activity in Severe Asthma

    Science.gov (United States)

    Ono, Emiko; Dutile, Stefanie; Kazani, Shamsah; Wechsler, Michael E.; Yang, Jun; Hammock, Bruce D.; Douda, David Nobuhiro; Tabet, Yacine; Khaddaj-Mallat, Rayan; Sirois, Marco; Sirois, Chantal; Rizcallah, Edmond; Rousseau, Éric; Martin, Richard; Sutherland, E. Rand; Castro, Mario; N. Jarjour, Nizar; Israel, Elliot

    2014-01-01

    Rationale: Severe asthma is characterized by airway inflammatory responses associated with aberrant metabolism of arachidonic acid. Lipoxins (LX) are arachidonate-derived pro-resolving mediators that are decreased in severe asthma, yet mechanisms for defective LX biosynthesis and a means to increase LXs in severe asthma remain to be established. Objectives: To determine if oxidative stress and soluble epoxide hydrolase (sEH) activity are linked to decreased LX biosynthesis in severe asthma. Methods: Aliquots of blood, sputum, and bronchoalveolar lavage fluid were obtained from asthma subjects for mediator determination. Select samples were exposed to t-butyl-hydroperoxide or sEH inhibitor (sEHI) before activation. Peripheral blood leukocyte–platelet aggregates were monitored by flow cytometry, and bronchial contraction was determined with cytokine-treated human lung sections. Measurements and Main Results: 8-Isoprostane levels in sputum supernatants were inversely related to LXA4 in severe asthma (r = −0.55; P = 0.03) and t-butyl-hydroperoxide decreased LXA4 and 15-epi-LXA4 biosynthesis by peripheral blood leukocytes. LXA4 and 15-epi-LXA4 levels were inversely related to sEH activity in sputum supernatants and sEHIs significantly increased 14,15-epoxy-eicosatrienoic acid and 15-epi-LXA4 generation by severe asthma whole blood and bronchoalveolar lavage fluid cells. The abundance of peripheral blood leukocyte–platelet aggregates was related to asthma severity. In a concentration-dependent manner, LXs significantly inhibited platelet-activating factor–induced increases in leukocyte–platelet aggregates (70.8% inhibition [LXA4 100 nM], 78.3% inhibition [15-epi-LXA4 100 nM]) and 15-epi-LXA4 markedly inhibited tumor necrosis factor-α–induced increases in bronchial contraction. Conclusions: LX levels were decreased by oxidative stress and sEH activity. Inhibitors of sEH increased LXs that mediated antiphlogistic actions, suggesting a new therapeutic approach

  8. Biosynthesis of intestinal microvillar proteins. Dimerization of aminopeptidase N and lactase-phlorizin hydrolase

    DEFF Research Database (Denmark)

    Danielsen, E M

    1990-01-01

    of dimers of this enzyme therefore occurs prior to the Golgi-associated processing, and the slow rate of dimerization may be the rate-limiting step in the transport from the endoplasmic reticulum to the Golgi complex. For lactase-phlorizin hydrolase, the posttranslational processing includes a proteolytic......The pig intestinal brush border enzymes aminopeptidase N (EC 3.4.11.2) and lactase-phlorizin hydrolase (EC 3.2.1.23-62) are present in the microvillar membrane as homodimers. Dimethyl adipimidate was used to cross-link the two [35S]methionine-labeled brush border enzymes from cultured mucosal...... explants. For aminopeptidase N, dimerization did not begin until 5-10 min after synthesis, and maximal dimerization by cross-linking of the transient form of the enzyme required 1 h, whereas the mature form of aminopeptidase N cross-linked with unchanged efficiency from 45 min to 3 h of labeling. Formation...

  9. Preparation, crystallization and preliminary X-ray crystallographic studies of diadenosine tetraphosphate hydrolase from Shigella flexneri 2a

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Wenxin; Wang, Qihai; Bi, Ruchang, E-mail: rcbi@sun5.ibp.ac.cn [Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101 (China)

    2005-12-01

    The 31.3 kDa Ap{sub 4}A hydrolase from Shigella flexneri 2a has been cloned, expressed and purified using an Escherichia coli expression system. Crystals of Ap{sub 4}A hydrolase have been obtained by the hanging-drop technique at 291 K using PEG 550 MME as precipitant. Diadenosine tetraphosphate (Ap{sub 4}A) hydrolase (EC 3.6.1.41) hydrolyzes Ap{sub 4}A symmetrically in prokaryotes. It plays a potential role in organisms by regulating the concentration of Ap{sub 4}A in vivo. To date, no three-dimensional structures of proteins with significant sequence homology to this protein have been determined. The 31.3 kDa Ap{sub 4}A hydrolase from Shigella flexneri 2a has been cloned, expressed and purified using an Escherichia coli expression system. Crystals of Ap{sub 4}A hydrolase have been obtained by the hanging-drop technique at 291 K using PEG 550 MME as precipitant. Ap{sub 4}A hydrolase crystals diffract X-rays to 3.26 Å and belong to space group P2{sub 1}, with unit-cell parameters a = 118.9, b = 54.6, c = 128.5 Å, β = 95.7°.

  10. Screening brazilian macrophomina phaseolina isolates for alkaline lipases and other extracellular hydrolases

    OpenAIRE

    Schinke, Cláudia; Germani, Jose Carlos

    2012-01-01

    Macrophomina phaseolina, phylum Ascomycota, is a phytopathogenic fungus distributed worldwide in hot dry areas. There are few studies on its secreted lipases and none on its colony radial growth rate, an indicator of fungal ability to use nutrients for growth, on media other than potato-dextrose agar. In this study, 13 M. phaseolina isolates collected in different Brazilian regions were screened for fast-growth and the production of hydrolases of industrial interest, especially alkaline lipas...

  11. Murein Hydrolase Activity in the Surface Layer of Lactobacillus acidophilus ATCC 4356▿

    OpenAIRE

    Prado Acosta, Mariano; Palomino, María Mercedes; Allievi, Mariana C.; Rivas, Carmen Sanchez; Ruzal, Sandra M.

    2008-01-01

    We describe a new enzymatic functionality for the surface layer (S-layer) of Lactobacillus acidophilus ATCC 4356, namely, an endopeptidase activity against the cell wall of Salmonella enterica serovar Newport, assayed via zymograms and identified by Western blotting. Based on amino acid sequence comparisons, the hydrolase activity was predicted to be located at the C terminus. Subsequent cloning and expression of the C-terminal domain in Bacillus subtilis resulted in the functional verificati...

  12. Functional analysis of the Escherichia coli genome for members of the alpha/beta hydrolase family.

    Science.gov (United States)

    Zhang, L; Godzik, A; Skolnick, J; Fetrow, J S

    1998-01-01

    Database-searching methods based on sequence similarity have become the most commonly used tools for characterizing newly sequenced proteins. Due to the often underestimated functional diversity in protein families and superfamilies, however, it is difficult to make the characterization specific and accurate. In this work, we have extended a method for active-site identification from predicted protein structures. The structural conservation and variation of the active sites of the alpha/beta hydrolases with known structures were studied. The similarities were incorporated into a three-dimensional motif that specifies essential requirements for the enzymatic functions. A threading algorithm was used to align 651 Escherichia coli open reading frames (ORFs) to one of the members of the alpha/beta hydrolase fold family. These ORFs were then screened according to our three-dimensional motif and with an extra requirement that demands conservation of the key active-site residues among the proteins that bear significant sequence similarity to the ORFs. 17 ORFs from E. coli were predicted to have hydrolase activity and their putative active-site residues were identified. Most were in agreement with the experiments and results of other database-searching methods. The study further suggests that YHET_ECOLI, a hypothetical protein classified as a member of the UPF0017 family (an uncharacterized protein family), bears all the hallmarks of the alpha/beta hydrolase family. The novel feature of our method is that it uses three-dimensional structural information for function prediction. The results demonstrate the importance and necessity of such a method to fill the gap between sequence alignment and function prediction; furthermore, the method provides a way to verify the structure predictions, which enables an expansion of the applicable scope of the threading algorithms.

  13. Phenotypic assessment of THC discriminative stimulus properties in fatty acid amide hydrolase knockout and wildtype mice

    OpenAIRE

    Walentiny, D. Matthew; Vann, Robert E.; Wiley, Jenny L.

    2015-01-01

    A number of studies have examined the ability of the endogenous cannabinoid anandamide to elicit Δ9 -tetrahydrocannabinol (THC)-like subjective effects, as modeled through the THC discrimination paradigm. In the present study, we compared transgenic mice lacking fatty acid amide hydrolase (FAAH), the enzyme primarily responsible for anandamide catabolism, to wildtype counterparts in a THC discrimination procedure. THC (5.6 mg/kg) served as a discriminative stimulus in both genotypes, with sim...

  14. Characterization of an epoxide hydrolase from the Florida red tide dinoflagellate, Karenia brevis.

    Science.gov (United States)

    Sun, Pengfei; Leeson, Cristian; Zhi, Xiaoduo; Leng, Fenfei; Pierce, Richard H; Henry, Michael S; Rein, Kathleen S

    2016-02-01

    Epoxide hydrolases (EH, EC 3.3.2.3) have been proposed to be key enzymes in the biosynthesis of polyether (PE) ladder compounds such as the brevetoxins which are produced by the dinoflagellate Karenia brevis. These enzymes have the potential to catalyze kinetically disfavored endo-tet cyclization reactions. Data mining of K. brevis transcriptome libraries revealed two classes of epoxide hydrolases: microsomal and leukotriene A4 (LTA4) hydrolases. A microsomal EH was cloned and expressed for characterization. The enzyme is a monomeric protein with molecular weight 44kDa. Kinetic parameters were evaluated using a variety of epoxide substrates to assess substrate selectivity and enantioselectivity, as well as its potential to catalyze the critical endo-tet cyclization of epoxy alcohols. Monitoring of EH activity in high and low toxin producing cultures of K. brevis over a three week period showed consistently higher activity in the high toxin producing culture implicating the involvement of one or more EH in brevetoxin biosynthesis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Purification, crystallization and preliminary crystallographic studies of plant S-adenosyl-l-homocysteine hydrolase (Lupinus luteus)

    International Nuclear Information System (INIS)

    Brzezinski, Krzysztof; Bujacz, Grzegorz; Jaskolski, Mariusz

    2008-01-01

    Single crystals of recombinant S-adenosyl-l-homocysteine hydrolase from L. luteus in complex with adenosine diffract X-rays to 1.17 Å resolution at 100 K. The crystals are tetragonal, space group P4 3 2 1 2, and contain one copy of the dimeric enzyme in the asymmetric unit. By degrading S-adenosyl-l-homocysteine, which is a byproduct of S-adenosyl-l-methionine-dependent methylation reactions, S-adenosyl-l-homocysteine hydrolase (SAHase) acts as a regulator of cellular methylation processes. S-Adenosyl-l-homocysteine hydrolase from the leguminose plant yellow lupin (Lupinus luteus), LlSAHase, which is composed of 485 amino acids and has a molecular weight of 55 kDa, has been cloned, expressed in Escherichia coli and purified. Crystals of LlSAHase in complex with adenosine were obtained by the hanging-drop vapour-diffusion method using 20%(w/v) PEG 4000 and 10%(v/v) 2-propanol as precipitants in 0.1 M Tris–HCl buffer pH 8.0. The crystals were tetragonal, space group P4 3 2 1 2, with unit-cell parameters a = 122.4, c = 126.5 Å and contained two protein molecules in the asymmetric unit, corresponding to the functional dimeric form of the enzyme. Atomic resolution (1.17 Å) X-ray diffraction data have been collected using synchrotron radiation

  16. Structure of a Trypanosoma brucei α/β-hydrolase fold protein with unknown function

    International Nuclear Information System (INIS)

    Merritt, Ethan A.; Holmes, Margaret; Buckner, Frederick S.; Van Voorhis, Wesley C.; Quartly, Erin; Phizicky, Eric M.; Lauricella, Angela; Luft, Joseph; DeTitta, George; Neely, Helen; Zucker, Frank; Hol, Wim G. J.

    2008-01-01

    T. brucei gene Tb10.6k15.0140 codes for an α/β-hydrolase fold protein of unknown function. The 2.2 Å crystal structure shows that members of this sequence family retain a conserved Ser residue at the expected site of a catalytic nucleophile, but that trypanosomatid sequences lack structural homologs for the other expected residues of the catalytic triad. The structure of a structural genomics target protein, Tbru020260AAA from Trypanosoma brucei, has been determined to a resolution of 2.2 Å using multiple-wavelength anomalous diffraction at the Se K edge. This protein belongs to Pfam sequence family PF08538 and is only distantly related to previously studied members of the α/β-hydrolase fold family. Structural superposition onto representative α/β-hydrolase fold proteins of known function indicates that a possible catalytic nucleophile, Ser116 in the T. brucei protein, lies at the expected location. However, the present structure and by extension the other trypanosomatid members of this sequence family have neither sequence nor structural similarity at the location of other active-site residues typical for proteins with this fold. Together with the presence of an additional domain between strands β6 and β7 that is conserved in trypanosomatid genomes, this suggests that the function of these homologs has diverged from other members of the fold family

  17. Purification, crystallization and preliminary crystallographic studies of plant S-adenosyl-l-homocysteine hydrolase (Lupinus luteus)

    Energy Technology Data Exchange (ETDEWEB)

    Brzezinski, Krzysztof [Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan (Poland); Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, Poznan (Poland); Bujacz, Grzegorz [Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan (Poland); Faculty of Food Chemistry and Biotechnology, Technical University of Lodz (Poland); Jaskolski, Mariusz, E-mail: mariuszj@amu.edu.pl [Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan (Poland); Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, Poznan (Poland)

    2008-07-01

    Single crystals of recombinant S-adenosyl-l-homocysteine hydrolase from L. luteus in complex with adenosine diffract X-rays to 1.17 Å resolution at 100 K. The crystals are tetragonal, space group P4{sub 3}2{sub 1}2, and contain one copy of the dimeric enzyme in the asymmetric unit. By degrading S-adenosyl-l-homocysteine, which is a byproduct of S-adenosyl-l-methionine-dependent methylation reactions, S-adenosyl-l-homocysteine hydrolase (SAHase) acts as a regulator of cellular methylation processes. S-Adenosyl-l-homocysteine hydrolase from the leguminose plant yellow lupin (Lupinus luteus), LlSAHase, which is composed of 485 amino acids and has a molecular weight of 55 kDa, has been cloned, expressed in Escherichia coli and purified. Crystals of LlSAHase in complex with adenosine were obtained by the hanging-drop vapour-diffusion method using 20%(w/v) PEG 4000 and 10%(v/v) 2-propanol as precipitants in 0.1 M Tris–HCl buffer pH 8.0. The crystals were tetragonal, space group P4{sub 3}2{sub 1}2, with unit-cell parameters a = 122.4, c = 126.5 Å and contained two protein molecules in the asymmetric unit, corresponding to the functional dimeric form of the enzyme. Atomic resolution (1.17 Å) X-ray diffraction data have been collected using synchrotron radiation.

  18. ClbS Is a Cyclopropane Hydrolase That Confers Colibactin Resistance.

    Science.gov (United States)

    Tripathi, Prabhanshu; Shine, Emilee E; Healy, Alan R; Kim, Chung Sub; Herzon, Seth B; Bruner, Steven D; Crawford, Jason M

    2017-12-13

    Certain commensal Escherichia coli contain the clb biosynthetic gene cluster that codes for small molecule prodrugs known as precolibactins. Precolibactins are converted to colibactins by N-deacylation; the latter are postulated to be genotoxic and to contribute to colorectal cancer formation. Though advances toward elucidating (pre)colibactin biosynthesis have been made, the functions and mechanisms of several clb gene products remain poorly understood. Here we report the 2.1 Å X-ray structure and molecular function of ClbS, a gene product that confers resistance to colibactin toxicity in host bacteria and which has been shown to be important for bacterial viability. The structure harbors a potential colibactin binding site and shares similarity to known hydrolases. In vitro studies using a synthetic colibactin analog and ClbS or an active site residue mutant reveal cyclopropane hydrolase activity that converts the electrophilic cyclopropane of the colibactins into an innocuous hydrolysis product. As the cyclopropane has been shown to be essential for genotoxic effects in vitro, this ClbS-catalyzed ring-opening provides a means for the bacteria to circumvent self-induced genotoxicity. Our study provides a molecular-level view of the first reported cyclopropane hydrolase and support for a specific mechanistic role of this enzyme in colibactin resistance.

  19. Regulation of catalytic behaviour of hydrolases through interactions with functionalized carbon-based nanomaterials

    International Nuclear Information System (INIS)

    Pavlidis, Ioannis V.; Vorhaben, Torge; Gournis, Dimitrios; Papadopoulos, George K.; Bornscheuer, Uwe T.; Stamatis, Haralambos

    2012-01-01

    The interaction of enzymes with carbon-based nanomaterials (CBNs) is crucial for the function of biomolecules and therefore for the design and development of effective nanobiocatalytic systems. In this study, the effect of functionalized CBNs, such as graphene oxide (GO) and multi-wall carbon nanotubes (CNTs), on the catalytic behaviour of various hydrolases of biotechnological interest was monitored and the interactions between CBNs and proteins were investigated. The enzyme–nanomaterial interactions significantly affect the catalytic behaviour of enzymes, resulting in an increase up to 60 % of the catalytic efficiency of lipases and a decrease up to 30 % of the esterase. Moreover, the use of CNTs and GO derivatives, especially those that are amine-functionalized, led to increased thermal stability of most the hydrolases tested. Fluorescence and circular dichroism studies indicated that the altered catalytic behaviour of enzymes in the presence of CBNs arises from specific enzyme–nanomaterial interactions, which can lead to significant conformational changes. In the case of lipases, the conformational changes led to a more active and rigid structure, while in the case of esterases this led to destabilization and unfolding. Kinetic and spectroscopic studies indicated that the extent of the interactions between CBNs and hydrolases can be mainly controlled by the functionalization of nanomaterials than by their geometry.

  20. Regulation of catalytic behaviour of hydrolases through interactions with functionalized carbon-based nanomaterials

    Energy Technology Data Exchange (ETDEWEB)

    Pavlidis, Ioannis V. [University of Ioannina, Laboratory of Biotechnology, Department of Biological Applications and Technologies (Greece); Vorhaben, Torge [Institute of Biochemistry, Greifswald University, Department of Biotechnology and Enzyme Catalysis (Germany); Gournis, Dimitrios [University of Ioannina, Department of Materials Science and Engineering (Greece); Papadopoulos, George K. [Epirus Institute of Technology, Laboratory of Biochemistry and Biophysics, Faculty of Agricultural Technology (Greece); Bornscheuer, Uwe T. [Institute of Biochemistry, Greifswald University, Department of Biotechnology and Enzyme Catalysis (Germany); Stamatis, Haralambos, E-mail: hstamati@cc.uoi.gr [University of Ioannina, Laboratory of Biotechnology, Department of Biological Applications and Technologies (Greece)

    2012-05-15

    The interaction of enzymes with carbon-based nanomaterials (CBNs) is crucial for the function of biomolecules and therefore for the design and development of effective nanobiocatalytic systems. In this study, the effect of functionalized CBNs, such as graphene oxide (GO) and multi-wall carbon nanotubes (CNTs), on the catalytic behaviour of various hydrolases of biotechnological interest was monitored and the interactions between CBNs and proteins were investigated. The enzyme-nanomaterial interactions significantly affect the catalytic behaviour of enzymes, resulting in an increase up to 60 % of the catalytic efficiency of lipases and a decrease up to 30 % of the esterase. Moreover, the use of CNTs and GO derivatives, especially those that are amine-functionalized, led to increased thermal stability of most the hydrolases tested. Fluorescence and circular dichroism studies indicated that the altered catalytic behaviour of enzymes in the presence of CBNs arises from specific enzyme-nanomaterial interactions, which can lead to significant conformational changes. In the case of lipases, the conformational changes led to a more active and rigid structure, while in the case of esterases this led to destabilization and unfolding. Kinetic and spectroscopic studies indicated that the extent of the interactions between CBNs and hydrolases can be mainly controlled by the functionalization of nanomaterials than by their geometry.

  1. Lysophosphatidic acids are new substrates for the phosphatase domain of soluble epoxide hydrolase.

    Science.gov (United States)

    Oguro, Ami; Imaoka, Susumu

    2012-03-01

    Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that has a C-terminus epoxide hydrolase domain and an N-terminus phosphatase domain. The endogenous substrates of epoxide hydrolase are known to be epoxyeicosatrienoic acids, but the endogenous substrates of the phosphatase activity are not well understood. In this study, to explore the substrates of sEH, we investigated the inhibition of the phosphatase activity of sEH toward 4-methylumbelliferyl phosphate by using lecithin and its hydrolyzed products. Although lecithin itself did not inhibit the phosphatase activity, the hydrolyzed lecithin significantly inhibited it, suggesting that lysophospholipid or fatty acid can inhibit it. Next, we investigated the inhibition of phosphatase activity by lysophosphatidyl choline, palmitoyl lysophosphatidic acid, monopalmitoyl glycerol, and palmitic acid. Palmitoyl lysophosphatidic acid and fatty acid efficiently inhibited phosphatase activity, suggesting that lysophosphatidic acids (LPAs) are substrates for the phosphatase activity of sEH. As expected, palmitoyl, stearoyl, oleoyl, and arachidonoyl LPAs were efficiently dephosphorylated by sEH (Km, 3-7 μM; Vmax, 150-193 nmol/min/mg). These results suggest that LPAs are substrates of sEH, which may regulate physiological functions of cells via their metabolism.

  2. Structure of the Cyanuric Acid Hydrolase TrzD Reveals Product Exit Channel.

    Science.gov (United States)

    Bera, Asim K; Aukema, Kelly G; Elias, Mikael; Wackett, Lawrence P

    2017-03-27

    Cyanuric acid hydrolases are of industrial importance because of their use in aquatic recreational facilities to remove cyanuric acid, a stabilizer for the chlorine. Degradation of excess cyanuric acid is necessary to maintain chlorine disinfection in the waters. Cyanuric acid hydrolase opens the cyanuric acid ring hydrolytically and subsequent decarboxylation produces carbon dioxide and biuret. In the present study, we report the X-ray structure of TrzD, a cyanuric acid hydrolase from Acidovorax citrulli. The crystal structure at 2.19 Å resolution shows a large displacement of the catalytic lysine (Lys163) in domain 2 away from the active site core, whereas the two other active site lysines from the two other domains are not able to move. The lysine displacement is proposed here to open up a channel for product release. Consistent with that, the structure also showed two molecules of the co-product, carbon dioxide, one in the active site and another trapped in the proposed exit channel. Previous data indicated that the domain 2 lysine residue plays a role in activating an adjacent serine residue carrying out nucleophilic attack, opening the cyanuric acid ring, and the mobile lysine guides products through the exit channel.

  3. Cloning, recombinant production, crystallization and preliminary X-ray diffraction studies of a family 84 glycoside hydrolase from Clostridium perfringens

    International Nuclear Information System (INIS)

    Ficko-Blean, Elizabeth; Boraston, Alisdair B.

    2005-01-01

    Crystallization of a family 84 glycoside hydrolase, a putative virulence factor, secreted by C. perfringens is reported. Clostridium perfringens is a ubiquitous environmental organism that is capable of causing a variety of diseases in mammals, including gas gangrene and necrotic enteritis in humans. The activity of a secreted hyaluronidase, attributed to the NagH protein, contributes to the pathogenicity of this organism. The family 84 catalytic module of one of the three homologues of NagH found in C. perfringens (ATCC 13124) has been cloned. The 69 kDa catalytic module of NagJ, here called GH84C, was overproduced in Escherichia coli and purified by immobilized metal-affinity chromatography (IMAC). Crystals belonging to space group I222 or I2 1 2 1 2 1 with unit-cell parameters a = 130.39, b = 150.05, c = 155.43 Å were obtained that diffracted to 2.1 Å. Selenomethionyl crystals have also been produced, leading to the possibility of solving the phase problem by MAD using synchrotron radiation

  4. Fatty Acid Amide Hydrolase Binding in Brain of Cannabis Users: Imaging with the Novel Radiotracer [11C]CURB

    Science.gov (United States)

    Boileau, Isabelle; Mansouri, Esmaeil; Williams, Belinda; Le Foll, Bernard; Rusjan, Pablo; Mizrahi, Romina; Tyndale, Rachel F.; Huestis, Marilyn A.; Payer, Doris E.; Wilson, Alan A.; Houle, Sylvain; Kish, Stephen J.; Tong, Junchao

    2016-01-01

    Background One of the major mechanisms for terminating the actions of the endocannabinoid anandamide is hydrolysis by fatty acid amide hydrolase (FAAH) and inhibitors of the enzyme were suggested as potential treatment for human cannabis dependence. However, the status of brain FAAH in cannabis use disorder is unknown. Methods Brain FAAH binding was measured with positron emission tomography and [11C]CURB in 22 healthy control subjects and ten chronic, frequent cannabis users during early abstinence. The FAAH genetic polymorphism (rs324420) and blood, urine and hair levels of cannabinoids and metabolites were determined. Results In cannabis users FAAH binding was significantly lower by 14–20% across the brain regions examined as compared to matched control subjects (overall Cohen’s d=0.96). Lower binding was negatively correlated with cannabinoid concentrations in blood and urine and was associated with higher trait impulsiveness. Conclusions Lower FAAH binding levels in the brain may be a consequence of chronic and recent cannabis exposure and could contribute to cannabis withdrawal. This effect should be considered in the development of novel treatment strategies for cannabis use disorder that target FAAH and endocannabinoids. Further studies are needed to examine possible changes in FAAH binding during prolonged cannabis abstinence and whether lower FAAH binding predates drug use. PMID:27345297

  5. Generation and characterization of epoxide hydrolase 3 (EPHX3-deficient mice.

    Directory of Open Access Journals (Sweden)

    Samantha L Hoopes

    Full Text Available Cytochrome P450 (CYP epoxygenases metabolize arachidonic acid into epoxyeicosatrienoic acids (EETs, which play an important role in blood pressure regulation, protection against ischemia-reperfusion injury, angiogenesis, and inflammation. Epoxide hydrolases metabolize EETs to their corresponding diols (dihydroxyeicosatrienoic acids; DHETs which are biologically less active. Microsomal epoxide hydrolase (EPHX1, mEH and soluble epoxide hydrolase (EPHX2, sEH were identified >30 years ago and are capable of hydrolyzing EETs to DHETs. A novel epoxide hydrolase, EPHX3, was recently identified by sequence homology and also exhibits epoxide hydrolase activity in vitro with a substrate preference for 9,10-epoxyoctadecamonoenoic acid (EpOME and 11,12-EET. EPHX3 is highly expressed in the skin, lung, stomach, esophagus, and tongue; however, its endogenous function is unknown. Therefore, we investigated the impact of genetic disruption of Ephx3 on fatty acid epoxide hydrolysis and EET-related physiology in mice. Ephx3-/- mice were generated by excising the promoter and first four exons of the Ephx3 gene using Cre-LoxP methodology. LC-MS/MS analysis of Ephx3-/- heart, lung, and skin lysates revealed no differences in endogenous epoxide:diol ratios compared to wild type (WT. Ephx3-/- mice also exhibited no change in plasma levels of fatty acid epoxides and diols relative to WT. Incubations of cytosolic and microsomal fractions prepared from Ephx3-/- and WT stomach, lung, and skin with synthetic 8,9-EET, 11,12-EET, and 9,10-EpOME revealed no significant differences in rates of fatty acid diol formation between the genotypes. Ephx3-/- hearts had similar functional recovery compared to WT hearts following ischemia/reperfusion injury. Following intranasal lipopolysaccharide (LPS exposure, Ephx3-/- mice were not different from WT in terms of lung histology, bronchoalveolar lavage fluid cell counts, or fatty acid epoxide and diol levels. We conclude that genetic

  6. The Differential Proteome of the Probiotic Lactobacillus acidophilus NCFM Grown on the Potential Prebiotic Cellobiose Shows Upregulation of Two beta-Glycoside Hydrolases

    DEFF Research Database (Denmark)

    van Zanten, Gabriella Christina; Sparding, Nadja; Majumder, Avishek

    2015-01-01

    Probiotics, prebiotics, and combinations there of, that is, synbiotics, are known to exert beneficial health effects in humans; however interactions between pro-and prebiotics remain poorly understood at the molecular level. The present study describes changes in abundance of different proteins...... of the probiotic bacterium Lactobacillus acidophilus NCFM (NCFM) when grown on the potential prebiotic cellobiose as compared to glucose. Cytosolic cell extract proteomes after harvest at late exponential phase of NCFM grown on cellobiose or glucose were analyzed by two dimensional difference gel electrophoresis....... Many of these proteins were associated with energy metabolism, including the cellobiose related glycoside hydrolases phospho-β-glucosidase (LBA0881) and phospho-β-galactosidase II (LBA0726). The data provide insight into the utilization of the candidate prebiotic cellobiose by the probiotic bacterium...

  7. Crystal structures of Mycobacterium tuberculosis S-adenosyl-L-homocysteine hydrolase in ternary complex with substrate and inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Reddy, Manchi C.M.; Kuppan, Gokulan; Shetty, Nishant D.; Owen, Joshua L.; Ioerger, Thomas R.; Sacchettini, James C. (TAM)

    2009-12-01

    S-adenosylhomocysteine hydrolase (SAHH) is a ubiquitous enzyme that plays a central role in methylation-based processes by maintaining the intracellular balance between S-adenosylhomocysteine (SAH) and S-adenosylmethionine. We report the first prokaryotic crystal structure of SAHH, from Mycobacterium tuberculosis (Mtb), in complex with adenosine (ADO) and nicotinamide adenine dinucleotide. Structures of complexes with three inhibitors are also reported: 3{prime}-keto aristeromycin (ARI), 2-fluoroadenosine, and 3-deazaadenosine. The ARI complex is the first reported structure of SAHH complexed with this inhibitor, and confirms the oxidation of the 3{prime} hydroxyl to a planar keto group, consistent with its prediction as a mechanism-based inhibitor. We demonstrate the in vivo enzyme inhibition activity of the three inhibitors and also show that 2-fluoradenosine has bactericidal activity. While most of the residues lining the ADO-binding pocket are identical between Mtb and human SAHH, less is known about the binding mode of the homocysteine (HCY) appendage of the full substrate. We report the 2.0 {angstrom} resolution structure of the complex of SAHH cocrystallized with SAH. The most striking change in the structure is that binding of HCY forces a rotation of His363 around the backbone to flip out of contact with the 5{prime} hydroxyl of the ADO and opens access to a nearby channel that leads to the surface. This complex suggests that His363 acts as a switch that opens up to permit binding of substrate, then closes down after release of the cleaved HCY. Differences in the entrance to this access channel between human and Mtb SAHH are identified.

  8. Mitochondrial NUDIX hydrolases: A metabolic link between NAD catabolism, GTP and mitochondrial dynamics.

    Science.gov (United States)

    Long, Aaron; Klimova, Nina; Kristian, Tibor

    2017-10-01

    NAD + catabolism and mitochondrial dynamics are important parts of normal mitochondrial function and are both reported to be disrupted in aging, neurodegenerative diseases, and acute brain injury. While both processes have been extensively studied there has been little reported on how the mechanisms of these two processes are linked. This review focuses on how downstream NAD + catabolism via NUDIX hydrolases affects mitochondrial dynamics under pathologic conditions. Additionally, several potential targets in mitochondrial dysfunction and fragmentation are discussed, including the roles of mitochondrial poly(ADP-ribose) polymerase 1(mtPARP1), AMPK, AMP, and intra-mitochondrial GTP metabolism. Mitochondrial and cytosolic NUDIX hydrolases (NUDT9α and NUDT9β) can affect mitochondrial and cellular AMP levels by hydrolyzing ADP- ribose (ADPr) and subsequently altering the levels of GTP and ATP. Poly (ADP-ribose) polymerase 1 (PARP1) is activated after DNA damage, which depletes NAD + pools and results in the PARylation of nuclear and mitochondrial proteins. In the mitochondria, ADP-ribosyl hydrolase-3 (ARH3) hydrolyzes PAR to ADPr, while NUDT9α metabolizes ADPr to AMP. Elevated AMP levels have been reported to reduce mitochondrial ATP production by inhibiting the adenine nucleotide translocase (ANT), allosterically activating AMPK by altering the cellular AMP: ATP ratio, and by depleting mitochondrial GTP pools by being phosphorylated by adenylate kinase 3 (AK3), which uses GTP as a phosphate donor. Recently, activated AMPK was reported to phosphorylate mitochondria fission factor (MFF), which increases Drp1 localization to the mitochondria and promotes mitochondrial fission. Moreover, the increased AK3 activity could deplete mitochondrial GTP pools and possibly inhibit normal activity of GTP-dependent fusion enzymes, thus altering mitochondrial dynamics. Published by Elsevier Ltd.

  9. Some hydrolase activities from the tick Hyalomma lusitanicum Koch, 1844 (Ixodoidea: Ixodida

    Directory of Open Access Journals (Sweden)

    Giménez-Pardo C.

    2008-12-01

    Full Text Available In this work has been made a detection and preliminary characterization of some hydrolases in whole extracts from unfed adult males and females of Hyalomma lusitanicum, one of the vectors for Theileria annulata that causes Mediterranean theileriosis in cattle. We have elected as targets, proteases as enzymes implicated in the nutritional processes of ticks, esterases that are usually implicated in resistance to organophosphates and phosphatises often implicated in protein phosphorilation and control of ticks salivary gland. The biological role and physiological significance are discussed in terms of the possibility of use these enzymes as possible in future anti-tick vaccination or acaricide resistance.

  10. Studies on whole cell fluorescence-based screening for epoxide hydrolases and Baeyer-Villiger monooxygenases

    International Nuclear Information System (INIS)

    Bicalho, Beatriz; Chen, Lu S.; Marsaioli, Anita J.; Grognux, Johann; Reymond, Jean-Louis

    2004-01-01

    Biocatalysis reactions were performed on microtiter plates (200 μL) aiming at the utilization of fluorogenic substrates (100 μmol L -1 ) for rapid whole cell screening for epoxide hydrolases (EHs) and Baeyer-Villiger monooxygenases (BVMOs). A final protocol was achieved for EHs, with 3 new enzymatic sources being detected (Agrobacterium tumefaciens, Pichia stipitis, Trichosporom cutaneum). The fluorogenic assay for BVMO did not work as expected. However, an approach to possible variables involved (aeration; pH) provided the first detection of a BVMO activity in T. cutaneum. (author)

  11. Protein features as determinants of wild-type glycoside hydrolase thermostability

    DEFF Research Database (Denmark)

    Geertz-Hansen, Henrik Marcus; Kiemer, Lars; Nielsen, Morten

    2017-01-01

    -silico methods guiding the discovery process would be of high value. To develop such an in-silico method and provide the data foundation of it, we determined the melting temperatures of 602 fungal glycoside hydrolases from the families GH5, 6, 7, 10, 11, 43 and AA9 (formerly GH61). We, then used sequence...... and homology modeled structure information of these enzymes to develop the ThermoP melting temperature prediction method. Futhermore, in the context of thermostability, we determined the relative importance of 160 molecular features, such as amino acid frequencies and spatial interactions, and exemplified...

  12. A proton wire and water channel revealed in the crystal structure of isatin hydrolase

    DEFF Research Database (Denmark)

    Bjerregaard-Andersen, Kaare; Sommer, Theis; Jensen, Jan Kristian

    2014-01-01

    to a novel family of metalloenzymes that include the bacterial kynurenine formamidase. The product state, mimicked by bound thioisatinate, reveals a water molecule that bridges the thioisatinate to a proton wire in an adjacent water channel and thus allows the proton released by the reaction to escape only...... when the product is formed. The functional proton wire present in IH-b represents a unique catalytic feature common to all hydrolases is here trapped and visualized for the first time. The local molecular environment required to coordinate thioisatinate allows stronger and more confident identification...

  13. Purification and characterization of RihC, a xanthosine-inosine-uridine-adenosine-preferring hydrolase from Salmonella enterica serovar Typhimurium

    DEFF Research Database (Denmark)

    Hansen, Michael Riis; Dandanell, Gert

    2005-01-01

    as the sole carbon and energy source. By functional complementation, we have isolated a nucleoside hydrolase (rihC) that can complement a xapA deletion in E. coli and we have overexpressed, purified and characterized this hydrolase. RihC is a heat stable homotetrameric enzyme with a molecular weight of 135 k...... the neutral form of xanthosine....

  14. Characterization of two novel bacterial type A exo-chitobiose hydrolases having C-terminal 5/12-type carbohydrate-binding modules

    DEFF Research Database (Denmark)

    Binti Jamek, Shariza; Nyffenegger, Christian; Muschiol, Jan

    2017-01-01

    "exo-chitobiose hydrolases." In this study, the chitinase type A from Serratia marcescens (SmaChiA) was used as a template for identifying two novel exo-chitobiose hydrolase type A enzymes, FbalChi18A and MvarChi18A, originating from the marine organisms Ferrimonas balearica and Microbulbifer...

  15. Cellular localization of peptide hydrolases in chicken embryo tissues and influence of gamma irradiation on their activity

    Energy Technology Data Exchange (ETDEWEB)

    Khristov, D; Marinopolski, G

    1975-01-01

    Studied was the influence of chicken embryo irradiation at 600 R and 1000 R gamma rays on the activity of tissue peptide hydrolases in mitochondrial-lysosomal, microsomal and supernatant (cell hyaloplasm) cell fractions. The investigation was performed 50 to 168 hours post irradiation. The wole tissue (of the whole embryo) was examined following irradiation of 4-day-old embryos whose liver, muscle and brain tissues were post irradiation examined on day 12 and 16 of incubation. Prior to treatment, the tissues were threfold rinsed with sucrose solution to eliminate proeinase inhibitors. Lysosome membranes were destroyed by adding 0.5 % desoxycholate. It was found that: Peptide hydrolase activity of mitochondrial-lysosomal cell fractions of tissues of whole 6-day chicken embryos is 4-5 times as high as that of cell hyaloplasm. Peptide hydrolase activity of mitochondrial-lysosomal fractions of liver tissues decreases on day 18 and 19 post incubation, while the same fraction of muscle and brain tissues shows high activity. Peptide hydrolase activity of microsomal fraction and of cell hyaloplasm rises during embryonal development and exceeds the activity of liver tissue mitochondrial fraction. Peptide hydrolase activity of mitochondrial-lysosomal fraction of tissue of whole 6-day-old embryos 50 hours post irradiation is higher than the activity of non-irradiated embryos. Later the activity of this fraction diminishes and on the 168 hr post irradiation it drops below the normal. Microsomal fraction and cell hyaloplasm activity likewise show deviation from the norm. Peptide hydrolase activity of mitochondrial-lysosomal fraction of liver, muscle and brain tissue of 14 and 18-day-old embryos is higher than the control 50 hours post irradiation and then declines. The activity of mitochondrial-lysosomal fraction of embryo brain tissue changes most strikingly on irradiation, while other brain cell fractions change less compared with liver and muscle fractions.

  16. Cellular localization of peptide hydrolases in chicken embryo tissues and influence of gamma irradiation on their activity

    International Nuclear Information System (INIS)

    Khristov, D.; Marinopolski, G.

    1975-01-01

    Studied was the influence of chicken embryo irradiation at 600 R and 1000 R gamma rays on the activity of tissue peptide hydrolases in mitochondrial-lysosomal, microsomal and supernatant (cell hyaloplasm) cell fractions. The investigation was performed 50 to 168 hours post irradiation. The wole tissue (of the whole embryo) was examined following irradiation of 4-day-old embryos whose liver, muscle and brain tissues were post irradiation examined on day 12 and 16 of incubation. Prior to treatment, the tissues were threfold rinsed with sucrose solution to eliminate proeinase inhibitors. Lysosome membranes were destroyed by adding 0.5 % desoxycholate. It was found that: Peptide hydrolase activity of mitochondrial-lysosomal cell fractions of tissues of whole 6-day chicken embryos is 4-5 times as high as that of cell hyaloplasm. Peptide hydrolase activity of mitochondrial-lysosomal fractions of liver tissues decreases on day 18 and 19 post incubation, while the same fraction of muscle and brain tissues shows high activity. Peptide hydrolase activity of microsomal fraction and of cell hyaloplasm rises during embryonal development and exceeds the activity of liver tissue mitochondrial fraction. Peptide hydrolase activity of mitochondrial-lysosomal fraction of tissue of whole 6-day-old embryos 50 hours post irradiation is higher than the activity of non-irradiated embryos. Later the activity of this fraction diminishes and on the 168 hr post irradiation it drops below the normal. Microsomal fraction and cell hyaloplasm activity likewise show deviation from the norm. Peptide hydrolase activity of mitochondrial-lysosomal fraction of liver, muscle and brain tissue of 14 and 18-day-old embryos is higher than the control 50 hours post irradiation and then declines. The activity of mitochondrial-lysosomal fraction of embryo brain tissue changes most strikingly on irradiation, while other brain cell fractions change less compared with liver and muscle fractions

  17. Mutations in Four Glycosyl Hydrolases Reveal a Highly Coordinated Pathway for Rhodopsin Biosynthesis and N-Glycan Trimming in Drosophila melanogaster

    Science.gov (United States)

    Rosenbaum, Erica E.; Vasiljevic, Eva; Brehm, Kimberley S.; Colley, Nansi Jo

    2014-01-01

    As newly synthesized glycoproteins move through the secretory pathway, the asparagine-linked glycan (N-glycan) undergoes extensive modifications involving the sequential removal and addition of sugar residues. These modifications are critical for the proper assembly, quality control and transport of glycoproteins during biosynthesis. The importance of N-glycosylation is illustrated by a growing list of diseases that result from defects in the biosynthesis and processing of N-linked glycans. The major rhodopsin in Drosophila melanogaster photoreceptors, Rh1, is highly unique among glycoproteins, as the N-glycan appears to be completely removed during Rh1 biosynthesis and maturation. However, much of the deglycosylation pathway for Rh1 remains unknown. To elucidate the key steps in Rh1 deglycosylation in vivo, we characterized mutant alleles of four Drosophila glycosyl hydrolases, namely α-mannosidase-II (α-Man-II), α-mannosidase-IIb (α-Man-IIb), a β-N-acetylglucosaminidase called fused lobes (Fdl), and hexosaminidase 1 (Hexo1). We have demonstrated that these four enzymes play essential and unique roles in a highly coordinated pathway for oligosaccharide trimming during Rh1 biosynthesis. Our results reveal that α-Man-II and α-Man-IIb are not isozymes like their mammalian counterparts, but rather function at distinct stages in Rh1 maturation. Also of significance, our results indicate that Hexo1 has a biosynthetic role in N-glycan processing during Rh1 maturation. This is unexpected given that in humans, the hexosaminidases are typically lysosomal enzymes involved in N-glycan catabolism with no known roles in protein biosynthesis. Here, we present a genetic dissection of glycoprotein processing in Drosophila and unveil key steps in N-glycan trimming during Rh1 biosynthesis. Taken together, our results provide fundamental advances towards understanding the complex and highly regulated pathway of N-glycosylation in vivo and reveal novel insights into the

  18. Mutations in four glycosyl hydrolases reveal a highly coordinated pathway for rhodopsin biosynthesis and N-glycan trimming in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Erica E Rosenbaum

    2014-05-01

    Full Text Available As newly synthesized glycoproteins move through the secretory pathway, the asparagine-linked glycan (N-glycan undergoes extensive modifications involving the sequential removal and addition of sugar residues. These modifications are critical for the proper assembly, quality control and transport of glycoproteins during biosynthesis. The importance of N-glycosylation is illustrated by a growing list of diseases that result from defects in the biosynthesis and processing of N-linked glycans. The major rhodopsin in Drosophila melanogaster photoreceptors, Rh1, is highly unique among glycoproteins, as the N-glycan appears to be completely removed during Rh1 biosynthesis and maturation. However, much of the deglycosylation pathway for Rh1 remains unknown. To elucidate the key steps in Rh1 deglycosylation in vivo, we characterized mutant alleles of four Drosophila glycosyl hydrolases, namely α-mannosidase-II (α-Man-II, α-mannosidase-IIb (α-Man-IIb, a β-N-acetylglucosaminidase called fused lobes (Fdl, and hexosaminidase 1 (Hexo1. We have demonstrated that these four enzymes play essential and unique roles in a highly coordinated pathway for oligosaccharide trimming during Rh1 biosynthesis. Our results reveal that α-Man-II and α-Man-IIb are not isozymes like their mammalian counterparts, but rather function at distinct stages in Rh1 maturation. Also of significance, our results indicate that Hexo1 has a biosynthetic role in N-glycan processing during Rh1 maturation. This is unexpected given that in humans, the hexosaminidases are typically lysosomal enzymes involved in N-glycan catabolism with no known roles in protein biosynthesis. Here, we present a genetic dissection of glycoprotein processing in Drosophila and unveil key steps in N-glycan trimming during Rh1 biosynthesis. Taken together, our results provide fundamental advances towards understanding the complex and highly regulated pathway of N-glycosylation in vivo and reveal novel insights

  19. Deconjugated bile salts produced by extracellular bile-salt hydrolase-like activities from the probiotic Lactobacillus johnsonii La1 inhibit Giardia duodenalis in vitro growth

    Directory of Open Access Journals (Sweden)

    Marie-Agnès Travers

    2016-09-01

    Full Text Available Giardiasis, currently considered a neglected disease, is caused by the intestinal protozoan parasite Giardia duodenalis and is widely spread in human as well as domestic and wild animals. The lack of appropriate medications and the spread of resistant parasite strains urgently call for the development of novel therapeutic strategies. Host microbiota or certain probiotic strains have the capacity to provide some protection against giardiasis. By combining biological and biochemical approaches, we have been able to decipher a molecular mechanism used by the probiotic strain Lactobacillus johnsonii La1 to prevent Giardia growth in vitro. We provide evidence that the supernatant of this strain contains active principle(s not directly toxic to Giardia but able to convert non-toxic components of bile into components highly toxic to Giardia. By using bile acid profiling, these components were identified as deconjugated bile-salts. A bacterial bile-salt-hydrolase of commercial origin was able to mimic the properties of the supernatant. Mass spectrometric analysis of the bacterial supernatant identified two of the three bile-salt-hydrolases encoded in the genome of this probiotic strain. These observations document a possible mechanism by which L. johnsonii La1, by secreting or releasing BSH-like activity(ies in the vicinity of replicating Giardia in an environment where bile is present and abundant, can fight this parasite. This discovery has both fundamental and applied outcomes to fight giardiasis, based on local delivery of deconjugated bile salts, enzyme deconjugation of bile components, or natural or recombinant probiotic strains that secrete or release such deconjugating activities in a compartment where both bile salts and Giardia are present.

  20. Identification of the chain-dispersing peptidoglycan hydrolase LytB of Streptococcus gordonii.

    Directory of Open Access Journals (Sweden)

    Riccardo Arrigucci

    Full Text Available Bacterial cell division ends with the separation of the daughter cells, a process that requires peptidoglycan hydrolases (PGHs. Bacteria lacking cell separating PGHs are impaired in cell separation with the formation of long chains or clusters. We identified a gene in Streptococcus gordonii encoding for a putative glucosaminidase (lytB. The lytB isogenic mutant grew in long bacterial chains and resulted in impaired biofilm formation. Purified recombinant LytB showed a murolytic activity on Micrococcus lysodeikticus cell suspension and was able to disperse the long chains of the mutant, restoring the wild type diplococci/short chain phenotype. LytB protein was localized only in culture supernatant cell fraction of S. gordonii, and co-cultures of wild type and lytB mutant showed a significant reduction of bacterial chain length, indicating that LytB is a secreted enzyme. Our results demonstrate that LytB is a secreted peptidoglycan hydrolase required for S. gordonii cell separation.

  1. Screening Brazilian Macrophomina phaseolina isolates for alkaline lipases and other extracellular hydrolases.

    Science.gov (United States)

    Schinke, Claudia; Germani, José C

    2012-03-01

    Macrophomina phaseolina, phylum Ascomycota, is a phytopathogenic fungus distributed worldwide in hot dry areas. There are few studies on its secreted lipases and none on its colony radial growth rate, an indicator of fungal ability to use nutrients for growth, on media other than potato-dextrose agar. In this study, 13 M. phaseolina isolates collected in different Brazilian regions were screened for fast-growth and the production of hydrolases of industrial interest, especially alkaline lipases. Hydrolase detection and growth rate determination were done on citric pectin, gelatin, casein, soluble starch, and olive oil as substrates. Ten isolates were found to be active on all substrates tested. The most commonly detected enzymes were pectinases, amylases, and lipases. The growth rate on pectin was significantly higher (P media identified CMM 2105, CMM 1091, and PEL as the fastest-growing isolates. The lipase activity of four isolates grown on olive oil was followed for 4 days by measuring the activity in the cultivation broth. The specific lipolytic activity of isolate PEL was significantly higher at 96 h (130 mU mg protein(-1)). The broth was active at 37 °C, pH 8, indicating the potential utility of the lipases of this isolate in mild alkaline detergents. There was a strong and positive correlation (0.86) between radial growth rate and specific lipolytic activity.

  2. Targeted discovery of glycoside hydrolases from a switchgrass-adapted compost community

    Energy Technology Data Exchange (ETDEWEB)

    Allgaier, M.; Reddy, A.; Park, J. I.; Ivanova, N.; D' haeseleer, P.; Lowry, S.; Sapra, R.; Hazen, T.C.; Simmons, B.A.; VanderGheynst, J. S.; Hugenholtz, P.

    2009-11-15

    Development of cellulosic biofuels from non-food crops is currently an area of intense research interest. Tailoring depolymerizing enzymes to particular feedstocks and pretreatment conditions is one promising avenue of research in this area. Here we added a green-waste compost inoculum to switchgrass (Panicum virgatum) and simulated thermophilic composting in a bioreactor to select for a switchgrass-adapted community and to facilitate targeted discovery of glycoside hydrolases. Small-subunit (SSU) rRNA-based community profiles revealed that the microbial community changed dramatically between the initial and switchgrass-adapted compost (SAC) with some bacterial populations being enriched over 20-fold. We obtained 225 Mbp of 454-titanium pyrosequence data from the SAC community and conservatively identified 800 genes encoding glycoside hydrolase domains that were biased toward depolymerizing grass cell wall components. Of these, {approx}10% were putative cellulases mostly belonging to families GH5 and GH9. We synthesized two SAC GH9 genes with codon optimization for heterologous expression in Escherichia coli and observed activity for one on carboxymethyl cellulose. The active GH9 enzyme has a temperature optimum of 50 C and pH range of 5.5 to 8 consistent with the composting conditions applied. We demonstrate that microbial communities adapt to switchgrass decomposition using simulated composting condition and that full-length genes can be identified from complex metagenomic sequence data, synthesized and expressed resulting in active enzyme.

  3. Targeted discovery of glycoside hydrolases from a switchgrass-adapted compost community.

    Directory of Open Access Journals (Sweden)

    Martin Allgaier

    Full Text Available Development of cellulosic biofuels from non-food crops is currently an area of intense research interest. Tailoring depolymerizing enzymes to particular feedstocks and pretreatment conditions is one promising avenue of research in this area. Here we added a green-waste compost inoculum to switchgrass (Panicum virgatum and simulated thermophilic composting in a bioreactor to select for a switchgrass-adapted community and to facilitate targeted discovery of glycoside hydrolases. Small-subunit (SSU rRNA-based community profiles revealed that the microbial community changed dramatically between the initial and switchgrass-adapted compost (SAC with some bacterial populations being enriched over 20-fold. We obtained 225 Mbp of 454-titanium pyrosequence data from the SAC community and conservatively identified 800 genes encoding glycoside hydrolase domains that were biased toward depolymerizing grass cell wall components. Of these, approximately 10% were putative cellulases mostly belonging to families GH5 and GH9. We synthesized two SAC GH9 genes with codon optimization for heterologous expression in Escherichia coli and observed activity for one on carboxymethyl cellulose. The active GH9 enzyme has a temperature optimum of 50 degrees C and pH range of 5.5 to 8 consistent with the composting conditions applied. We demonstrate that microbial communities adapt to switchgrass decomposition using simulated composting condition and that full-length genes can be identified from complex metagenomic sequence data, synthesized and expressed resulting in active enzyme.

  4. Extracellular Xylanolytic and Pectinolytic Hydrolase Production by Aspergillus flavus Isolates Contributes to Crop Invasion

    Directory of Open Access Journals (Sweden)

    Jay E. Mellon

    2015-08-01

    Full Text Available Several atoxigenic Aspergillus flavus isolates, including some being used as biocontrol agents, and one toxigenic isolate were surveyed for the ability to produce extracellular xylanolytic and pectinolytic hydrolases. All of the tested isolates displayed good production of endoxylanases when grown on a medium utilizing larch xylan as a sole carbon substrate. Four of the tested isolates produced reasonably high levels of esterase activity, while the atoxigenic biocontrol agent NRRL 21882 isolate esterase level was significantly lower than the others. Atoxigenic A. flavus isolates 19, 22, K49, AF36 (the latter two are biocontrol agents and toxigenic AF13 produced copious levels of pectinolytic activity when grown on a pectin medium. The pectinolytic activity levels of the atoxigenic A. flavus 17 and NRRL 21882 isolates were significantly lower than the other tested isolates. In addition, A. flavus isolates that displayed high levels of pectinolytic activity in the plate assay produced high levels of endopolygalacturonase (pectinase P2c, as ascertained by isoelectric focusing electrophoresis. Isolate NRRL 21882 displayed low levels of both pectinase P2c and pectin methyl esterase. A. flavus appears capable of producing these hydrolytic enzymes irrespective of aflatoxin production. This ability of atoxigenic isolates to produce xylanolytic and pectinolytic hydrolases mimics that of toxigenic isolates and, therefore, contributes to the ability of atoxigenic isolates to occupy the same niche as A. flavus toxigenic isolates.

  5. Differential recognition and hydrolysis of host carbohydrate antigens by Streptococcus pneumoniae family 98 glycoside hydrolases.

    Science.gov (United States)

    Higgins, Melanie A; Whitworth, Garrett E; El Warry, Nahida; Randriantsoa, Mialy; Samain, Eric; Burke, Robert D; Vocadlo, David J; Boraston, Alisdair B

    2009-09-18

    The presence of a fucose utilization operon in the Streptococcus pneumoniae genome and its established importance in virulence indicates a reliance of this bacterium on the harvesting of host fucose-containing glycans. The identities of these glycans, however, and how they are harvested is presently unknown. The biochemical and high resolution x-ray crystallographic analysis of two family 98 glycoside hydrolases (GH98s) from distinctive forms of the fucose utilization operon that originate from different S. pneumoniae strains reveal that one enzyme, the predominant type among pneumococcal isolates, has a unique endo-beta-galactosidase activity on the LewisY antigen. Altered active site topography in the other species of GH98 enzyme tune its endo-beta-galactosidase activity to the blood group A and B antigens. Despite their different specificities, these enzymes, and by extension all family 98 glycoside hydrolases, use an inverting catalytic mechanism. Many bacterial and viral pathogens exploit host carbohydrate antigens for adherence as a precursor to colonization or infection. However, this is the first evidence of bacterial endoglycosidase enzymes that are known to play a role in virulence and are specific for distinct host carbohydrate antigens. The strain-specific distribution of two distinct types of GH98 enzymes further suggests that S. pneumoniae strains may specialize to exploit host-specific antigens that vary from host to host, a factor that may feature in whether a strain is capable of colonizing a host or establishing an invasive infection.

  6. α/β-Hydrolase Domain 6 in the Ventromedial Hypothalamus Controls Energy Metabolism Flexibility

    Directory of Open Access Journals (Sweden)

    Alexandre Fisette

    2016-10-01

    Full Text Available α/β-Hydrolase domain 6 (ABHD6 is a monoacylglycerol hydrolase that degrades the endocannabinoid 2-arachidonoylglycerol (2-AG. Although complete or peripheral ABHD6 loss of function is protective against diet-induced obesity and insulin resistance, the role of ABHD6 in the central control of energy balance is unknown. Using a viral-mediated knockout approach, targeted endocannabinoid measures, and pharmacology, we discovered that mice lacking ABHD6 from neurons of the ventromedial hypothalamus (VMHKO have higher VMH 2-AG levels in conditions of endocannabinoid recruitment and fail to physiologically adapt to key metabolic challenges. VMHKO mice exhibited blunted fasting-induced feeding and reduced food intake, energy expenditure, and adaptive thermogenesis in response to cold exposure, high-fat feeding, and dieting (transition to a low-fat diet. Our findings identify ABHD6 as a regulator of the counter-regulatory responses to major metabolic shifts, including fasting, nutrient excess, cold, and dieting, thereby highlighting the importance of ABHD6 in the VMH in mediating energy metabolism flexibility.

  7. The Serine Hydrolase ABHD6 Is a Critical Regulator of the Metabolic Syndrome

    Directory of Open Access Journals (Sweden)

    Gwynneth Thomas

    2013-10-01

    Full Text Available The serine hydrolase α/β hydrolase domain 6 (ABHD6 has recently been implicated as a key lipase for the endocannabinoid 2-arachidonylglycerol (2-AG in the brain. However, the biochemical and physiological function for ABHD6 outside of the central nervous system has not been established. To address this, we utilized targeted antisense oligonucleotides (ASOs to selectively knock down ABHD6 in peripheral tissues in order to identify in vivo substrates and understand ABHD6’s role in energy metabolism. Here, we show that selective knockdown of ABHD6 in metabolic tissues protects mice from high-fat-diet-induced obesity, hepatic steatosis, and systemic insulin resistance. Using combined in vivo lipidomic identification and in vitro enzymology approaches, we show that ABHD6 can hydrolyze several lipid substrates, positioning ABHD6 at the interface of glycerophospholipid metabolism and lipid signal transduction. Collectively, these data suggest that ABHD6 inhibitors may serve as therapeutics for obesity, nonalcoholic fatty liver disease, and type II diabetes.

  8. Screening and evaluation of the glucoside hydrolase activity in Saccharomyces and Brettanomyces brewing yeasts.

    Science.gov (United States)

    Daenen, L; Saison, D; Sterckx, F; Delvaux, F R; Verachtert, H; Derdelinckx, G

    2008-02-01

    The aim of this study was to select and examine Saccharomyces and Brettanomyces brewing yeasts for hydrolase activity towards glycosidically bound volatile compounds. A screening for glucoside hydrolase activity of 58 brewing yeasts belonging to the genera Saccharomyces and Brettanomyces was performed. The studied Saccharomyces brewing yeasts did not show 1,4-beta-glucosidase activity, but a strain dependent beta-glucanase activity was observed. Some Brettanomyces species did show 1,4-beta-glucosidase activity. The highest constitutive activity was found in Brettanomyces custersii. For the most interesting strains the substrate specificity was studied and their activity was evaluated in fermentation experiments with added hop glycosides. Fermentations with Br. custersii led to the highest release of aglycones. Pronounced exo-beta-glucanase activity in Saccharomyces brewing yeasts leads to a higher release of certain aglycones. Certain Brettanomyces brewing yeasts, however, are more interesting for hydrolysis of glycosidically bound volatiles of hops. The release of flavour active compounds from hop glycosides opens perspectives for the bioflavouring and product diversification of beverages like beer. The release can be enhanced by using Saccharomyces strains with high exo-beta-glucanase activity. Higher activities can be found in Brettanomyces species with beta-glucosidase activity.

  9. Malbranchea cinnamomea: A thermophilic fungal source of catalytically efficient lignocellulolytic glycosyl hydrolases and metal dependent enzymes.

    Science.gov (United States)

    Mahajan, Chhavi; Basotra, Neha; Singh, Surender; Di Falco, Marcos; Tsang, Adrian; Chadha, B S

    2016-01-01

    This study reports thermophilic fungus Malbranchea cinnamomea as an important source of lignocellulolytic enzymes. The secretome analysis using LC-MS/MS orbitrap showed that fungus produced a spectrum of glycosyl hydrolases (cellulase/hemicellulase), polysaccharide lyases (PL) and carbohydrate esterases (CE) in addition to cellobiose dehydrogenase (CDH) indicating the presence of functional classical and oxidative cellulolytic mechanisms. The protein fractions in the secretome resolved by ion exchange chromatography were analyzed for ability to hydrolyze alkali treated carrot grass (ATCG) in the presence of Mn(2+)/Cu(2+). This strategy in tandem with peptide mass fingerprinting led to identification of metal dependent protein hydrolases with no apparent hydrolytic activity, however, showed 5.7 folds higher saccharification in presence of Mn(2+). Furthermore, adding different protein fractions to commercial cellulase (Novozymes: Cellic CTec2) resulted in enhanced hydrolysis of ATCG ranging between 1.57 and 3.43 folds indicating the enzymes from M. cinnamomea as catalytically efficient. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Novel Strategies for Upstream and Downstream Processing of Tannin Acyl Hydrolase

    Directory of Open Access Journals (Sweden)

    Luis V. Rodríguez-Durán

    2011-01-01

    Full Text Available Tannin acyl hydrolase also referred as tannase is an enzyme with important applications in several science and technology fields. Due to its hydrolytic and synthetic properties, tannase could be used to reduce the negative effects of tannins in beverages, food, feed, and tannery effluents, for the production of gallic acid from tannin-rich materials, the elucidation of tannin structure, and the synthesis of gallic acid esters in nonaqueous media. However, industrial applications of tannase are still very limited due to its high production cost. Thus, there is a growing interest in the production, recovery, and purification of this enzyme. Recently, there have been published a number of papers on the improvement of upstream and downstream processing of the enzyme. These papers dealt with the search for new tannase producing microorganisms, the application of novel fermentation systems, optimization of culture conditions, the production of the enzyme by recombinant microorganism, and the design of efficient protocols for tannase recovery and purification. The present work reviews the state of the art of basic and biotechnological aspects of tannin acyl hydrolase, focusing on the recent advances in the upstream and downstream processing of the enzyme.

  11. Novel strategies for upstream and downstream processing of tannin acyl hydrolase.

    Science.gov (United States)

    Rodríguez-Durán, Luis V; Valdivia-Urdiales, Blanca; Contreras-Esquivel, Juan C; Rodríguez-Herrera, Raúl; Aguilar, Cristóbal N

    2011-01-01

    Tannin acyl hydrolase also referred as tannase is an enzyme with important applications in several science and technology fields. Due to its hydrolytic and synthetic properties, tannase could be used to reduce the negative effects of tannins in beverages, food, feed, and tannery effluents, for the production of gallic acid from tannin-rich materials, the elucidation of tannin structure, and the synthesis of gallic acid esters in nonaqueous media. However, industrial applications of tannase are still very limited due to its high production cost. Thus, there is a growing interest in the production, recovery, and purification of this enzyme. Recently, there have been published a number of papers on the improvement of upstream and downstream processing of the enzyme. These papers dealt with the search for new tannase producing microorganisms, the application of novel fermentation systems, optimization of culture conditions, the production of the enzyme by recombinant microorganism, and the design of efficient protocols for tannase recovery and purification. The present work reviews the state of the art of basic and biotechnological aspects of tannin acyl hydrolase, focusing on the recent advances in the upstream and downstream processing of the enzyme.

  12. Microbial biodegradation of biuret: defining biuret hydrolases within the isochorismatase superfamily.

    Science.gov (United States)

    Robinson, Serina L; Badalamenti, Jonathan P; Dodge, Anthony G; Tassoulas, Lambros J; Wackett, Lawrence P

    2018-03-12

    Biuret is a minor component of urea fertilizer and an intermediate in s-triazine herbicide biodegradation. The microbial metabolism of biuret has never been comprehensively studied. Here, we enriched and isolated bacteria from a potato field that grew on biuret as a sole nitrogen source. We sequenced the genome of the fastest-growing isolate, Herbaspirillum sp. BH-1 and identified genes encoding putative biuret hydrolases (BHs). We purified and characterized a functional BH enzyme from Herbaspirillum sp. BH-1 and two other bacteria from divergent phyla. The BH enzymes reacted exclusively with biuret in the range of 2-11 µmol min -1 mg -1 protein. We then constructed a global protein superfamily network to map structure-function relationships in the BH subfamily and used this to mine > 7000 genomes. High-confidence BH sequences were detected in Actinobacteria, Alpha- and Beta-proteobacteria, and some fungi, archaea and green algae, but not animals or land plants. Unexpectedly, no cyanuric acid hydrolase homologs were detected in > 90% of genomes with BH homologs, suggesting BHs may have arisen independently of s-triazine ring metabolism. This work links genotype to phenotype by enabling accurate genome-mining to predict microbial utilization of biuret. Importantly, it advances understanding of the microbial capacity for biuret biodegradation in agricultural systems. © 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

  13. COMPARATIVE MODELLING AND LIGAND BINDING SITE PREDICTION OF A FAMILY 43 GLYCOSIDE HYDROLASE FROM Clostridium thermocellum

    Directory of Open Access Journals (Sweden)

    Shadab Ahmed

    2012-06-01

    Full Text Available The phylogenetic analysis of Clostridium thermocellum family 43 glycoside hydrolase (CtGH43 showed close evolutionary relation with carbohydrate binding family 6 proteins from C. cellulolyticum, C. papyrosolvens, C. cellulyticum, and A. cellulyticum. Comparative modeling of CtGH43 was performed based on crystal structures with PDB IDs 3C7F, 1YIF, 1YRZ, 2EXH and 1WL7. The structure having lowest MODELLER objective function was selected. The three-dimensional structure revealed typical 5-fold beta–propeller architecture. Energy minimization and validation of predicted model with VERIFY 3D indicated acceptability of the proposed atomic structure. The Ramachandran plot analysis by RAMPAGE confirmed that family 43 glycoside hydrolase (CtGH43 contains little or negligible segments of helices. It also showed that out of 301 residues, 267 (89.3% were in most favoured region, 23 (7.7% were in allowed region and 9 (3.0% were in outlier region. IUPred analysis of CtGH43 showed no disordered region. Active site analysis showed presence of two Asp and one Glu, assumed to form a catalytic triad. This study gives us information about three-dimensional structure and reaffirms the fact that it has the similar core 5-fold beta–propeller architecture and so probably has the same inverting mechanism of action with the formation of above mentioned catalytic triad for catalysis of polysaccharides.

  14. Cloning, expression and mutation of a triazophos hydrolase gene from Burkholderia sp. SZL-1.

    Science.gov (United States)

    Zhang, Hao; Li, Qiang; Guo, Su-Hui; Cheng, Ming-Gen; Zhao, Meng-Jun; Hong, Qing; Huang, Xing

    2016-06-01

    Triazophos is a broad-spectrum and highly effective insecticide, and the residues of triazophos have been frequently detected in the environment. A triazophos-degrading bacterium, Burkholderia sp. SZL-1, was isolated from a long-term triazophos-polluted soil. Strain SZL-1 could hydrolyze triazophos to 1-phenyl-3-hydroxy-1,2,4-triazole, which was further utilized as the carbon sources for growth. The triazophos hydrolase gene trhA, cloned from strain SZL-1, was expressed and homogenously purified using Ni-nitrilotriacetic acid affinity chromatography. TrhA is 55 kDa and displays maximum activity at 25°C, pH 8.0. This enzyme still has nearly 60% activity at the range of 15°C-50°C for 30 min. TrhA was mutated by sequential error prone PCR and screened for improved activity for triazophos degradation. One purified variant protein (Val89-Gly89) named TrhA-M1 showed up to 3-fold improvement in specific activity against triazophos, and the specificity constants of Kcat and Kcat/Km for TrhA-M1 were improved up to 2.3- and 8.28-fold, respectively, compared to the wild-type enzyme. The results in this paper provided potential material for the contaminated soil remediation and hydrolase genetic structure research. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  15. Identification of the Gene Encoding Isoprimeverose-producing Oligoxyloglucan Hydrolase in Aspergillus oryzae*

    Science.gov (United States)

    Matsuzawa, Tomohiko; Mitsuishi, Yasushi; Kameyama, Akihiko

    2016-01-01

    Aspergillus oryzae produces a unique β-glucosidase, isoprimeverose-producing oligoxyloglucan hydrolase (IPase), that recognizes and releases isoprimeverose (α-d-xylopyranose-(1→6)-d-glucopyranose) units from the non-reducing ends of oligoxyloglucans. A gene encoding A. oryzae IPase, termed ipeA, was identified and expressed in Pichia pastoris. With the exception of cellobiose, IpeA hydrolyzes a variety of oligoxyloglucans and is a member of the glycoside hydrolase family 3. Xylopyranosyl branching at the non-reducing ends was vital for IPase activity, and galactosylation at a α-1,6-linked xylopyranosyl side chain completely abolished IpeA activity. Hepta-oligoxyloglucan saccharide (Xyl3Glc4) substrate was preferred over tri- (Xyl1Glc2) and tetra- (Xyl2Glc2) oligoxyloglucan saccharides substrates. IpeA transferred isoprimeverose units to other saccharides, indicating transglycosylation activity. The ipeA gene was expressed in xylose and xyloglucan media and was strongly induced in the presence of xyloglucan endo-xyloglucanase-hydrolyzed products. This is the first study to report the identification of a gene encoding IPase in eukaryotes. PMID:26755723

  16. Systematic Survey of Serine Hydrolase Activity in Mycobacterium tuberculosis Defines Changes Associated with Persistence

    Energy Technology Data Exchange (ETDEWEB)

    Ortega, Corrie; Anderson, Lindsey N.; Frando, Andrew; Sadler, Natalie C.; Brown, Robert W.; Smith, Richard D.; Wright, Aaron T.; Grundner, Christoph

    2016-02-01

    The transition between replication and non-replication underlies much of Mycobacterium tuberculosis (Mtb) pathogenicity, as non- or slowly replicating Mtb are responsible for persistence and poor treatment outcomes. Therapeutic targeting of non-replicating, persistent populations is a priority for tuberculosis treatment, but only few drug targets in non-replicating Mtb are currently known. Here, we directly measure the activity of the highly diverse and druggable serine hydrolases (SHs) during active replication and non-replication by activity-based proteomics. We predict serine hydrolase activity for 78 proteins, including 27 proteins with previously unknown function, and identify 37 SHs that remain active even in the absence of replication, providing a set of candidate persistence targets. Non-replication was associated with large shifts in the activity of the majority of SHs. These activity changes were largely independent of SH abundance, indicating extensive post-translational regulation. By probing a large cross-section of druggable Mtb enzyme space during replication and non-replication, we identify new SHs and suggest new persistence targets.

  17. Targeted Discovery of Glycoside Hydrolases from a Switchgrass-Adapted Compost Community

    Energy Technology Data Exchange (ETDEWEB)

    Reddy, Amitha; Allgaier, Martin; Park, Joshua I.; Ivanoval, Natalia; Dhaeseleer, Patrik; Lowry, Steve; Sapra, Rajat; Hazen, Terry C.; Simmons, Blake A.; VanderGheynst, Jean S.; Hugenholtz, Philip

    2011-05-11

    Development of cellulosic biofuels from non-food crops is currently an area of intense research interest. Tailoring depolymerizing enzymes to particular feedstocks and pretreatment conditions is one promising avenue of research in this area. Here we added a green-waste compost inoculum to switchgrass (Panicum virgatum) and simulated thermophilic composting in a bioreactor to select for a switchgrass-adapted community and to facilitate targeted discovery of glycoside hydrolases. Smallsubunit (SSU) rRNA-based community profiles revealed that the microbial community changed dramatically between the initial and switchgrass-adapted compost (SAC) with some bacterial populations being enriched over 20-fold. We obtained 225 Mbp of 454-titanium pyrosequence data from the SAC community and conservatively identified 800 genes encoding glycoside hydrolase domains that were biased toward depolymerizing grass cell wall components. Of these, ,10percent were putative cellulasesmostly belonging to families GH5 and GH9. We synthesized two SAC GH9 genes with codon optimization for heterologous expression in Escherichia coli and observed activity for one on carboxymethyl cellulose. The active GH9 enzyme has a temperature optimum of 50uC and pH range of 5.5 to 8 consistent with the composting conditions applied. We demonstrate that microbial communities adapt to switchgrass decomposition using simulated composting condition and that full-length genes can be identified from complex metagenomic sequence data, synthesized and expressed resulting in active enzyme.

  18. Inhibition of soluble epoxide hydrolase contributes to the anti-inflammatory effect of antimicrobial triclocarban in a murine model

    International Nuclear Information System (INIS)

    Liu Junyan; Qiu Hong; Morisseau, Christophe; Hwang, Sung Hee; Tsai, Hsing-Ju; Ulu, Arzu; Chiamvimonvat, Nipavan; Hammock, Bruce D.

    2011-01-01

    The increasing use of the antimicrobial triclocarban (TCC) in personal care products (PCPs) has resulted in concern regarding environmental pollution. TCC is a potent inhibitor of soluble epoxide hydrolase (sEH). Inhibitors of sEH (sEHIs) are anti-inflammatory, anti-hypertensive and cardio-protective in multiple animal models. However, the in vivo effects anticipated from a sEHI have not been reported for TCC. Here we demonstrated the anti-inflammatory effects in vivo of TCC in a murine model. TCC was employed in a lipopolysaccharide (LPS)-challenged murine model. Systolic blood pressure, plasma levels of several inflammatory cytokines and chemokine, and metabolomic profile of plasma oxylipins were determined. TCC significantly reversed LPS-induced morbid hypotension in a time-dependent manner. TCC significantly repressed the increased release of inflammatory cytokines and chemokine caused by LPS. Furthermore, TCC significantly shifted the oxylipin profile in vivo in a time-dependent manner towards resolution of inflammation as expected from a sEHI. These results demonstrated that at the doses used TCC is anti-inflammatory in the murine model. This study suggests that TCC may provide some benefits in humans in addition to its antimicrobial activities due to its potent inhibition of sEH. It may be a promising starting point for developing new low volume high value applications of TCC. However these biological effects also caution against the general over use of TCC in PCPs. - Graphical abstract: Display Omitted Research Highlights: → Anti-microbial triclocarban (TCC) is anti-inflammatory in a murine model. → TCC significantly shifted the oxylipin profile in vivo as expected from a sEHI. → TCC significantly reversed LPS-induced morbid hypotension in a time-dependent manner. → TCC significantly repressed LPS-induced increased release of inflammatory cytokines.

  19. IS30-related transposon mediated insertional inactivation of bile salt hydrolase (bsh1) gene of Lactobacillus plantarum strain Lp20.

    Science.gov (United States)

    Kumar, Rajesh; Grover, Sunita; Kaushik, Jai K; Batish, Virender Kumar

    2014-01-01

    Lactobacillus plantarum is a flexible and versatile microorganism that inhabits a variety of niches, and its genome may express up to four bsh genes to maximize its survival in the mammalian gut. However, the ecological significance of multiple bsh genes in L. plantarum is still not clearly understood. Hence, this study demonstrated the disruption of bile salt hydrolase (bsh1) gene due to the insertion of a transposable element in L. plantarum Lp20 - a wild strain of human fecal origin. Surprisingly, L. plantarum strain Lp20 produced a ∼2.0 kb bsh1 amplicon against the normal size (∼1.0 kb) bsh1 amplicon of Bsh(+)L. plantarum Lp21. Strain Lp20 exhibited minimal Bsh activity in spite of having intact bsh2, bsh3 and bsh4 genes in its genome and hence had a Bsh(-) phenotype. Cloning and sequence characterization of Lp20 bsh1 gene predicted four individual open reading frames (ORFs) within this region. BLAST analysis of ORF1 and ORF2 revealed significant sequence similarity to the L. plantarum bsh1 gene while ORF3 and ORF4 showed high sequence homology to IS30-family transposases. Since, IS30-related transposon element was inserted within Lp20 bsh1 gene in reverse orientation (3'-5'), it introduced several stop codons and disrupted the protein reading frames of both Bsh1 and transposase. Inverted terminal repeats (GGCAGATTG) of transposon, mediated its insertion at 255-263 nt and 1301-1309 nt positions of Lp20 bsh1 gene. In conclusion, insertion of IS30 related-transposon within the bsh1 gene sequence of L. plantarum strain Lp20 demolished the integrity and functionality of Bsh1 enzyme. Additionally, this transposon DNA sequence remains active among various Lactobacillus spp. and hence harbors the potential to be explored in the development of efficient insertion mutagenesis system. Copyright © 2013 Elsevier GmbH. All rights reserved.

  20. Diverse modes of galacto-specific carbohydrate recognition by a family 31 glycoside hydrolase from Clostridium perfringens.

    Directory of Open Access Journals (Sweden)

    Julie M Grondin

    Full Text Available Clostridium perfringens is a commensal member of the human gut microbiome and an opportunistic pathogen whose genome encodes a suite of putative large, multi-modular carbohydrate-active enzymes that appears to play a role in the interaction of the bacterium with mucin-based carbohydrates. Among the most complex of these is an enzyme that contains a presumed catalytic module belonging to glycoside hydrolase family 31 (GH31. This large enzyme, which based on its possession of a GH31 module is a predicted α-glucosidase, contains a variety of non-catalytic ancillary modules, including three CBM32 modules that to date have not been characterized. NMR-based experiments demonstrated a preference of each module for galacto-configured sugars, including the ability of all three CBM32s to recognize the common mucin monosaccharide GalNAc. X-ray crystal structures of the CpGH31 CBM32s, both in apo form and bound to GalNAc, revealed the finely-tuned molecular strategies employed by these sequentially variable CBM32s in coordinating a common ligand. The data highlight that sequence similarities to previously characterized CBMs alone are insufficient for identifying the molecular mechanism of ligand binding by individual CBMs. Furthermore, the overlapping ligand binding profiles of the three CBMs provide a fail-safe mechanism for the recognition of GalNAc among the dense eukaryotic carbohydrate networks of the colonic mucosa. These findings expand our understanding of ligand targeting by large, multi-modular carbohydrate-active enzymes, and offer unique insights into of the expanding ligand-binding preferences and binding site topologies observed in CBM32s.

  1. Oxidoreductases provide a more generic response to metallic stressors (Cu and Cd) than hydrolases in soil fungi: new ecotoxicological insights.

    Science.gov (United States)

    Lebrun, Jérémie D; Demont-Caulet, Nathalie; Cheviron, Nathalie; Laval, Karine; Trinsoutrot-Gattin, Isabelle; Mougin, Christian

    2016-02-01

    The present study investigates the effect of metals on the secretion of enzymes from 12 fungal strains maintained in liquid cultures. Hydrolases (acid phosphatase, β-glucosidase, β-galactosidase, and N-acetyl-β-glucosaminidase) and ligninolytic oxidoreductases (laccase, Mn, and lignin peroxidases) activities, as well as biomass production, were measured in culture fluids from fungi exposed to Cu or Cd. Our results showed that all fungi secreted most of the selected hydrolases and that about 50% of them produced a partial oxidative system in the absence of metals. Then, exposure of fungi to metals led to the decrease in biomass production. At the enzymatic level, Cu and Cd modified the secretion profiles of soil fungi. The response of hydrolases to metals was contrasted and complex and depended on metal, enzyme, and fungal strain considered. By contrast, the metals always stimulated the activity of ligninolytic oxidoreductases in fungal strains. In some of them, oxidoreductases were specifically produced following metal exposure. Fungal oxidoreductases provide a more generic response than hydrolases, constituting thus a physiological basis for their use as biomarkers of metal exposure in soils.

  2. Crystal Structure of α-1,4-Glucan Lyase, a Unique Glycoside Hydrolase Family Member with a Novel Catalytic Mechanism

    NARCIS (Netherlands)

    Rozeboom, Henriëtte J.; Yu, Shukun; Madrid, Susan; Kalk, Kor H.; Zhang, Ran; Dijkstra, Bauke W.

    2013-01-01

    α-1,4-Glucan lyase (EC 4.2.2.13) from the red seaweed Gracilariopsis lemaneiformis cleaves α-1,4-glucosidic linkages in glycogen, starch, and malto-oligosaccharides, yielding the keto-monosaccharide 1,5-anhydro-D-fructose. The enzyme belongs to glycoside hydrolase family 31 (GH31) but degrades

  3. AMPEROMETRIC THICK-FILM STRIP ELECTRODES FOR MONITORING ORGANOPHOSPHATE NERVE AGENTS BASED ON IMMOBILIZED ORGANOPHOSPHORUS HYDROLASE. (R823663)

    Science.gov (United States)

    An amperometric biosensor based on the immobilization of organophosphorus hydrolase(OPH) onto screen-printed carbon electrodes is shown useful for the rapid, sensitive, and low-costdetection of organophosphate (OP) nerve agents. The sensor relies upon the sensitive and ra...

  4. Construction and characterisation of a genetically engineered Escherichia coli strain for the epoxide hydrolase-catalysed kinetic resolution of epoxides

    NARCIS (Netherlands)

    Visser, H.; Oliveira Vil Filho, de M.; Liese, A.; Weijers, C.A.G.M.; Verdoes, J.C.

    2003-01-01

    The Rhodotorula glutinis epoxide hydrolase, Eph1, was produced in the heterologous host Escherichia coli BL21(DE3) in order to develop a highly effective epoxide hydrolysis system. A 138-fold increase in Eph1 activity was found in cell extracts of the recombinant E. coli when compared to cell

  5. Cocaine Hydrolase Gene Transfer Demonstrates Cardiac Safety and Efficacy against Cocaine-Induced QT Prolongation in Mice

    OpenAIRE

    Murthy, Vishakantha; Reyes, Santiago; Geng, Liyi; Gao, Yang; Brimijoin, Stephen

    2016-01-01

    Cocaine addiction is associated with devastating medical consequences, including cardiotoxicity and risk-conferring prolongation of the QT interval. Viral gene transfer of cocaine hydrolase engineered from butyrylcholinesterase offers therapeutic promise for treatment-seeking drug users. Although previous preclinical studies have demonstrated benefits of this strategy without signs of toxicity, the specific cardiac safety and efficacy of engineered butyrylcholinesterase viral delivery remains...

  6. Genetic and biochemical characterization of a novel monoterpene epsilon-lactone hydrolase from Rhodococcus erythropolis DCL14

    NARCIS (Netherlands)

    Vlugt-Bergmans, van der C.J.B.; Werf, van der M.J.

    2001-01-01

    A monoterpene ε-lactone hydrolase (MLH) from Rhodococcus erythropolis DCL14, catalyzing the ring opening of lactones which are formed during degradation of several monocyclic monoterpenes, including carvone and menthol, was purified to apparent homogeneity. It is a monomeric enzyme of 31 kDa that is

  7. Genetic and biochemical characterization of a novel monoterpene e-lactone hydrolase from Rhodococcus erythropolis DCL14

    NARCIS (Netherlands)

    Vlugt-Bergmans, C.J.B. van der; Werf, M.J. van der

    2001-01-01

    A monoterpene ε-lactone hydrolase (MLH) from Rhodococcus erythropolis DCL14, catalyzing the ring opening of lactones which are formed during degradation of several monocyclic monoterpenes, including carvone and menthol, was purified to apparent homogeneity. It is a monomeric enzyme of 31 kDa that is

  8. Biotechnological potential of novel glycoside hydrolase family 70 enzymes synthesizing α-glucans from starch and sucrose

    NARCIS (Netherlands)

    Gangoiti, Joana; Pijning, Tjaard; Dijkhuizen, Lubbert

    Transglucosidases belonging to the glycoside hydrolase (GH) family 70 are promising enzymatic tools for the synthesis of α-glucans with defined structures from renewable sucrose and starch substrates. Depending on the GH70 enzyme specificity, α-glucans with different structures and physicochemical

  9. Acetobacter turbidans α-Amino Acid Ester Hydrolase. How a Single Mutation Improves an Antibiotic-Producing Enzyme

    NARCIS (Netherlands)

    Barends, Thomas R.M.; Polderman-Tijmes, Jolanda J.; Jekel, Peter A.; Williams, Christopher; Wybenga, Gjalt; Janssen, Dick B.; Dijkstra, Bauke W.

    2006-01-01

    The α-amino acid ester hydrolase (AEH) from Acetobacter turbidans is a bacterial enzyme catalyzing the hydrolysis and synthesis of β-lactam antibiotics. The crystal structures of the native enzyme, both unliganded and in complex with the hydrolysis product D-phenylglycine are reported, as well as

  10. Genetic variation in the bleomycin hydrolase gene and bleomycin-induced pulmonary toxicity in germ cell cancer patients

    NARCIS (Netherlands)

    Nuver, J; Lutke-Holzik, MF; van Zweeden, M; Hoekstra, HJ; Meijer, C; Suurmeijer, AJH; Hofstra, RM; Sluiter, WJ; Sleijfer, D; Gietema, JA; Groen, Hendricus; Groen, Herman

    Objective Use of bleomycin as a cytotoxic agent is limited by its pulmonary toxicity. Bleomycin is mainly excreted by the kidneys, but can also be inactivated by bleomycin hydrolase (BMH). An 1450A > G polymorphic site in the BMH gene results in an amino acid substitution in the C-terminal domain of

  11. Fatty Acid Amide Hydrolase (FAAH) Inhibition Enhances Memory Acquisition through Activation of PPAR-alpha Nuclear Receptors

    Science.gov (United States)

    Mazzola, Carmen; Medalie, Julie; Scherma, Maria; Panlilio, Leigh V.; Solinas, Marcello; Tanda, Gianluigi; Drago, Filippo; Cadet, Jean Lud; Goldberg, Steven R.; Yasar, Sevil

    2009-01-01

    Inhibitors of fatty acid amide hydrolase (FAAH) increase endogenous levels of anandamide (a cannabinoid CB[subscript 1]-receptor ligand) and oleoylethanolamide and palmitoylethanolamide (OEA and PEA, ligands for alpha-type peroxisome proliferator-activated nuclear receptors, PPAR-alpha) when and where they are naturally released in the brain.…

  12. Bacterial Cell Enlargement Requires Control of Cell Wall Stiffness Mediated by Peptidoglycan Hydrolases.

    Science.gov (United States)

    Wheeler, Richard; Turner, Robert D; Bailey, Richard G; Salamaga, Bartłomiej; Mesnage, Stéphane; Mohamad, Sharifah A S; Hayhurst, Emma J; Horsburgh, Malcolm; Hobbs, Jamie K; Foster, Simon J

    2015-07-28

    Most bacterial cells are enclosed in a single macromolecule of the cell wall polymer, peptidoglycan, which is required for shape determination and maintenance of viability, while peptidoglycan biosynthesis is an important antibiotic target. It is hypothesized that cellular enlargement requires regional expansion of the cell wall through coordinated insertion and hydrolysis of peptidoglycan. Here, a group of (apparent glucosaminidase) peptidoglycan hydrolases are identified that are together required for cell enlargement and correct cellular morphology of Staphylococcus aureus, demonstrating the overall importance of this enzyme activity. These are Atl, SagA, ScaH, and SagB. The major advance here is the explanation of the observed morphological defects in terms of the mechanical and biochemical properties of peptidoglycan. It was shown that cells lacking groups of these hydrolases have increased surface stiffness and, in the absence of SagB, substantially increased glycan chain length. This indicates that, beyond their established roles (for example in cell separation), some hydrolases enable cellular enlargement by making peptidoglycan easier to stretch, providing the first direct evidence demonstrating that cellular enlargement occurs via modulation of the mechanical properties of peptidoglycan. Understanding bacterial growth and division is a fundamental problem, and knowledge in this area underlies the treatment of many infectious diseases. Almost all bacteria are surrounded by a macromolecule of peptidoglycan that encloses the cell and maintains shape, and bacterial cells must increase the size of this molecule in order to enlarge themselves. This requires not only the insertion of new peptidoglycan monomers, a process targeted by antibiotics, including penicillin, but also breakage of existing bonds, a potentially hazardous activity for the cell. Using Staphylococcus aureus, we have identified a set of enzymes that are critical for cellular enlargement. We

  13. Identification and characterization of carprofen as a multitarget fatty acid amide hydrolase/cyclooxygenase inhibitor.

    Science.gov (United States)

    Favia, Angelo D; Habrant, Damien; Scarpelli, Rita; Migliore, Marco; Albani, Clara; Bertozzi, Sine Mandrup; Dionisi, Mauro; Tarozzo, Glauco; Piomelli, Daniele; Cavalli, Andrea; De Vivo, Marco

    2012-10-25

    Pain and inflammation are major therapeutic areas for drug discovery. Current drugs for these pathologies have limited efficacy, however, and often cause a number of unwanted side effects. In the present study, we identify the nonsteroidal anti-inflammatory drug carprofen as a multitarget-directed ligand that simultaneously inhibits cyclooxygenase-1 (COX-1), COX-2, and fatty acid amide hydrolase (FAAH). Additionally, we synthesized and tested several derivatives of carprofen, sharing this multitarget activity. This may result in improved analgesic efficacy and reduced side effects (Naidu et al. J. Pharmacol. Exp. Ther.2009, 329, 48-56; Fowler, C. J.; et al. J. Enzyme Inhib. Med. Chem.2012, in press; Sasso et al. Pharmacol. Res.2012, 65, 553). The new compounds are among the most potent multitarget FAAH/COX inhibitors reported so far in the literature and thus may represent promising starting points for the discovery of new analgesic and anti-inflammatory drugs.

  14. Soluble Epoxide Hydrolase Inhibitory Activity of Selaginellin Derivatives from Selaginella tamariscina

    Directory of Open Access Journals (Sweden)

    Jang Hoon Kim

    2015-12-01

    Full Text Available Selaginellin derivatives 1–3 isolated from Selaginella tamariscina were evaluated for their inhibition of soluble epoxide hydrolase (sEH to demonstrate their potential for the treatment of cardiovascular disease. All selaginellin derivatives (1–3 inhibited sEH enzymatic activity and PHOME hydrolysis, in a dose-dependent manner, with IC50 values of 3.1 ± 0.1, 8.2 ± 2.2, and 4.2 ± 0.2 μM, respectively. We further determined that the derivatives function as non-competitive inhibitors. Moreover, the predicted that binding sites and interaction between 1–3 and sEH were solved by docking simulations. According to quantitative analysis, 1–3 were confirmed to have high content in the roots of S. tamariscina; among them, selaginellin 3 exhibited the highest content of 189.3 ± 0.0 μg/g.

  15. N (6-substituted AMPs inhibit mammalian deoxynucleotide N-hydrolase DNPH1.

    Directory of Open Access Journals (Sweden)

    Claire Amiable

    Full Text Available The gene dnph1 (or rcl encodes a hydrolase that cleaves the 2'-deoxyribonucleoside 5'-monophosphate (dNMP N-glycosidic bond to yield a free nucleobase and 2-deoxyribose 5-phosphate. Recently, the crystal structure of rat DNPH1, a potential target for anti-cancer therapies, suggested that various analogs of AMP may inhibit this enzyme. From this result, we asked whether N (6-substituted AMPs, and among them, cytotoxic cytokinin riboside 5'-monophosphates, may inhibit DNPH1. Here, we characterized the structural and thermodynamic aspects of the interactions of these various analogs with DNPH1. Our results indicate that DNPH1 is inhibited by cytotoxic cytokinins at concentrations that inhibit cell growth.

  16. Development and Properties of a Wax Ester Hydrolase in the Cotyledons of Jojoba Seedlings 1

    Science.gov (United States)

    Huang, Anthony H. C.; Moreau, Robert A.; Liu, Kitty D. F.

    1978-01-01

    The activity of a wax ester hydrolase in the cotyledons of jojoba (Simmondsia chinensis) seedlings increased drastically during germination, parallel to the development of the gluconeogenic process. The enzyme at its peak of development was obtained in association with the wax body membrane, and its properties were studied. It had an optimal activity at alkaline pH (8.5-9). The apparent Km value for N-methylindoxylmyristate was 93 μM. It was stable at 40 C for 30 min but was inactivated at higher temperature. Various divalent cations and ethylenediaminetetraacetate had little effect on the activity. p-Chloromercuribenzoate was a strong inhibitor of the enzyme activity, and its effect was reversed by subsequent addition of dithiothreitol. It had a broad substrate specificity with highest activities on monoglycerides, wax esters, and the native substrate (jojoba wax). PMID:16660288

  17. Development and properties of a wax ester hydrolase in the cotyledons of jojoba seedlings.

    Science.gov (United States)

    Huang, A H; Moreau, R A; Liu, K D

    1978-03-01

    The activity of a wax ester hydrolase in the cotyledons of jojoba (Simmondsia chinensis) seedlings increased drastically during germination, parallel to the development of the gluconeogenic process. The enzyme at its peak of development was obtained in association with the wax body membrane, and its properties were studied. It had an optimal activity at alkaline pH (8.5-9). The apparent K(m) value for N-methylindoxylmyristate was 93 muM. It was stable at 40 C for 30 min but was inactivated at higher temperature. Various divalent cations and ethylenediaminetetraacetate had little effect on the activity. p-Chloromercuribenzoate was a strong inhibitor of the enzyme activity, and its effect was reversed by subsequent addition of dithiothreitol. It had a broad substrate specificity with highest activities on monoglycerides, wax esters, and the native substrate (jojoba wax).

  18. Use of nanostructure initiator mass spectrometry (NIMS to deduce selectivity of reaction in glycoside hydrolases

    Directory of Open Access Journals (Sweden)

    Kai eDeng

    2015-10-01

    Full Text Available Chemically synthesized nanostructure-initiator mass spectrometry (NIMS probes derivatized with tetrasaccharides were used to study the reactivity of representative Clostridium thermocellum β-glucosidase, endoglucanases and cellobiohydrolase. Diagnostic patterns for reactions of these different classes of enzymes were observed. Results show sequential removal of glucose by the β-glucosidase and a progressive increase in specificity of reaction from endoglucanases to cellobiohydrolase. Time-dependent reactions of these polysaccharide-selective enzymes were modeled by numerical integration, which provides a quantitative basis to make functional distinctions among a continuum of naturally evolved catalytic properties. Consequently, our method, which combines automated protein translation with high-sensitivity and time-dependent detection of multiple products, provides a new approach to annotate glycoside hydrolase phylogenetic trees with functional measurements.

  19. Chitosanases from Family 46 of Glycoside Hydrolases: From Proteins to Phenotypes

    Directory of Open Access Journals (Sweden)

    Pascal Viens

    2015-10-01

    Full Text Available Chitosanases, enzymes that catalyze the endo-hydrolysis of glycolytic links in chitosan, are the subject of numerous studies as biotechnological tools to generate low molecular weight chitosan (LMWC or chitosan oligosaccharides (CHOS from native, high molecular weight chitosan. Glycoside hydrolases belonging to family GH46 are among the best-studied chitosanases, with four crystallography-derived structures available and more than forty enzymes studied at the biochemical level. They were also subjected to numerous site-directed mutagenesis studies, unraveling the molecular mechanisms of hydrolysis. This review is focused on the taxonomic distribution of GH46 proteins, their multi-modular character, the structure-function relationships and their biological functions in the host organisms.

  20. Selective inhibition of plant serine hydrolases by agrochemicals revealed by competitive ABPP.

    Science.gov (United States)

    Kaschani, Farnusch; Nickel, Sabrina; Pandey, Bikram; Cravatt, Benjamin F; Kaiser, Markus; van der Hoorn, Renier A L

    2012-01-15

    Organophosphate and -phosphonates and their thio derivatives are often used in agroindustry as herbicides and insecticides, but their potential off-targets in the plant are poorly investigated. Here, we use competitive activity-based protein profiling (ABPP) of serine hydrolases (SHs) to detect targets of these agrochemicals and other compounds in Arabidopsis thaliana. Using broad-range and specific probes, and by overexpression of various SHs in planta, we are able to confirm eight SH-compound interactions, including selective inhibition of carboxylesterase CXE12, prolyloligopeptidase, methylesterase MES2 and tripeptidyl peptidase TPP2. These observations can be used for the design of novel probes and selective inhibitors and may help to assess physiological effects of agrochemicals on crop plants. Copyright © 2011 Elsevier Ltd. All rights reserved.

  1. ETHANOL PRECIPITATION OF GLYCOSYL HYDROLASES PRODUCED BY Trichoderma harzianum P49P11

    Directory of Open Access Journals (Sweden)

    M. A. Mariño

    2015-06-01

    Full Text Available AbstractThis study aimed to concentrate glycosyl hydrolases produced by Trichoderma harzianum P49P11 by ethanol precipitation. The variables tested besides ethanol concentration were temperature and pH. The precipitation with 90% (v/v ethanol at pH 5.0 recovered more than 98% of the xylanase activity, regard less of the temperature (5.0, 15.0, or 25.0 °C. The maximum recovery of cellulase activity as FPase was 77% by precipitation carried out at this same pH and ethanol concentration but at 5.0 °C. Therefore, ethanol precipitation can be considered to be an efficient technique for xylanase concentration and, to a certain extent, also for the cellulase complex.

  2. Discovery and characterization of thermophilic limonene-1,2-epoxide hydrolases from hot spring metagenomic libraries

    DEFF Research Database (Denmark)

    Ferrandi, Erica Elisa; Sayer, Christopher; Isupov, Michail N.

    2015-01-01

    thermophilic sources, have higher optimal temperatures and apparent melting temperatures than Re-LEH. The new LEH enzymes have been crystallized and their structures solved to high resolution in the native form and in complex with the inhibitor valpromide for Tomsk-LEH and poly(ethylene glycol) for CH55-LEH......,2-epoxide hydrolase (LEH) family of enzymes. These two LEHs (Tomsk-LEH and CH55-LEH) show EH activities towards different epoxide substrates, differing in most cases from those previously identified for Rhodococcus erythropolis (Re-LEH) in terms of stereoselectivity. Tomsk-LEH and CH55-LEH, both from....... The structural analysis has provided insights into the LEH mechanism, substrate specificity and stereoselectivity of these new LEH enzymes, which has been supported by mutagenesis studies....

  3. Synthesis of novel bioactive lactose-derived oligosaccharides by microbial glycoside hydrolases

    Science.gov (United States)

    Díez-Municio, Marina; Herrero, Miguel; Olano, Agustín; Moreno, F Javier

    2014-01-01

    Prebiotic oligosaccharides are increasingly demanded within the Food Science domain because of the interesting healthy properties that these compounds may induce to the organism, thanks to their beneficial intestinal microbiota growth promotion ability. In this regard, the development of new efficient, convenient and affordable methods to obtain this class of compounds might expand even further their use as functional ingredients. This review presents an overview on the most recent interesting approaches to synthesize lactose-derived oligosaccharides with potential prebiotic activity paying special focus on the microbial glycoside hydrolases that can be effectively employed to obtain these prebiotic compounds. The most notable advantages of using lactose-derived carbohydrates such as lactosucrose, galactooligosaccharides from lactulose, lactulosucrose and 2-α-glucosyl-lactose are also described and commented. PMID:24690139

  4. The use of neutron scattering to determine the functional structure of glycoside hydrolase.

    Science.gov (United States)

    Nakamura, Akihiko; Ishida, Takuya; Samejima, Masahiro; Igarashi, Kiyohiko

    2016-10-01

    Neutron diffraction provides different information from X-ray diffraction, because neutrons are scattered by atomic nuclei, whereas X-rays are scattered by electrons. One of the key advantages of neutron crystallography is the ability to visualize hydrogen and deuterium atoms, making it possible to observe the protonation state of amino acid residues, hydrogen bonds, networks of water molecules and proton relay pathways in enzymes. But, because of technical difficulties, less than 100 enzyme structures have been evaluated by neutron crystallography to date. In this review, we discuss the advantages and disadvantages of neutron crystallography as a tool to investigate the functional structure of glycoside hydrolases, with some examples. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Studies on sterol-ester hydrolase from Fusarium oxysporum. I. Partial purification and properties.

    Science.gov (United States)

    Okawa, Y; Yamaguchi, T

    1977-05-01

    1. A search for a long chain fatty acyl sterol-ester hydrolase in microorganisms led to the isolation from soil of five strains belonging to Fusarium sp. which produced strong activity in the culture medium. 2. The cholesterol esterase from Fusarium oxysporum IGH-2 was purified about 270-fold by means of CaCl2 precipitation and Sephadex G-75 column chromatography. 3. The cholesterol esterase was activated by adekatol and Triton X-100. It was inhibited by lecithin and lysolecithin, and completely inactivated by heat treatment (60 degrees C for 30 min, at pH 7.0). 4. The optimum pH of the enzyme was found to be around 7.0. 5. Among various cholesterol esters tested, cholesterol linoleate was the most suitable substrate. 6. Cholesterol esters in serum were also hydrolyzed by this enzyme.

  6. Alternative strategy for converting an inverting glycoside hydrolase into a glycosynthase.

    Science.gov (United States)

    Honda, Yuji; Fushinobu, Shinya; Hidaka, Masafumi; Wakagi, Takayoshi; Shoun, Hirofumi; Taniguchi, Hajime; Kitaoka, Motomitsu

    2008-04-01

    The tyrosine residue Y198 is known to support a nucleophilic water molecule with the general base residue, D263, in the reducing-end xylose-releasing exo-oligoxylanase (Rex). A mutation in the tyrosine residue changing it into phenylalanine caused a drastic decrease in the hydrolytic activity and a small increase in the F(-) releasing activity from alpha-xylobiosyl fluoride in the presence of xylose. In contrast, mutations at D263 resulted in the decreased F(-) releasing activity. As a result of the high F(-) releasing activity and low hydrolytic activity, Y198F of Rex accumulates a large amount of product during the glycosynthase reaction. We propose a novel method for producing a glycosynthase from an inverting glycoside hydrolase by mutating a residue that holds the nucleophilic water molecule with the general base residue while keeping the general base residue intact.

  7. Luciferin Amides Enable in Vivo Bioluminescence Detection of Endogenous Fatty Acid Amide Hydrolase Activity.

    Science.gov (United States)

    Mofford, David M; Adams, Spencer T; Reddy, G S Kiran Kumar; Reddy, Gadarla Randheer; Miller, Stephen C

    2015-07-15

    Firefly luciferase is homologous to fatty acyl-CoA synthetases. We hypothesized that the firefly luciferase substrate d-luciferin and its analogs are fatty acid mimics that are ideally suited to probe the chemistry of enzymes that release fatty acid products. Here, we synthesized luciferin amides and found that these molecules are hydrolyzed to substrates for firefly luciferase by the enzyme fatty acid amide hydrolase (FAAH). In the presence of luciferase, these molecules enable highly sensitive and selective bioluminescent detection of FAAH activity in vitro, in live cells, and in vivo. The potency and tissue distribution of FAAH inhibitors can be imaged in live mice, and luciferin amides serve as exemplary reagents for greatly improved bioluminescence imaging in FAAH-expressing tissues such as the brain.

  8. Crystal structure of glycoside hydrolase family 127 β-L-arabinofuranosidase from Bifidobacterium longum

    Energy Technology Data Exchange (ETDEWEB)

    Ito, Tasuku; Saikawa, Kyo [Department of Biotechnology, The University of Tokyo, Tokyo (Japan); Kim, Seonah [National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO (United States); Fujita, Kiyotaka [Faculty of Agriculture, Kagoshima University, Korimoto, Kagoshima (Japan); Ishiwata, Akihiro [Synthetic Cellular Chemistry Laboratory, RIKEN (Japan); Kaeothip, Sophon [ERATO Glycotrilogy Project, JST, Wako, Saitama (Japan); Arakawa, Takatoshi; Wakagi, Takayoshi [Department of Biotechnology, The University of Tokyo, Tokyo (Japan); Beckham, Gregg T., E-mail: Gregg.Beckham@nrel.gov [National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO (United States); Ito, Yukishige [Synthetic Cellular Chemistry Laboratory, RIKEN (Japan); ERATO Glycotrilogy Project, JST, Wako, Saitama (Japan); Fushinobu, Shinya, E-mail: asfushi@mail.ecc.u-tokyo.ac.jp [Department of Biotechnology, The University of Tokyo, Tokyo (Japan)

    2014-04-25

    Graphical abstract: - Highlights: • HypBA1 β-L-arabinofuranosidase belongs to glycoside hydrolase family 127. • Crystal structure of HypBA1 was determined. • HypBA1 consists of a catalytic barrel and two additional β-sandwich domains. • The active site contains a Zn{sup 2+} coordinated by glutamate and three cysteines. • A possible reaction mechanism involving cysteine as the nucleophile is proposed. - Abstract: Enzymes acting on β-linked arabinofuranosides have been unknown until recently, in spite of wide distribution of β-L-arabinofuranosyl oligosaccharides in plant cells. Recently, a β-L-arabinofuranosidase from the glycoside hydrolase family 127 (HypBA1) was discovered in the newly characterized degradation system of hydroxyproline-linked β-L-arabinooligosaccharides in the bacterium Bifidobacterium longum. Here, we report the crystal structure of HypBA1 in the ligand-free and β-L-arabinofuranose complex forms. The structure of HypBA1 consists of a catalytic barrel domain and two additional β-sandwich domains, with one β-sandwich domain involved in the formation of a dimer. Interestingly, there is an unprecedented metal-binding motif with Zn{sup 2+} coordinated by glutamate and three cysteines in the active site. The glutamate residue is located far from the anomeric carbon of the β-L-arabinofuranose ligand, but one cysteine residue is appropriately located for nucleophilic attack for glycosidic bond cleavage. The residues around the active site are highly conserved among GH127 members. Based on biochemical experiments and quantum mechanical calculations, a possible reaction mechanism involving cysteine as the nucleophile is proposed.

  9. Dysregulation of soluble epoxide hydrolase and lipidomic profiles in anorexia nervosa

    KAUST Repository

    Shih, P. B.

    2015-03-31

    Individuals with anorexia nervosa (AN) restrict eating and become emaciated. They tend to have an aversion to foods rich in fat. Because epoxide hydrolase 2 (EPHX2) was identified as a novel AN susceptibility gene, and because its protein product, soluble epoxide hydrolase (sEH), converts bioactive epoxides of polyunsaturated fatty acid (PUFA) to the corresponding diols, lipidomic and metabolomic targets of EPHX2 were assessed to evaluate the biological functions of EPHX2 and their role in AN. Epoxide substrates of sEH and associated oxylipins were measured in ill AN, recovered AN and gender- and race-matched controls. PUFA and oxylipin markers were tested as potential biomarkers for AN. Oxylipin ratios were calculated as proxy markers of in vivo sEH activity. Several free- and total PUFAs were associated with AN diagnosis and with AN recovery. AN displayed elevated n-3 PUFAs and may differ from controls in PUFA elongation and desaturation processes. Cytochrome P450 pathway oxylipins from arachidonic acid, linoleic acid, alpha-linolenic acid and docosahexaenoic acid PUFAs are associated with AN diagnosis. The diol:epoxide ratios suggest the sEH activity is higher in AN compared with controls. Multivariate analysis illustrates normalization of lipidomic profiles in recovered ANs. EPHX2 influences AN risk through in vivo interaction with dietary PUFAs. PUFA composition and concentrations as well as sEH activity may contribute to the pathogenesis and prognosis of AN. Our data support the involvement of EPHX2-associated lipidomic and oxylipin dysregulations in AN, and reveal their potential as biomarkers to assess responsiveness to future intervention or treatment.

  10. Overexpression of fatty acid amide hydrolase induces early flowering in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Neal D. Teaster

    2012-02-01

    Full Text Available N-Acylethanolamines (NAEs are bioactive lipids derived from the hydrolysis of the membrane phospholipid N-acylphosphatidylethanolamine (NAPE. In animal systems this reaction is part of the endocannabinoid signaling pathway, which regulates a variety of physiological processes. The signaling function of NAE is terminated by fatty acid amide hydrolase (FAAH, which hydrolyzes NAE to ethanolamine and free fatty acid. Our previous work in Arabidopsis thaliana showed that overexpression of AtFAAH (At5g64440 lowered endogenous levels of NAEs in seeds, consistent with its role in NAE signal termination. Reduced NAE levels were accompanied by an accelerated growth phenotype, increased sensitivity to abscisic acid (ABA, enhanced susceptibility to bacterial pathogens, and early flowering. Here we investigated the nature of the early flowering phenotype of AtFAAH overexpression. AtFAAH overexpressors flowered several days earlier than wild type and AtFAAH knockouts under both non-inductive short day (SD and inductive long day (LD conditions. Microarray analysis revealed that the FLOWERING LOCUS T (FT gene, which plays a major role in regulating flowering time, and one target MADS box transcription factor, SEPATALLA3 (SEP3, were elevated in AtFAAH overexpressors. Furthermore, AtFAAH overexpressors, with the early flowering phenotype had lower endogenous NAE levels in leaves compared to wild type prior to flowering. Exogenous application of NAE 12:0, which was reduced by up to 30% in AtFAAH overexpressors, delayed the onset of flowering in wild type plants. We conclude that the early flowering phenotype of AtFAAH overexpressors is, in part, explained by elevated FT gene expression resulting from the enhanced NAE hydrolase activity of AtFAAH, suggesting that NAE metabolism may participate in floral signaling pathways.

  11. A screening method for β-glucan hydrolase employing Trypan Blue-coupled β-glucan agar plate and β-glucan zymography.

    Science.gov (United States)

    Park, Chang-Su; Yang, Hee-Jong; Kim, Dong-Ho; Kang, Dae-Ook; Kim, Min-Soo; Choi, Nack-Shick

    2012-06-01

    A new screening method for β-(1,3-1,6) glucan hydrolase was developed using a pure β-glucan from Aureobaisidum pullulans by zymography and an LB-agar plate. Paenibacillus sp. was screened as a producer a β-glucan hydrolase on the Trypan Blue-coupled β-glucan LB-agar plate and the activity of the enzyme was analyzed by SDS-β-glucan zymography. The β-glucan was not hydrolyzed by Bacillus spp. strains, which exhibit cellulolytic activity on CMC zymography. The gene, obtaining by shotgun cloning and encoding the β-glucan hydrolase of Paenibacillus sp. was sequenced.

  12. Interspecies differences in the enantioselectivity of epoxide hydrolases in Cryptococcus laurentii (Kufferath) C.E. Skinner and Cryptococcus podzolicus (Bab'jeva & Reshetova) Golubev

    CSIR Research Space (South Africa)

    Botes, AL

    2005-01-01

    Full Text Available Isolates representing Cryptococcus laurentii and Cryptococcus podzolicus, originating from soil of a heath land indigenous to South Africa, were screened for the presence of enantioselective epoxide hydrolases for 2, 2-disubstituted epoxides...

  13. Epoxide hydrolase-catalyzed enantioselective conversion of trans-stilbene oxide: Insights into the reaction mechanism from steady-state and pre-steady-state enzyme kinetics

    Czech Academy of Sciences Publication Activity Database

    Archelas, A.; Zhao, W.; Faure, B.; Iacazio, G.; Kotík, Michael

    2016-01-01

    Roč. 591, FEB 2016 (2016), s. 66-75 ISSN 0003-9861 Institutional support: RVO:61388971 Keywords : Catalytic mechanism * Epoxide hydrolase * Electrophilic catalysis Subject RIV: CE - Biochemistry Impact factor: 3.165, year: 2016

  14. Colloid-based multiplexed method for screening plant biomass-degrading glycoside hydrolase activities in microbial communities

    Energy Technology Data Exchange (ETDEWEB)

    Reindl, W.; Deng, K.; Gladden, J.M.; Cheng, G.; Wong, A.; Singer, S.W.; Singh, S.; Lee, J.-C.; Yao, J.-S.; Hazen, T.C.; Singh, A.K; Simmons, B.A.; Adams, P.D.; Northen, T.R.

    2011-05-01

    The enzymatic hydrolysis of long-chain polysaccharides is a crucial step in the conversion of biomass to lignocellulosic biofuels. The identification and characterization of optimal glycoside hydrolases is dependent on enzyme activity assays, however existing methods are limited in terms of compatibility with a broad range of reaction conditions, sample complexity, and especially multiplexity. The method we present is a multiplexed approach based on Nanostructure-Initiator Mass Spectrometry (NIMS) that allowed studying several glycolytic activities in parallel under diverse assay conditions. Although the substrate analogs carried a highly hydrophobic perfluorinated tag, assays could be performed in aqueous solutions due colloid formation of the substrate molecules. We first validated our method by analyzing known {beta}-glucosidase and {beta}-xylosidase activities in single and parallel assay setups, followed by the identification and characterization of yet unknown glycoside hydrolase activities in microbial communities.

  15. Optimization of the fermentation conditions and substrate specifity of mycelium-bound ester hydrolases of Aspergillus oryzae Cs007

    Directory of Open Access Journals (Sweden)

    de Hong Yan

    2015-01-01

    Full Text Available In order to improve mycelium-bound ester hydrolases activities of Aspergillus oryzae Cs007, the main production conditions were investigated. The ester hydrolases activities were simultaneously determined by titration assay and spectrophotometric assay methods, using olive oil and p-nitrophenyl esters as substrates, respectively. The optimum carbon source and nitrogen source were olive oil and peptone, with the concentrations of 1% and 2.2%, respectively. The effects of carbon source, nitrogen source and their concentrations on the production of enzymes were identical when the enzymes activities were assayed by the two methods. The mycelium-bound enzymes showed hydrolytic activity toward all the tested p-nitrophenyl esters, triglycerides and fatty acid ethyl esters. But it showed greater preference for long-chain triglycerides and short-chain p-nitrophenyl esters.

  16. Biodegradation of phthalic acid esters (PAEs) and in silico structural characterization of mono-2-ethylhexyl phthalate (MEHP) hydrolase on the basis of close structural homolog.

    Science.gov (United States)

    Singh, Neha; Dalal, Vikram; Mahto, Jai Krishna; Kumar, Pravindra

    2017-09-15

    Three bacterial strains capable of degrading phthalates namely Pseudomonas sp. PKDM2, Pseudomonas sp. PKDE1 and Pseudomonas sp. PKDE2 were isolated and characterized for their degradative potential. These strains efficiently degraded 77.4%-84.4% of DMP, 75.0%-75.7% of DEP and 71.7%-74.7% of DEHP, initial amount of each phthalate is 500mgL -1 of each phthalate, after 44h of incubation. GC-MS results reveal the tentative DEHP degradation pathway, where hydrolases mediate the breakdown of DEHP to phthalic acid (PA) via an intermediate MEHP. MEHP hydrolase is a serine hydrolase which is involved in the reduction of the MEHP to PA. The predicted 3D model of MEHP hydrolase from Pseudomonas mosselii was docked with phthalate monoesters (PMEs) such as MEHP, mono-n-hexyl phthalate (MHP), mono-n-butyl phthalate (MBP) and mono-n-ethyl phthalate (MEP), respectively. Docking results show the distance between the carbonyl carbon of respective phthalate monoester and the hydroxyl group of catalytic serine lies in the range of 2.9 to 3.3Å, which is similar to the ES complex of other serine hydrolases. This structural study highlights the interaction and the role of catalytic residues of MEHP hydrolase involved in the biodegradation of PMEs to phthalate. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Microsomal epoxide hydrolase gene polymorphisms and risk of chronic obstructive pulmonary disease: A comprehensive meta-analysis

    OpenAIRE

    LI, HUI; FU, WEI-PING; HONG, ZE-HUI

    2012-01-01

    Microsomal epoxide hydrolase (EPHX1) is an enzyme involved in the detoxification the products of smoking and is proposed to be a genetic factor for the development of chronic obstructive pulmonary disease (COPD). Two functional polymorphisms of EPHX1, T113C and A139G, have been analyzed in numerous studies to assess the COPD risk attributed to these variants. However, the conclusions were controversial. We performed a comprehensive meta-analysis to clarify these findings. A total of 24 studie...

  18. 4,3-α-Glucanotransferase, a novel reaction specificity in glycoside hydrolase family 70 and clan GH-H

    NARCIS (Netherlands)

    Gangoiti Muñecas, Joana; van Leeuwen, Sander S; Gerwig, Gerrit J; Duboux, Stéphane; Vafiadi, Christina; Pijning, Tjaard; Dijkhuizen, Lubbert

    2017-01-01

    Lactic acid bacteria possess a diversity of glucansucrase (GS) enzymes that belong to glycoside hydrolase family 70 (GH70) and convert sucrose into α-glucan polysaccharides with (α1 → 2)-, (α1 → 3)-, (α1 → 4)- and/or (α1 → 6)-glycosidic bonds. In recent years 3 novel subfamilies of GH70 enzymes,

  19. The Natural Product Acivicin as a Tool for ABPP and the Activity of Serine Hydrolases in Uterine Fibroids

    OpenAIRE

    Kreuzer, Johannes

    2015-01-01

    The target proteins of acivicin and structure derived probes in tumor cells were identified using activity-based protein profiling. The target proteins were further characterized and their relation to the antitumor activity of acivicin pointed out. In a further project, the activity of serine hydrolases in myoma and myometrium was examined from tissue samples. This revealed a different activity of mast cell proteases. Mittels Activity-based Protein Profiling wurde eine Identifikation der Z...

  20. Structure of XC6422 from Xanthomonas campestris at 1.6 Å resolution: a small serine α/β-hydrolase

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Chao-Yu; Chin, Ko-Hsin [Institute of Biochemistry, National Chung-Hsing University, Taichung 40227,Taiwan (China); Chou, Chia-Cheng; Wang, Andrew H.-J. [Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei,Taiwan (China); Core Facility for Protein Crystallography, Academia Sinica, Nankang, Taipei,Taiwan (China); Chou, Shan-Ho, E-mail: shchou@nchu.edu.tw [Institute of Biochemistry, National Chung-Hsing University, Taichung 40227,Taiwan (China)

    2006-06-01

    The crystal structure of a conserved hypothetical protein from X. campestris has been determined to a resolution of 1.6 Å. The determined X. campestris structure shows that it belongs to the superfamily of serine α/β hydrolase, with an extra strand preceding the first β-strand to lead to extensive subunit interactions in the crystal. XC6422 is a conserved hypothetical protein from Xanthomonas campestris pathovar campestris (Xcc), a Gram-negative yellow-pigmented pathogenic bacterium that causes black rot, one of the major worldwide diseases of cruciferous crops. The protein consists of 220 amino acids and its structure has been determined to 1.6 Å resolution using the multi-wavelength anomalous dispersion (MAD) method. Although it has very low sequence identity to protein sequences in the PDB (less than 20%), the determined structure nevertheless shows that it belongs to the superfamily of serine α/β-hydrolases, with an active site that is fully accessible to solvent owing to the absence of a lid domain. Modelling studies with the serine esterase inhibitor E600 indicate that XC6422 adopts a conserved Ser-His-Asp catalytic triad common to this superfamily and has a preformed oxyanion hole for catalytic activation. These structural features suggest that XC6422 is most likely to be a hydrolase active on a soluble ester or a small lipid. An extra strand preceding the first β-strand in the canonical α/β-hydrolase fold leads to extensive subunit interactions between XC6422 monomers, which may explain why XC6422 crystals of good diffraction quality can grow to dimensions of up to 1.5 mm in a few days.

  1. Simultaneous Inhibition of Fatty Acid Amide Hydrolase and Monoacylglycerol Lipase Shares Discriminative Stimulus Effects with Δ9-Tetrahydrocannabinol in Mice

    OpenAIRE

    Hruba, Lenka; Seillier, Alexandre; Zaki, Armia; Cravatt, Benjamin F.; Lichtman, Aron H.; Giuffrida, Andrea; McMahon, Lance R.

    2015-01-01

    Monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) inhibitors exert preclinical effects indicative of therapeutic potential (i.e., analgesia). However, the extent to which MAGL and FAAH inhibitors produce unwanted effects remains unclear. Here, FAAH and MAGL inhibition was examined separately and together in a Δ9-tetrahydrocannabinol (Δ9-THC; 5.6 mg/kg i.p.) discrimination assay predictive of subjective effects associated with cannabis use, and the relative contribution of N...

  2. Determination of frequencies of alleles, associated with the pseudodeficiency of lysosomal hydrolases, in population of Ukraine.

    Science.gov (United States)

    Olkhovych, N V; Gorovenko, N G

    2016-01-01

    The pseudodeficiency of lysosomal hydrolases described as a significant reduction in enzyme activi­ty in vitro in clinically healthy individuals, can lead to diagnostic errors in the process of biochemical analysis of lysosomal storage disease in case of its combination with pathology of another origin. Pseudodeficiency is mostly caused by some non-pathogenic changes in the corresponding gene. These changes lead to the in vitro lability of the enzyme molecule, whereas in vivo the enzyme retains its functional activity. To assess the prevalence of the most common lysosomal hydrolases pseudodeficiency alleles in Ukraine, we have determined the frequency of alleles c.1055A>G and c.* 96A>G in the ARSA gene, substitutions c.739C>T (R247W) and c.745C>T (R249W) in the HEXA gene, c.1726G>A (G576S) and c.2065G>A (E689K) in the GAA gene, c.937G>T (D313Y) in the GLA1 gene and c.898G>A (A300T) in the IDUA gene in a group of 117 healthy individuals from different regions of the country and 14 heterozygous carriers of pathogenic mutations in the HEXA gene (parents of children with confirmed diagnosis of Tay-Sachs disease). The total frequency of haplotypes, associated with arylsulfatase A pseudodeficiency, in healthy people in Ukraine (c.1055G/c.*96G and c.1055G/c.*96A haplotypes) was 10.3%. The frequency of c.739C>T (R247W) allele, associated with hexo­saminidase A pseudodeficiency, among Tay-Sachs carriers from Ukraine was 7.1%. The total frequency of α-glucosidase pseudodeficiency haplotypes in healthy individuals in Ukraine (c.1726A/c.2065A and c.1726G/c.2065A haplotypes) was 2.6%. No person among examined individuals with the substitution c.937G>T (D313Y) in the GLA1 gene and c.898G>A (A300T) in the IDUA gene was found. The differential diagnostics of lysosomal storage diseases requires obligatory determination of the presence of the pseudodeficiency alleles, particularly the ones with high incidence in the total population. Ignoring phenomenon of pseudodeficiency may

  3. Modulation of redox homeostasis under suboptimal conditions by Arabidopsis nudix hydrolase 7

    Directory of Open Access Journals (Sweden)

    Jambunathan Niranjani

    2010-08-01

    Full Text Available Abstract Background Nudix hydrolases play a key role in maintaining cellular homeostasis by hydrolyzing various nuceloside diphosphate derivatives and capped mRNAs. Several independent studies have demonstrated that Arabidopsis nudix hydrolase 7 (AtNUDT7 hydrolyzes NADH and ADP-ribose. Loss of function Atnudt7-1 mutant plants (SALK_046441 exhibit stunted growth, higher levels of reactive oxygen species, enhanced resistance to pathogens. However, using the same T-DNA line, two other groups reported that mutant plants do not exhibit any visible phenotypes. In this study we analyze plausible factors that account for differences in the observed phenotypes in Atnudt7. Secondly, we evaluate the biochemical and molecular consequences of increased NADH levels due to loss of function of AtNUDT7 in Arabidopsis. Results We identified a novel conditional phenotype of Atnudt7-1 knockout plants that was contingent upon nutrient composition of potting mix. In nutrient-rich Metro-Mix, there were no phenotypic differences between mutant and wild-type (WT plants. In the nutrient-poor mix (12 parts vermiculite: 3 parts Redi-earth and 1 part sand, mutant plants showed the characteristic stunted phenotype. Compared with WT plants, levels of glutathione, NAD+, NADH, and in turn NADH:NAD+ ratio were higher in Atnudt7-1 plants growing in 12:3:1 potting mix. Infiltrating NADH and ADP-ribose into WT leaves was sufficient to induce AtNUDT7 protein. Constitutive over-expression of AtNudt7 did not alter NADH levels or resistance to pathogens. Transcriptome analysis identified nearly 700 genes differentially expressed in the Atnudt7-1 mutant compared to WT plants grown in 12:3:1 potting mix. In the Atnudt7-1 mutant, genes associated with defense response, proteolytic activities, and systemic acquired resistance were upregulated, while gene ontologies for transcription and phytohormone signaling were downregulated. Conclusions Based on these observations, we conclude that the

  4. Soluble epoxide hydrolase activity and pharmacologic inhibition in horses with chronic severe laminitis.

    Science.gov (United States)

    Guedes, A; Galuppo, L; Hood, D; Hwang, S H; Morisseau, C; Hammock, B D

    2017-05-01

    The roles of soluble epoxide hydrolase and lipid mediators in inflammatory and neuropathic pain could be relevant in laminitis pain management. To determine soluble epoxide hydrolase (sEH) activity in the digital laminae, sEH inhibitor potency in vitro, and efficacy of a sEH inhibitor as an adjunct analgesic therapy in chronic laminitic horses. In vitro experiments and clinical case series. sEH activity was measured in digital laminae from euthanised healthy and laminitic horses (n = 5-6/group). Potency of 7 synthetic sEH inhibitors was determined in vitro using equine liver cytosol. One of them (t-TUCB; 0.1 mg/kg bwt i.v. every 24 h) was selected based on potency and stability, and used as adjunct therapy in 10 horses with severe chronic laminitis (Obel grades 2, one horse; 3-4, nine horses). Daily assessments of forelimb lifts, pain scores, physiologic and laboratory examinations were performed before (baseline) and during t-TUCB treatment. Data are presented as mean ± s.d. and 95% confidence intervals (CI). sEH activity in the digital laminae from laminitic horses (0.9±0.6 nmol/min/mg; 95% CI 0.16-1.55 nmol/min/mg) was significantly greater (P = 0.01) than in healthy horses (0.17±0.09 nmol/min/mg; CI 0.07-0.26 nmol/min/mg). t-TUCB as an adjunct analgesic up to 10 days (4.3±3 days) in laminitic horses was associated with significant reduction in forelimb lifts (36±22%; 95% CI 9-64%) and in pain scores (18±23%; 95% CI 2-35%) compared with baseline (P = 0.04). One horse developed gas colic and another corneal vascularisation in a blind eye during treatment. No other significant changes were observed. Absence of control group and evaluator blinding in case series. sEH activity is significantly higher in the digital laminae of actively laminitic compared with healthy horses, and use of a potent inhibitor of equine sEH as adjunct analgesic therapy appears to decrease signs of pathologic pain in laminitic horses. © 2016 EVJ Ltd.

  5. Determination of frequencies of alleles, associated with the pseudodeficiency of lysosomal hydrolases, in population of Ukraine

    Directory of Open Access Journals (Sweden)

    N. V. Olkhovych

    2016-10-01

    Full Text Available The pseudodeficiency of lysosomal hydrolases described as a significant reduction in enzyme activi­ty in vitro in clinically healthy individuals, can lead to diagnostic errors in the process of biochemical analysis of lysosomal storage disease in case of its combination with pathology of another origin. Pseudodeficiency is mostly caused by some non-pathogenic changes in the corresponding gene. These changes lead to the in vitro lability of the enzyme molecule, whereas in vivo the enzyme retains its functional activity. To assess the prevalence of the most common lysosomal hydrolases pseudodeficiency alleles in Ukraine, we have determined the frequency of alleles c.1055A>G and c.* 96A>G in the ARSA gene, substitutions c.739C>T (R247W and c.745C>T (R249W in the HEXA gene, c.1726G>A (G576S and c.2065G>A (E689K in the GAA gene, c.937G>T (D313Y in the GLA1 gene and c.898G>A (A300T in the IDUA gene in a group of 117 healthy individuals from different regions of the country and 14 heterozygous carriers of pathogenic mutations in the HEXA gene (parents of children with confirmed diagnosis of Tay-Sachs disease. The total frequency of haplotypes, associated with arylsulfatase A pseudodeficiency, in healthy people in Ukraine (c.1055G/c.*96G and c.1055G/c.*96A haplotypes was 10.3%. The frequency of c.739C>T (R247W allele, associated with hexo­saminidase A pseudodeficiency, among Tay-Sachs carriers from Ukraine was 7.1%. The total frequency of α-glucosidase pseudodeficiency haplotypes in healthy individuals in Ukraine (c.1726A/c.2065A and c.1726G/c.2065A haplotypes was 2.6%. No person among examined individuals with the substitution c.937G>T (D313Y in the GLA1 gene and c.898G>A (A300T in the IDUA gene was found. The differential diagnostics of lysosomal storage diseases requires obligatory determination of the presence of the pseudodeficiency alleles, particularly the ones with high incidence in the total population. Ignoring phenomenon of

  6. An appraisal of eighteen commonly consumed edible plants as functional food based on their antioxidant and starch hydrolase inhibitory activities.

    Science.gov (United States)

    Lee, Yian Hoon; Choo, Candy; Watawana, Mindani I; Jayawardena, Nilakshi; Waisundara, Viduranga Y

    2015-11-01

    Eighteen edible plants were assessed for their antioxidant potential based on oxygen radical absorbance capacity (ORAC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, total phenolics, vitamin C content and various lipophilic antioxidants. The inhibitory activities of the plant extracts against the enzymatic activities of α-amylase and α-glucosidase were also evaluated. The antioxidant and starch hydrolase activities of the plants varied widely across a single batch of analysis. The ORAC and DPPH radical scavenging EC50 values varied between 298 and 1984 Trolox equivalents g(-1) fresh weight and between 91 and 533 mg kg(-1) fresh weight, respectively. The total phenolics and vitamin C contents varied between 32 and 125 mg gallic acid equivalents g(-1) fresh weight and between 96 and 285 µg g(-1) fresh weight, respectively. All the plants contained neoxanthin, violaxanthin, and α- and β-carotene in varying amounts. Coccinia grandis, Asparagus racemosus, Costus speciosus, Amaranthus viridis and Annona muricata displayed the highest inhibitory activities against starch hydrolases. They were the most efficient against the breakdown of seven starches exposed to the two enzymes as well. Overall, the edible plants were observed to display a high antioxidant potential with starch hydrolase inhibitory properties, which were beneficial in their being recognized as functional food. © 2014 Society of Chemical Industry.

  7. Synergistic function of four novel thermostable glycoside hydrolases from a long-term enriched thermophilic methanogenic digester

    Directory of Open Access Journals (Sweden)

    Meng eWang

    2015-05-01

    Full Text Available In biofuel production from lignocellulose, low thermostability and product inhibition strongly restrict the enzyme activities and production process. Application of multiple thermostable glycoside hydrolases, forming an enzyme cocktail, can result in a synergistic action and therefore improve production efficiency and reduce operational costs. Therefore, increasing enzyme thermostabilities and compatibility are important for the biofuel industry. In this study, we reported the screening, cloning and biochemical characterization of four novel thermostable lignocellulose hydrolases from a metagenomic library of a long-term dry thermophilic methanogenic digester community, which were highly compatible with optimal conditions and specific activities. The optimal temperatures of the four enzymes, β-xylosidase, xylanase, β-glucosidase, and cellulase ranged from 60°C to 75°C, and over 80% residual activities were observed after 2 h incubation at 50°C. Mixtures of these hydrolases retained high residual synergistic activities after incubation with cellulose, xylan, and steam-exploded corncob at 50°C for 72 h. In addition, about 55% dry weight of steam-exploded corncob was hydrolyzed to glucose and xylose by the synergistic action of the four enzymes at 50°C for 48 h. This work suggested that since different enzymes from a same ecosystem could be more compatible, screening enzymes from a long-term enriching community could be a favorable strategy.

  8. Identification, structure, and function of a novel type VI secretion peptidoglycan glycoside hydrolase effector-immunity pair.

    Science.gov (United States)

    Whitney, John C; Chou, Seemay; Russell, Alistair B; Biboy, Jacob; Gardiner, Taylor E; Ferrin, Michael A; Brittnacher, Mitchell; Vollmer, Waldemar; Mougous, Joseph D

    2013-09-13

    Bacteria employ type VI secretion systems (T6SSs) to facilitate interactions with prokaryotic and eukaryotic cells. Despite the widespread identification of T6SSs among Gram-negative bacteria, the number of experimentally validated substrate effector proteins mediating these interactions remains small. Here, employing an informatics approach, we define novel families of T6S peptidoglycan glycoside hydrolase effectors. Consistent with the known intercellular self-intoxication exhibited by the T6S pathway, we observe that each effector gene is located adjacent to a hypothetical open reading frame encoding a putative periplasmically localized immunity determinant. To validate our sequence-based approach, we functionally investigate a representative family member from the soil-dwelling bacterium Pseudomonas protegens. We demonstrate that this protein is secreted in a T6SS-dependent manner and that it confers a fitness advantage in growth competition assays with Pseudomonas putida. In addition, we determined the 1.4 Å x-ray crystal structure of this effector in complex with its cognate immunity protein. The structure reveals the effector shares highest overall structural similarity to a glycoside hydrolase family associated with peptidoglycan N-acetylglucosaminidase activity, suggesting that T6S peptidoglycan glycoside hydrolase effector families may comprise significant enzymatic diversity. Our structural analyses also demonstrate that self-intoxication is prevented by the immunity protein through direct occlusion of the effector active site. This work significantly expands our current understanding of T6S effector diversity.

  9. Identification, Structure, and Function of a Novel Type VI Secretion Peptidoglycan Glycoside Hydrolase Effector-Immunity Pair*

    Science.gov (United States)

    Whitney, John C.; Chou, Seemay; Russell, Alistair B.; Biboy, Jacob; Gardiner, Taylor E.; Ferrin, Michael A.; Brittnacher, Mitchell; Vollmer, Waldemar; Mougous, Joseph D.

    2013-01-01

    Bacteria employ type VI secretion systems (T6SSs) to facilitate interactions with prokaryotic and eukaryotic cells. Despite the widespread identification of T6SSs among Gram-negative bacteria, the number of experimentally validated substrate effector proteins mediating these interactions remains small. Here, employing an informatics approach, we define novel families of T6S peptidoglycan glycoside hydrolase effectors. Consistent with the known intercellular self-intoxication exhibited by the T6S pathway, we observe that each effector gene is located adjacent to a hypothetical open reading frame encoding a putative periplasmically localized immunity determinant. To validate our sequence-based approach, we functionally investigate a representative family member from the soil-dwelling bacterium Pseudomonas protegens. We demonstrate that this protein is secreted in a T6SS-dependent manner and that it confers a fitness advantage in growth competition assays with Pseudomonas putida. In addition, we determined the 1.4 Å x-ray crystal structure of this effector in complex with its cognate immunity protein. The structure reveals the effector shares highest overall structural similarity to a glycoside hydrolase family associated with peptidoglycan N-acetylglucosaminidase activity, suggesting that T6S peptidoglycan glycoside hydrolase effector families may comprise significant enzymatic diversity. Our structural analyses also demonstrate that self-intoxication is prevented by the immunity protein through direct occlusion of the effector active site. This work significantly expands our current understanding of T6S effector diversity. PMID:23878199

  10. NatB domain-containing CRA-1 antagonizes hydrolase ACER-1 linking acetyl-CoA metabolism to the initiation of recombination during C. elegans meiosis.

    Directory of Open Access Journals (Sweden)

    Jinmin Gao

    2015-03-01

    Full Text Available The formation of DNA double-strand breaks (DSBs must take place during meiosis to ensure the formation of crossovers, which are required for accurate chromosome segregation, therefore avoiding aneuploidy. However, DSB formation must be tightly regulated to maintain genomic integrity. How this regulation operates in the context of different chromatin architectures and accessibility, and how it is linked to metabolic pathways, is not understood. We show here that global histone acetylation levels undergo changes throughout meiotic progression. Moreover, perturbations to global histone acetylation levels are accompanied by changes in the frequency of DSB formation in C. elegans. We provide evidence that the regulation of histone acetylation requires CRA-1, a NatB domain-containing protein homologous to human NAA25, which controls the levels of acetyl-Coenzyme A (acetyl-CoA by antagonizing ACER-1, a previously unknown and conserved acetyl-CoA hydrolase. CRA-1 is in turn negatively regulated by XND-1, an AT-hook containing protein. We propose that this newly defined protein network links acetyl-CoA metabolism to meiotic DSB formation via modulation of global histone acetylation.

  11. A new s-adenosylhomocysteine hydrolase-linked method for adenosine detection based on DNA-templated fluorescent Cu/Ag nanoclusters.

    Science.gov (United States)

    Ahn, Jun Ki; Kim, Hyo Yong; Baek, Songyi; Park, Hyun Gyu

    2017-07-15

    We herein describe a novel fluorescent method for the rapid and selective detection of adenosine by utilizing DNA-templated Cu/Ag nanoclusters (NCs) and employing s-adenosylhomocysteine hydrolase (SAHH). SAHH is allowed to promote hydrolysis reaction of s-adenosylhomocysteine (SAH) and consequently produces homocysteine, which would quench the fluorescence signal from DNA-templated Cu/Ag nanoclusters employed as a signaling probe in this study. On the other hand, adenosine significantly inhibits the hydrolysis reaction and prevent the formation of homocysteine. Consequently, highly enhanced fluorescence signal from DNA-Cu/Ag NCs is retained, which could be used to identify the presence of adenosine. By employing this design principle, adenosine was sensitively detected down to 19nM with high specificity over other adenosine analogs such as AMP, ADP, ATP, cAMP, guanosine, cytidine, and urine. Finally, the diagnostic capability of this method was successfully verified by reliably detecting adenosine present in a real human serum sample. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. The Immunoreactive Exo-1,3-β-Glucanase from the Pathogenic Oomycete Pythium insidiosum Is Temperature Regulated and Exhibits Glycoside Hydrolase Activity.

    Directory of Open Access Journals (Sweden)

    Angsana Keeratijarut

    Full Text Available The oomycete organism, Pythium insidiosum, is the etiologic agent of the life-threatening infectious disease called "pythiosis". Diagnosis and treatment of pythiosis is difficult and challenging. Novel methods for early diagnosis and effective treatment are urgently needed. Recently, we reported a 74-kDa immunodominant protein of P. insidiosum, which could be a diagnostic target, vaccine candidate, and virulence factor. The protein was identified as a putative exo-1,3-ß-glucanase (Exo1. This study reports on genetic, immunological, and biochemical characteristics of Exo1. The full-length exo1 coding sequence (2,229 bases was cloned. Phylogenetic analysis showed that exo1 is grouped with glucanase-encoding genes of other oomycetes, and is far different from glucanase-encoding genes of fungi. exo1 was up-regulated upon exposure to body temperature, and its gene product is predicted to contain BglC and X8 domains, which are involved in carbohydrate transport, binding, and metabolism. Based on its sequence, Exo1 belongs to the Glycoside Hydrolase family 5 (GH5. Exo1, expressed in E. coli, exhibited ß-glucanase and cellulase activities. Exo1 is a major intracellular immunoreactive protein that can trigger host immune responses during infection. Since GH5 enzyme-encoding genes are not present in human genomes, Exo1 could be a useful target for drug and vaccine development against this pathogen.

  13. Effects of anti-cocaine vaccine and viral gene transfer of cocaine hydrolase in mice on cocaine toxicity including motor strength and liver damage.

    Science.gov (United States)

    Gao, Yang; Geng, Liyi; Orson, Frank; Kinsey, Berma; Kosten, Thomas R; Shen, Xiaoyun; Brimijoin, Stephen

    2013-03-25

    In developing an vivo drug-interception therapy to treat cocaine abuse and hinder relapse into drug seeking provoked by re-encounter with cocaine, two promising agents are: (1) a cocaine hydrolase enzyme (CocH) derived from human butyrylcholinesterase and delivered by gene transfer; (2) an anti-cocaine antibody elicited by vaccination. Recent behavioral experiments showed that antibody and enzyme work in a complementary fashion to reduce cocaine-stimulated locomotor activity in rats and mice. Our present goal was to test protection against liver damage and muscle weakness in mice challenged with massive doses of cocaine at or near the LD50 level (100-120 mg/kg, i.p.). We found that, when the interceptor proteins were combined at doses that were only modestly protective in isolation (enzyme, 1mg/kg; antibody, 8 mg/kg), they provided complete protection of liver tissue and motor function. When the enzyme levels were ~400-fold higher, after in vivo transduction by adeno-associated viral vector, similar protection was observed from CocH alone. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  14. The Differential Proteome of the Probiotic Lactobacillus acidophilus NCFM Grown on the Potential Prebiotic Cellobiose Shows Upregulation of Two β-Glycoside Hydrolases

    Directory of Open Access Journals (Sweden)

    Gabriella C. van Zanten

    2015-01-01

    Full Text Available Probiotics, prebiotics, and combinations thereof, that is, synbiotics, are known to exert beneficial health effects in humans; however interactions between pro- and prebiotics remain poorly understood at the molecular level. The present study describes changes in abundance of different proteins of the probiotic bacterium Lactobacillus acidophilus NCFM (NCFM when grown on the potential prebiotic cellobiose as compared to glucose. Cytosolic cell extract proteomes after harvest at late exponential phase of NCFM grown on cellobiose or glucose were analyzed by two dimensional difference gel electrophoresis (2D-DIGE in the acidic (pH 4–7 and the alkaline (pH 6–11 regions showing a total of 136 spots to change in abundance. Proteins were identified by MS or MS/MS from 81 of these spots representing 49 unique proteins and either increasing 1.5–13.9-fold or decreasing 1.5–7.8-fold in relative abundance. Many of these proteins were associated with energy metabolism, including the cellobiose related glycoside hydrolases phospho-β-glucosidase (LBA0881 and phospho-β-galactosidase II (LBA0726. The data provide insight into the utilization of the candidate prebiotic cellobiose by the probiotic bacterium NCFM. Several of the upregulated or downregulated identified proteins associated with utilization of cellobiose indicate the presence of carbon catabolite repression and regulation of enzymes involved in carbohydrate metabolism.

  15. NatB domain-containing CRA-1 antagonizes hydrolase ACER-1 linking acetyl-CoA metabolism to the initiation of recombination during C. elegans meiosis.

    Science.gov (United States)

    Gao, Jinmin; Kim, Hyun-Min; Elia, Andrew E; Elledge, Stephen J; Colaiácovo, Monica P

    2015-03-01

    The formation of DNA double-strand breaks (DSBs) must take place during meiosis to ensure the formation of crossovers, which are required for accurate chromosome segregation, therefore avoiding aneuploidy. However, DSB formation must be tightly regulated to maintain genomic integrity. How this regulation operates in the context of different chromatin architectures and accessibility, and how it is linked to metabolic pathways, is not understood. We show here that global histone acetylation levels undergo changes throughout meiotic progression. Moreover, perturbations to global histone acetylation levels are accompanied by changes in the frequency of DSB formation in C. elegans. We provide evidence that the regulation of histone acetylation requires CRA-1, a NatB domain-containing protein homologous to human NAA25, which controls the levels of acetyl-Coenzyme A (acetyl-CoA) by antagonizing ACER-1, a previously unknown and conserved acetyl-CoA hydrolase. CRA-1 is in turn negatively regulated by XND-1, an AT-hook containing protein. We propose that this newly defined protein network links acetyl-CoA metabolism to meiotic DSB formation via modulation of global histone acetylation.

  16. Hydrolase stabilization via entanglement in poly(propylene sulfide) nanoparticles: stability towards reactive oxygen species

    International Nuclear Information System (INIS)

    Allen, Brett L; Johnson, Jermaine D; Walker, Jeremy P

    2012-01-01

    In the advancement of green syntheses and sustainable reactions, enzymatic biocatalysis offers extremely high reaction rates and selectivity that goes far beyond the reach of chemical catalysts; however, these enzymes suffer from typical environmental constraints, e.g. operational temperature, pH and tolerance to oxidative environments. A common hydrolase enzyme, diisopropylfluorophosphatase (DFPase, EC 3.1.8.2), has demonstrated a pronounced efficacy for the hydrolysis of a variety of substrates for potential toxin remediation, but suffers from the aforementioned limitations. As a means to enhance DFPase’s stability in oxidative environments, enzymatic covalent immobilization within the polymeric matrix of poly(propylene sulfide) (PPS) nanoparticles was performed. By modifying the enzyme’s exposed lysine residues via thiolation, DFPase is utilized as a comonomer/crosslinker in a mild emulsion polymerization. The resultant polymeric polysulfide shell acts as a ‘sacrificial barrier’ by first oxidizing to polysulfoxides and polysulfones, rendering DFPase in an active state. DFPase–PPS nanoparticles thus retain activity upon exposure to as high as 50 parts per million (ppm) of hypochlorous acid (HOCl), while native DFPase is observed as inactive at 500 parts per billion (ppb). This trend is also confirmed by enzyme-generated (chloroperoxidase (CPO), EC 1.11.1.10) reactive oxygen species (ROS) including both HOCl (3 ppm) and ClO 2 (100 ppm). (paper)

  17. New insights into plant glycoside hydrolase family 32 in Agave species.

    Science.gov (United States)

    Avila de Dios, Emmanuel; Gomez Vargas, Alan D; Damián Santos, Maura L; Simpson, June

    2015-01-01

    In order to optimize the use of agaves for commercial applications, an understanding of fructan metabolism in these species at the molecular and genetic level is essential. Based on transcriptome data, this report describes the identification and molecular characterization of cDNAs and deduced amino acid sequences for genes encoding fructosyltransferases, invertases and fructan exohydrolases (FEH) (enzymes belonging to plant glycoside hydrolase family 32) from four different agave species (A. tequilana, A. deserti, A. victoriae-reginae, and A. striata). Conserved amino acid sequences and a hypervariable domain allowed classification of distinct isoforms for each enzyme type. Notably however neither 1-FFT nor 6-SFT encoding cDNAs were identified. In silico analysis revealed that distinct isoforms for certain enzymes found in a single species, showed different levels and tissue specific patterns of expression whereas in other cases expression patterns were conserved both within the species and between different species. Relatively high levels of in silico expression for specific isoforms of both invertases and fructosyltransferases were observed in floral tissues in comparison to vegetative tissues such as leaves and stems and this pattern was confirmed by Quantitative Real Time PCR using RNA obtained from floral and leaf tissue of A. tequilana. Thin layer chromatography confirmed the presence of fructans with degree of polymerization (DP) greater than DP three in both immature buds and fully opened flowers also obtained from A. tequilana.

  18. New insights into plant glycoside hydrolase family 32 in Agave species

    Directory of Open Access Journals (Sweden)

    Emmanuel eAvila-de Dios

    2015-08-01

    Full Text Available In order to optimize the use of agaves for commercial applications, an understanding of fructan metabolism in these species at the molecular and genetic level is essential. Based on transcriptome data, this report describes the identification and molecular characterization of cDNAs and deduced amino acid sequences for genes encoding fructosyltransferases, invertases and fructan exohydrolases (enzymes belonging to plant glycoside hydrolase family 32 from four different agave species (A. tequilana, A. deserti, A. victoriae-reginae and A. striata. Conserved amino acid sequences and a hypervariable domain allowed classification of distinct isoforms for each enzyme type. Notably however neither 1-FFT nor 6-SFT encoding cDNAs were identified. In silico analysis revealed that distinct isoforms for certain enzymes found in a single species, showed different levels and tissue specific patterns of expression whereas in other cases expression patterns were conserved both within the species and between different species. Relatively high levels of in silico expression for specific isoforms of both invertases and fructosyltransferases were observed in floral tissues in comparison to vegetative tissues such as leaves and stems and this pattern was confirmed by Quantitative Real Time PCR using RNA obtained from floral and leaf tissue of A. tequilana. Thin layer chromatography confirmed the presence of fructans with degree of polymerization (DP greater than DP three in both immature buds and fully opened flowers also obtained from A. tequilana.

  19. [Substrate specificities of bile salt hydrolase 1 and its mutants from Lactobacillus salivarius].

    Science.gov (United States)

    Bi, Jie; Fang, Fang; Qiu, Yuying; Yang, Qingli; Chen, Jian

    2014-03-01

    In order to analyze the correlation between critical residues in the catalytic centre of BSH and the enzyme substrate specificity, seven mutants of Lactobacillus salivarius bile salt hydrolase (BSH1) were constructed by using the Escherichia coli pET-20b(+) gene expression system, rational design and site-directed mutagenesis. These BSH1 mutants exhibited different hydrolytic activities against various conjugated bile salts through substrate specificities comparison. Among the residues being tested, Cys2 and Thr264 were deduced as key sites for BSH1 to catalyze taurocholic acid and glycocholic acid, respectively. Moreover, Cys2 and Thr264 were important for keeping the catalytic activity of BSH1. The high conservative Cys2 was not the only active site, other mutant amino acid sites were possibly involved in substrate binding. These mutant residues might influence the space and shape of the substrate-binding pockets or the channel size for substrate passing through and entering active site of BSH1, thus, the hydrolytic activity of BSH1 was changed to different conjugated bile salt.

  20. Purification and Characterization of Tannin Acyl Hydrolase from Aspergillus niger ATCC 16620

    Directory of Open Access Journals (Sweden)

    Abdulhameed Sabu

    2005-01-01

    Full Text Available Tannin acyl hydrolase produced extracellularly by the fungal strain Aspergillus niger ATTC 16620 in solid state fermentation was purified from the cell free culture broth by ammonium sulphate fractionation followed by DEAE–Sephadex A-50 chromatography. SDS-PAGE analysis indicated that the enzyme protein molecular mass was 168 kDa. Enzyme activity was stable up to the temperature of 40 °C and the enzyme activity was optimal at pH=6. Tannase activity was maximal at 0.01 M concentration of the substrate. The addition of metal ions like Zn2+, Mn2+, Cu2+, Ca2+, Mg2+and Fe2+ inhibited the enzyme activity. Only K+ ions enhanced tannase activity, and an activity of 4.31 U/mL was reported here. Enzyme activity was maximal after 15–20 min of incubation time, with an activity of 3.9 U/mL. Km was found to be 1.03 mM and Vmax=4.25 mmol/min. Since the enzyme is active over a wide range of pH and temperature it could find potential use in the food-processing industry.

  1. Immunotherapy against visceral leishmaniasis with the nucleoside hydrolase-DNA vaccine of Leishmania donovani.

    Science.gov (United States)

    Gamboa-León, R; Paraguai de Souza, E; Borja-Cabrera, G P; Santos, F N; Myashiro, L M; Pinheiro, R O; Dumonteil, E; Palatnik-de-Sousa, C B

    2006-05-29

    The nucleoside hydrolase (NH36) of Leishmania (L.) donovani is a vital enzyme which releases purines or pyrimidines of foreign DNA to be used in the synthesis of parasite DNA. As a bivalent DNA vaccine, the VR1012-NH36 was immunoprotective against visceral and cutaneous murine leishmaniasis. In this work we tested the immunotherapy against Leishmania (L.) chagasi infection, using two doses of 100 or 20 microg VR1012-NH36 vaccine (i.m. route), and, as a possible immunomodulator, aqueous garlic extract (8 mg/kg/day by the i.p. route), which was effective in immunotherapy of cutaneous murine leishmaniasis. Liver parasitic load was significantly reduced following treatment with 100 microg (91%) and 20 microg (77%) of the DNA vaccine, and by 20 microg DNA vaccine and garlic extract (76%) (p=0.023). Survival was 33% for saline controls, 100% for the 100 microg vaccine, and 83 and 67% for the 20 microg vaccine with and without garlic extract addition, respectively. Garlic treatment alone did not reduce parasite load (p>0.05), but increased survival (100%). The NH36-DNA vaccine was highly effective as a new tool for the therapy and control of visceral leishmaniasis, while the mild protective effect of garlic might be related to an unspecific enhancement of IFN-gamma secretion.

  2. The response to selection in Glycoside Hydrolase Family 13 structures: A comparative quantitative genetics approach.

    Directory of Open Access Journals (Sweden)

    Jose Sergio Hleap

    Full Text Available The Glycoside Hydrolase Family 13 (GH13 is both evolutionarily diverse and relevant to many industrial applications. Its members hydrolyze starch into smaller carbohydrates and members of the family have been bioengineered to improve catalytic function under industrial environments. We introduce a framework to analyze the response to selection of GH13 protein structures given some phylogenetic and simulated dynamic information. We find that the TIM-barrel (a conserved protein fold consisting of eight α-helices and eight parallel β-strands that alternate along the peptide backbone, common to all amylases is not selectable since it is under purifying selection. We also show a method to rank important residues with higher inferred response to selection. These residues can be altered to effect change in properties. In this work, we define fitness as inferred thermodynamic stability. We show that under the developed framework, residues 112Y, 122K, 124D, 125W, and 126P are good candidates to increase the stability of the truncated α-amylase protein from Geobacillus thermoleovorans (PDB code: 4E2O; α-1,4-glucan-4-glucanohydrolase; EC 3.2.1.1. Overall, this paper demonstrates the feasibility of a framework for the analysis of protein structures for any other fitness landscape.

  3. Regulation of synaptic structure by ubiquitin C-terminal hydrolase L1.

    Science.gov (United States)

    Cartier, Anna E; Djakovic, Stevan N; Salehi, Afshin; Wilson, Scott M; Masliah, Eliezer; Patrick, Gentry N

    2009-06-17

    Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a deubiquitinating enzyme that is selectively and abundantly expressed in the brain, and its activity is required for normal synaptic function. Here, we show that UCH-L1 functions in maintaining normal synaptic structure in hippocampal neurons. We found that UCH-L1 activity is rapidly upregulated by NMDA receptor activation, which leads to an increase in the levels of free monomeric ubiquitin. Conversely, pharmacological inhibition of UCH-L1 significantly reduces monomeric ubiquitin levels and causes dramatic alterations in synaptic protein distribution and spine morphology. Inhibition of UCH-L1 activity increases spine size while decreasing spine density. Furthermore, there is a concomitant increase in the size of presynaptic and postsynaptic protein clusters. Interestingly, however, ectopic expression of ubiquitin restores normal synaptic structure in UCH-L1-inhibited neurons. These findings point to a significant role of UCH-L1 in synaptic remodeling, most likely by modulating free monomeric ubiquitin levels in an activity-dependent manner.

  4. Effects of Cu, Zn and Pb Combined Pollution on Soil Hydrolase Activities

    Directory of Open Access Journals (Sweden)

    FENG Dan

    2015-08-01

    Full Text Available To study the relations between soil enzyme activities and heavy metal pollution, the combined effects of Cu, Zn and Pb on the three hydrolase activities, including invertase(IN, urease(Uand alkaline phosphatase(ALPwere investigated via an orthogonal experiment. Results showed as the following: When the concentration of Cu was 400 mg·kg-1, the U and ALP activities were decreased 51% and 44%, separately; When Zn was at 500 mg·kg-1, IN and ALP activities were only decreased 3% and 9%, while U activity was increased; When Pb was at 500 mg·kg-1, IN and U activities were increased, while ALP activity was decreased 13%. As a whole, Cu was considered as the most remarkable influence factor for IN, U and ALP activity regardless of interactions among the heavy metals, Zn came second, and Pb mainly showed activation. Considering interactions, Cu×Zn could significantly influence U activity(P<0.05, effects of Cu×Pb and Cu×Zn on ALP activity were remarkable(95% confidence interval. The response of ALP activity was more sensitive than the other two enzymes. Soil ALP activity might be a sensitive tool for assessing the pollution degree of Cu.

  5. Catalytic Characteristics of New Antibacterials Based on Hexahistidine-Containing Organophosphorus Hydrolase

    Directory of Open Access Journals (Sweden)

    Olga Maslova

    2017-09-01

    Full Text Available Catalytic characteristics of hexahistidine-containing organophosphorus hydrolase (His6-OPH and its enzyme-polyelectrolyte complexes with poly-l-glutamic acid or poly-l-aspartic acid (His6-OPH/PLD50, hydrolyzing organophosphorous compounds, and N-acyl homoserine lactones were studied in the presence of various antibiotics (ampicillin, gentamicin, kanamycin, and rifampicin. The antibiotics at concentrations below 1 g·L−1 had a negligible inhibiting effect on the His6-OPH activity. Mixed inhibition of His6-OPH was established for higher antibiotic concentrations, and rifampicin was the most potent inhibitor. Stabilization of the His6-OPH activity was observed in the presence of antibiotics at a concentration of 0.2 g·L−1 during exposure at 25–41 °C. Molecular docking of antibiotics to the surface of His6-OPH dimer revealed the antibiotics binding both to the area near active centers of the enzyme subunits and to the region of contact between subunits of the dimer. Such interactions between antibiotics and His6-OPH were verified with Fourier-transform infrared (FTIR spectroscopy. Considering all the results of the study, the combination of His6-OPH/PLD50 with β-lactam antibiotic ampicillin was established as the optimal one in terms of exhibition and persistence of maximal lactonase activity of the enzyme.

  6. Allelic variation of bile salt hydrolase genes in Lactobacillus salivarius does not determine bile resistance levels.

    LENUS (Irish Health Repository)

    Fang, Fang

    2009-09-01

    Commensal lactobacilli frequently produce bile salt hydrolase (Bsh) enzymes whose roles in intestinal survival are unclear. Twenty-six Lactobacillus salivarius strains from different sources all harbored a bsh1 allele on their respective megaplasmids. This allele was related to the plasmid-borne bsh1 gene of the probiotic strain UCC118. A second locus (bsh2) was found in the chromosomes of two strains that had higher bile resistance levels. Four Bsh1-encoding allele groups were identified, defined by truncations or deletions involving a conserved residue. In vitro analyses showed that this allelic variation was correlated with widely varying bile deconjugation phenotypes. Despite very low activity of the UCC118 Bsh1 enzyme, a mutant lacking this protein had significantly lower bile resistance, both in vitro and during intestinal transit in mice. However, the overall bile resistance phenotype of this and other strains was independent of the bsh1 allele type. Analysis of the L. salivarius transcriptome upon exposure to bile and cholate identified a multiplicity of stress response proteins and putative efflux proteins that appear to broadly compensate for, or mask, the effects of allelic variation of bsh genes. Bsh enzymes with different bile-degrading kinetics, though apparently not the primary determinants of bile resistance in L. salivarius, may have additional biological importance because of varying effects upon bile as a signaling molecule in the host.

  7. Glycoside Hydrolases from a targeted Compost Metagenome, activity-screening and functional characterization

    Directory of Open Access Journals (Sweden)

    Dougherty Michael J

    2012-07-01

    Full Text Available Abstract Background Metagenomics approaches provide access to environmental genetic diversity for biotechnology applications, enabling the discovery of new enzymes and pathways for numerous catalytic processes. Discovery of new glycoside hydrolases with improved biocatalytic properties for the efficient conversion of lignocellulosic material to biofuels is a critical challenge in the development of economically viable routes from biomass to fuels and chemicals. Results Twenty-two putative ORFs (open reading frames were identified from a switchgrass-adapted compost community based on sequence homology to related gene families. These ORFs were expressed in E. coli and assayed for predicted activities. Seven of the ORFs were demonstrated to encode active enzymes, encompassing five classes of hemicellulases. Four enzymes were over expressed in vivo, purified to homogeneity and subjected to detailed biochemical characterization. Their pH optima ranged between 5.5 - 7.5 and they exhibit moderate thermostability up to ~60-70°C. Conclusions Seven active enzymes were identified from this set of ORFs comprising five different hemicellulose activities. These enzymes have been shown to have useful properties, such as moderate thermal stability and broad pH optima, and may serve as the starting points for future protein engineering towards the goal of developing efficient enzyme cocktails for biomass degradation under diverse process conditions.

  8. Analysis of Domain Architecture and Phylogenetics of Family 2 Glycoside Hydrolases (GH2.

    Directory of Open Access Journals (Sweden)

    David Talens-Perales

    Full Text Available In this work we report a detailed analysis of the topology and phylogenetics of family 2 glycoside hydrolases (GH2. We distinguish five topologies or domain architectures based on the presence and distribution of protein domains defined in Pfam and Interpro databases. All of them share a central TIM barrel (catalytic module with two β-sandwich domains (non-catalytic at the N-terminal end, but differ in the occurrence and nature of additional non-catalytic modules at the C-terminal region. Phylogenetic analysis was based on the sequence of the Pfam Glyco_hydro_2_C catalytic module present in most GH2 proteins. Our results led us to propose a model in which evolutionary diversity of GH2 enzymes is driven by the addition of different non-catalytic domains at the C-terminal region. This model accounts for the divergence of β-galactosidases from β-glucuronidases, the diversification of β-galactosidases with different transglycosylation specificities, and the emergence of bicistronic β-galactosidases. This study also allows the identification of groups of functionally uncharacterized protein sequences with potential biotechnological interest.

  9. Pharmacological inhibition of soluble epoxide hydrolase or genetic deletion reduces diclofenac-induced gastric ulcers.

    Science.gov (United States)

    Goswami, Sumanta Kumar; Rand, Amelia Ann; Wan, Debin; Yang, Jun; Inceoglu, Bora; Thomas, Melany; Morisseau, Christophe; Yang, Guang-Yu; Hammock, Bruce D

    2017-07-01

    This research was conducted to evaluate the hypothesis that gastric ulcers caused by the NSAID diclofenac sodium (DCF) can be prevented by the soluble epoxide hydrolase inhibitor TPPU. Mice were administered a single dose of 10, 30 or 100mg/kg of DCF. Once an ulcerative dose of DCF was chosen, mice were pretreated with TPPU for 7days at 0.1mg/kg to evaluate anti-ulcer effects of the sEH inhibitor on anatomy, histopathology, pH, inflammatory markers and epithelial apoptosis of stomachs. Diclofenac caused ulceration of the stomach at a dose of 100mg/kg and a time post dose of 6h. Ulcers generated under these conditions were associated with a significant increase in the levels of TNF-α and IL-6 in serum and increased apoptosis compared to control mice. Pretreatment with TPPU resulted in a decrease of ulceration in mice treated with DCF with a significant decrease in the level of apoptosis, TNF-α and IL-6 in the serum in comparison to diclofenac-treated mice. TPPU did not affect the pH of the stomach, whereas omeprazole elevated the pH of the stomach as expected. A similar anti-ulcer effect was observed in sEH gene knockout mice treated with DCF. The sEH inhibitor TPPU decreases the NSAID-induced stomach ulcers. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Intensification of Organophosphorus Hydrolase Synthesis by Using Substances with Gas-Transport Function

    Directory of Open Access Journals (Sweden)

    Olga Senko

    2017-12-01

    Full Text Available We have performed studies and comparative analysis of the biosynthesis characteristics of intracellular recombinant enzyme, such as hexahistidine-containing organophosphorus hydrolase (His6-OPH in Escherichia coli SG13009[pREP4] cells when various perfluorocarbon compounds (PFC were introduced into the medium for cell cultivation. The PFC were found to facilitate the biosynthesis of His6-OPH: increased levels of the total OPH-activity (up to 37% were measured upon introduction of 1,1,1,2,2,3,3,4,4,5,5,6,6,6-tetradecafluorohexane (PFH and 4,7,10,13,16,19,22,25,28,31-decaoxaperfluoro-5,8,11,14,17,18,21,24,27,30-decamethyl tetratriacontane (Polyether II into culture medium. We have demonstrated the possibility of effective and multiple (at least five-fold use of PFH for biosynthesis of intracellular recombinant protein His6-OPH, which catalyzes the hydrolysis of organophosphorus pesticides (OP, is widely used in agriculture and can be applied as new antidote for OP-detoxification in vivo. The multiple use of PFH was achieved through recycling of this substance: sediment of Escherichia coli SG13009[pREP4] cell biomass was collected at the end of each culture growing step and disintegrated with ultrasound, and obtained residue containing almost all of the initially introduced PFC was then added to the medium at the start of the following culture growing step.

  11. Soluble epoxide hydrolase inhibitors of indolinone alkaloids and phenolic derivatives from Cimicifuga dahurica (Turcz.) Maxim.

    Science.gov (United States)

    Thao, Nguyen Phuong; Luyen, Bui Thi Thuy; Lee, Ji Sun; Kim, Jang Hoon; Kim, Young Ho

    2017-04-15

    The aim of this study was to search for potential therapeutic agents by identifying novel inhibitors of soluble epoxide hydrolase (sEH) from natural plants using an in silico approach. We found that an ethanolic extract from the roots of Cimicifuga dahurica (Turcz.) Maxim. significantly inhibited sEH in vitro. In a phytochemical investigation using assay-guided fractionation of the dichloromethane extract of C. dahurica, we isolated two new indolinone alkaloids (5 and 6) and five related constituents (1-4, and 7) and established their structures based on an extensive analysis using 1D and 2D NMR, and MS methods. All of the isolated compounds inhibited sEH enzymatic activity in a dose-dependent manner, with IC 50 values ranging from 0.8±0.0 to 2.8±0.4μM. A kinetic analysis of compounds 1-7 revealed that compound 2 was non-competitive; 1, 3, and 7 were mixed-type; and 4-6 were competitive inhibitors. Molecular docking was employed to further elucidate their receptor-ligand binding characteristics. These results demonstrated that compounds from C. dahurica are potential sEH inhibitors. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Identification of Mur, an atypical peptidoglycan hydrolase derived from Leuconostoc citreum.

    Science.gov (United States)

    Cibik, R; Tailliez, P; Langella, P; Chapot-Chartier, M P

    2001-02-01

    A gene encoding a protein homologous to known bacterial N-acetyl-muramidases has been cloned from Leuconostoc citreum by a PCR-based approach. The encoded protein, Mur, consists of 209 amino acid residues with a calculated molecular mass of 23,821 Da including a 31-amino-acid putative signal peptide. In contrast to most of the other known peptidoglycan hydrolases, L. citreum Mur protein does not contain amino acid repeats involved in cell wall binding. The purified L. citreum Mur protein was shown to exhibit peptidoglycan-hydrolyzing activity by renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An active chimeric protein was constructed by fusion of L. citreum Mur to the C-terminal repeat-containing domain (cA) of AcmA, the major autolysin of Lactococcus lactis. Expression of the Mur-cA fusion protein was able to complement an acmA mutation in L. lactis; normal cell separation after cell division was restored by Mur-cA expression.

  13. A Comprehensive Genome Survey Provides Novel Insights into Bile Salt Hydrolase (BSH in Lactobacillaceae

    Directory of Open Access Journals (Sweden)

    Lifeng Liang

    2018-05-01

    Full Text Available Bile salt hydrolase (BSH is a well-known enzyme that has been commonly characterized in probiotic bacteria, as it has cholesterol-lowering effects. However, its molecular investigations are scarce. Here, we build a local database of BSH sequences from Lactobacillaceae (BSH–SDL, and phylogenetic analysis and homology searches were employed to elucidate their comparability and distinctiveness among species. Evolutionary study demonstrates that BSH sequences in BSH–SDL are divided into five groups, named BSH A, B, C, D and E here, which can be the genetic basis for BSH classification and nomenclature. Sequence analysis suggests the differences between BSH-active and BSH-inactive proteins clearly, especially on site 82. In addition, a total of 551 BSHs from 107 species are identified from 451 genomes of 158 Lactobacillaceae species. Interestingly, those bacteria carrying various copies of BSH A or B can be predicted to be potential cholesterol-lowering probiotics, based on the results of phylogenetic analysis and the subtypes that those previously reported BSH-active probiotics possess. In summary, this study elaborates the molecular basis of BSH in Lactobacillaceae systematically, and provides a novel methodology as well as a consistent standard for the identification of the BSH subtype. We believe that high-throughput screening can be efficiently applied to the selection of promising candidate BSH-active probiotics, which will advance the development of healthcare products in cholesterol metabolism.

  14. Inhibition of fatty acid amide hydrolase by kaempferol and related naturally occurring flavonoids

    Science.gov (United States)

    Thors, L; Belghiti, M; Fowler, C J

    2008-01-01

    Background and purpose: Recent studies have demonstrated that the naturally occurring isoflavone compounds genistein and daidzein inhibit the hydrolysis of anandamide by fatty acid amide hydrolase (FAAH) in the low micromolar concentration range. The purpose of the present study was to determine whether this property is shared by flavonoids. Experimental approach: The hydrolysis of anandamide in homogenates and intact cells was measured using the substrate labelled in the ethanolamine part of the molecule. Key results: Twenty compounds were tested. Among the commonly occurring flavonoids, kaempferol was the most potent, inhibiting FAAH in a competitive manner with a Ki value of 5 μM. Among flavonoids with a more restricted distribution in nature, the two most active toward FAAH were 7-hydroxyflavone (IC50 value of 0.5–1 μM depending on the solvent used) and 3,7-dihydroxyflavone (IC50 value 2.2 μM). All three compounds reduced the FAAH-dependent uptake of anandamide and its metabolism by intact RBL2H3 basophilic leukaemia cells. Conclusions and implications: Inhibition of FAAH is an additional in vitro biochemical property of flavonoids. Kaempferol, 7-hydroxyflavone and 3,7-dihydroxyflavone may be useful as templates for the synthesis of novel compounds, which target several systems that are involved in the control of inflammation and cancer. PMID:18552875

  15. Halotolerant bacteria in the São Paulo Zoo composting process and their hydrolases and bioproducts

    Science.gov (United States)

    Oliveira, Lilian C.G.; Ramos, Patricia Locosque; Marem, Alyne; Kondo, Marcia Y.; Rocha, Rafael C.S.; Bertolini, Thiago; Silveira, Marghuel A.V.; da Cruz, João Batista; de Vasconcellos, Suzan Pantaroto; Juliano, Luiz; Okamoto, Debora N.

    2015-01-01

    Halophilic microorganisms are able to grow in the presence of salt and are also excellent source of enzymes and biotechnological products, such as exopolysaccharides (EPSs) and polyhydroxyalkanoates (PHAs). Salt-tolerant bacteria were screened in the Organic Composting Production Unit (OCPU) of São Paulo Zoological Park Foundation, which processes 4 ton/day of organic residues including plant matter from the Atlantic Rain Forest, animal manure and carcasses and mud from water treatment. Among the screened microorganisms, eight halotolerant bacteria grew at NaCl concentrations up to 4 M. These cultures were classified based on phylogenetic characteristics and comparative partial 16S rRNA gene sequence analysis as belonging to the genera Staphylococcus, Bacillus and Brevibacterium. The results of this study describe the ability of these halotolerant bacteria to produce some classes of hydrolases, namely, lipases, proteases, amylases and cellulases, and biopolymers. The strain characterized as of Brevibacterium avium presented cellulase and amylase activities up to 4 M NaCl and also produced EPSs and PHAs. These results indicate the biotechnological potential of certain microorganisms recovered from the composting process, including halotolerant species, which have the ability to produce enzymes and biopolymers, offering new perspectives for environmental and industrial applications. PMID:26273248

  16. Identification and characterisation of a novel acylpeptide hydrolase from Sulfolobus solfataricus: structural and functional insights.

    Directory of Open Access Journals (Sweden)

    Marta Gogliettino

    Full Text Available A novel acylpeptide hydrolase, named APEH-3(Ss, was isolated from the hypertermophilic archaeon Sulfolobus solfataricus. APEH is a member of the prolyl oligopeptidase family which catalyzes the removal of acetylated amino acid residues from the N terminus of oligopeptides. The purified enzyme shows a homotrimeric structure, unique among the associate partners of the APEH cluster and, in contrast to the archaeal APEHs which show both exo/endo peptidase activities, it appears to be a "true" aminopeptidase as exemplified by its mammalian counterparts, with which it shares a similar substrate specificity. Furthermore, a comparative study on the regulation of apeh gene expression, revealed a significant but divergent alteration in the expression pattern of apeh-3(Ss and apeh(Ss (the gene encoding the previously identified APEH(Ss from S. solfataricus, which is induced in response to various stressful growth conditions. Hence, both APEH enzymes can be defined as stress-regulated proteins which play a complementary role in enabling the survival of S. solfataricus cells under different conditions. These results provide new structural and functional insights into S. solfataricus APEH, offering a possible explanation for the multiplicity of this enzyme in Archaea.

  17. Genetic Deletion of Soluble Epoxide Hydrolase Attenuates Inflammation and Fibrosis in Experimental Obstructive Nephropathy

    Directory of Open Access Journals (Sweden)

    Chin-Wei Chiang

    2015-01-01

    Full Text Available Soluble epoxide hydrolase (sEH is abundantly expressed in kidney and plays a potent role in regulating inflammatory response in inflammatory diseases. However, the role of sEH in progression of chronic kidney diseases such as obstructive nephropathy is still elusive. In current study, wild-type (WT and sEH deficient (sEH−/− mice were subjected to the unilateral ureteral obstruction (UUO surgery and the kidney injury was evaluated by histological examination, western blotting, and ELISA. The protein level of sEH in kidney was increased in UUO-treated mice group compared to nonobstructed group. Additionally, UUO-induced hydronephrosis, renal tubular injury, inflammation, and fibrosis were ameliorated in sEH−/− mice with the exception of glomerulosclerosis. Moreover, sEH−/− mice with UUO showed lower levels of inflammation-related and fibrosis-related protein such as monocyte chemoattractant protein-1, macrophage inflammatory protein-2, interleukin-1β (IL-1β, IL-6, inducible nitric oxide synthase, collagen 1A1, and α-actin. The levels of superoxide anion radical and hydrogen peroxide as well as NADPH oxidase activity were also decreased in UUO kidneys of sEH−/− mice compared to that observed in WT mice. Collectively, our findings suggest that sEH plays an important role in the pathogenesis of experimental obstructive nephropathy and may be a therapeutic target for the treatment of obstructive nephropathy-related diseases.

  18. Mode of action of xylogalacturonan hydrolase towards xylogalacturonan and xylogalacturonan oligosaccharides

    Science.gov (United States)

    2004-01-01

    XGH (xylogalacturonan hydrolase; GH 28) is an enzyme that is capable of degrading XGA (xylogalacturonan), which is a polymer of α-D-galacturonic acid, highly substituted with β-D-xylose. XGA is present in cell walls of various plants and exudates, such as gum tragacanth. XGA oligosaccharides were derived from an XGH digestion of gum tragacanth, then fractionated, and analysed for their sugar composition and structure by matrix-assisted laser-desorption ionization–time-of-flight MS and nanospray MS. Several oligosaccharides from XGA were identified with different galacturonic acid/xylose ratios including five oligosaccharide isomers. Although XGH can act as an endo-enzyme, product-progression profiling showed that the disaccharide GalAXyl was predominantly produced from XGA by XGH, which indicated also an exolytic action. The latter was further supported by degradation studies of purified oligosaccharide GalA4Xyl3. It was shown that XGH acted from the non-reducing end towards the reducing end of this oligosaccharide, and showed the processive character of XGH. The results from this study further show that although XGH prefers to act between two xylosidated GalA units, it tolerates unsubstituted GalA units in its −1 and +1 subsites. PMID:15560751

  19. Expression pattern of glycoside hydrolase genes in Lutzomyia longipalpis reveals key enzymes involved in larval digestion

    Directory of Open Access Journals (Sweden)

    Caroline da Silva Moraes

    2014-08-01

    Full Text Available The sand fly Lutzomyia longipalpis is the most important vector of American Visceral Leishmaniasis. Adults are phytophagous (males and females or blood feeders (females only, and larvae feed on solid detritus. Digestion in sand fly larvae has scarcely been studied, but some glycosidase activities putatively involved in microorganism digestion were already described. Nevertheless, the molecular nature of these enzymes, as the corresponding genes and transcripts, were not explored yet. Catabolism of microbial carbohydrates in insects generally involves β-1,3-glucanases, chitinases and digestive lysozymes. In this work, the transcripts of digestive β-1,3-glucanase and chitinases were identified in the L. longipalpis larvae throughout analysis of sequences and expression patterns of glycoside hydrolases families 16, 18 and 22. The activity of one i-type lysozyme was also registered. Interestingly, this lysozyme seems to play a role in immunity, rather than digestion. This is the first attempt to identify the molecular nature of sand fly larval digestive enzymes.

  20. Using directed evolution to probe the substrate specificity of mandelamide hydrolase.

    Science.gov (United States)

    Wang, Pan-Fen; Yep, Alejandra; Kenyon, George L; McLeish, Michael J

    2009-02-01

    Mandelamide hydrolase (MAH), a member of the amidase signature family, catalyzes the hydrolysis of mandelamide to mandelate and ammonia. X-ray structures of several members of this family, but not that of MAH, have been reported. These reveal nearly superimposable conformations of the unusual Ser-cisSer-Lys catalytic triad. Conversely, the residues involved in substrate recognition are not conserved, implying that the binding pocket could be modified to change the substrate specificity, perhaps by directed evolution. Here we show that MAH is able to hydrolyze small aliphatic substrates such as lactamide, albeit with low efficiency. A selection method to monitor changes in mandelamide/lactamide preference was developed and used to identify several mutations affecting substrate binding. A homology model places some of these mutations close to the catalytic triad, presumably in the MAH active site. In particular, Gly202 appears to control the preference for aromatic substrates as the G202A variant showed three orders of magnitude decrease in k(cat)/K(m) for (R)- and (S)-mandelamide. This reduction in activity increased to six orders of magnitude for the G202V variant.

  1. Meta-analysis of microsomal epoxide hydrolase gene polymorphism and risk of hepatocellular carcinoma.

    Science.gov (United States)

    Zhong, Jian-Hong; Xiang, Bang-De; Ma, Liang; You, Xue-Mei; Li, Le-Qun; Xie, Gui-Sheng

    2013-01-01

    Hepatocarcinogenesis is a complex process that may be influenced by many factors, including polymorphism in microsomal epoxide hydrolase (mEH). Previous work suggests an association between the Tyr113His and His139Arg mEH polymorphisms and susceptibility to hepatocellular carcinoma (HCC), but the results have been inconsistent. PubMed, EMBASE, Google Scholar and the Chinese National Knowledge Infrastructure databases were systematically searched to identify relevant studies. A meta-analysis was performed to examine the association between Tyr113His and His139Arg mEH polymorphism and susceptibility to HCC. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated. Eleven studies were included in the meta-analysis, involving 1,696 HCC cases and 3,600 controls. The 113His- mEH allele was significantly associated with increased risk of HCC based on allelic contrast (OR = 1.35, 95% CI = 1.04-1.75, p = 0.02), homozygote comparison (OR = 1.65, 95% CI = 1.07-2.54, p = 0.02) and a recessive genetic model (OR = 1.54, 95% CI = 1.21-1.96, penvironment to modulate risk of HCC. Further large and well-designed studies are needed to confirm these conclusions.

  2. Expression pattern of glycoside hydrolase genes in Lutzomyia longipalpis reveals key enzymes involved in larval digestion

    Science.gov (United States)

    Moraes, Caroline da Silva; Diaz-Albiter, Hector M.; Faria, Maiara do Valle; Sant'Anna, Maurício R. V.; Dillon, Rod J.; Genta, Fernando A.

    2014-01-01

    The sand fly Lutzomyia longipalpis is the most important vector of American Visceral Leishmaniasis. Adults are phytophagous (males and females) or blood feeders (females only), and larvae feed on solid detritus. Digestion in sand fly larvae has scarcely been studied, but some glycosidase activities putatively involved in microorganism digestion were already described. Nevertheless, the molecular nature of these enzymes, as the corresponding genes and transcripts, were not explored yet. Catabolism of microbial carbohydrates in insects generally involves β-1,3-glucanases, chitinases, and digestive lysozymes. In this work, the transcripts of digestive β-1,3-glucanase and chitinases were identified in the L. longipalpis larvae throughout analysis of sequences and expression patterns of glycoside hydrolases families 16, 18, and 22. The activity of one i-type lysozyme was also registered. Interestingly, this lysozyme seems to play a role in immunity, rather than digestion. This is the first attempt to identify the molecular nature of sand fly larval digestive enzymes. PMID:25140153

  3. Molecular characterization of a family 5 glycoside hydrolase suggests an induced-fit enzymatic mechanism

    Science.gov (United States)

    Liberato, Marcelo V.; Silveira, Rodrigo L.; Prates, Érica T.; de Araujo, Evandro A.; Pellegrini, Vanessa O. A.; Camilo, Cesar M.; Kadowaki, Marco A.; Neto, Mario De O.; Popov, Alexander; Skaf, Munir S.; Polikarpov, Igor

    2016-04-01

    Glycoside hydrolases (GHs) play fundamental roles in the decomposition of lignocellulosic biomaterials. Here, we report the full-length structure of a cellulase from Bacillus licheniformis (BlCel5B), a member of the GH5 subfamily 4 that is entirely dependent on its two ancillary modules (Ig-like module and CBM46) for catalytic activity. Using X-ray crystallography, small-angle X-ray scattering and molecular dynamics simulations, we propose that the C-terminal CBM46 caps the distal N-terminal catalytic domain (CD) to establish a fully functional active site via a combination of large-scale multidomain conformational selection and induced-fit mechanisms. The Ig-like module is pivoting the packing and unpacking motions of CBM46 relative to CD in the assembly of the binding subsite. This is the first example of a multidomain GH relying on large amplitude motions of the CBM46 for assembly of the catalytically competent form of the enzyme.

  4. Diversity of bacteria and glycosyl hydrolase family 48 genes in cellulolytic consortia enriched from thermophilic biocompost.

    Science.gov (United States)

    Izquierdo, Javier A; Sizova, Maria V; Lynd, Lee R

    2010-06-01

    The enrichment from nature of novel microbial communities with high cellulolytic activity is useful in the identification of novel organisms and novel functions that enhance the fundamental understanding of microbial cellulose degradation. In this work we identify predominant organisms in three cellulolytic enrichment cultures with thermophilic compost as an inoculum. Community structure based on 16S rRNA gene clone libraries featured extensive representation of clostridia from cluster III, with minor representation of clostridial clusters I and XIV and a novel Lutispora species cluster. Our studies reveal different levels of 16S rRNA gene diversity, ranging from 3 to 18 operational taxonomic units (OTUs), as well as variability in community membership across the three enrichment cultures. By comparison, glycosyl hydrolase family 48 (GHF48) diversity analyses revealed a narrower breadth of novel clostridial genes associated with cultured and uncultured cellulose degraders. The novel GHF48 genes identified in this study were related to the novel clostridia Clostridium straminisolvens and Clostridium clariflavum, with one cluster sharing as little as 73% sequence similarity with the closest known relative. In all, 14 new GHF48 gene sequences were added to the known diversity of 35 genes from cultured species.

  5. The Structural Basis of Exopolygalacturonase Activity in a Family 28 Glycoside Hydrolase

    Energy Technology Data Exchange (ETDEWEB)

    Abbott,D.; Boraston, A.

    2007-01-01

    Family 28 glycoside hydrolases (polygalacturonases) are found in organisms across the plant, fungal and bacterial kingdoms, where they are central to diverse biological functions such as fruit ripening, biomass recycling and plant pathogenesis. The structures of several polygalacturonases have been reported; however, all of these enzymes utilize an endo-mode of digestion, which generates a spectrum of oligosaccharide products with varying degrees of polymerization. The structure of a complementary exo-acting polygalacturonase and an accompanying explanation of the molecular determinants for its specialized activity have been noticeably lacking. We present the structure of an exopolygalacturonase from Yersinia enterocolitica, YeGH28 in a native form (solved to 2.19 {angstrom} resolution) and a digalacturonic acid product complex (solved to 2.10 {angstrom} resolution). The activity of YeGH28 is due to inserted stretches of amino acid residues that transform the active site from the open-ended channel observed in the endopolygalacturonases to a closed pocket that restricts the enzyme to the exclusive attack of the non-reducing end of oligogalacturonide substrates. In addition, YeGH28 possesses a fused FN3 domain with unknown function, the first such structure described in pectin active enzymes.

  6. S-Inosyl-L-Homocysteine Hydrolase, a Novel Enzyme Involved in S-Adenosyl-L-Methionine Recycling.

    Science.gov (United States)

    Miller, Danielle; Xu, Huimin; White, Robert H

    2015-07-01

    S-Adenosyl-L-homocysteine, the product of S-adenosyl-L-methionine (SAM) methyltransferases, is known to be a strong feedback inhibitor of these enzymes. A hydrolase specific for S-adenosyl-L-homocysteine produces L-homocysteine, which is remethylated to methionine and can be used to regenerate SAM. Here, we show that the annotated S-adenosyl-L-homocysteine hydrolase in Methanocaldococcus jannaschii is specific for the hydrolysis and synthesis of S-inosyl-L-homocysteine, not S-adenosyl-L-homocysteine. This is the first report of an enzyme specific for S-inosyl-L-homocysteine. As with S-adenosyl-L-homocysteine hydrolase, which shares greater than 45% sequence identity with the M. jannaschii homologue, the M. jannaschii enzyme was found to copurify with bound NAD(+) and has Km values of 0.64 ± 0.4 mM, 0.0054 ± 0.006 mM, and 0.22 ± 0.11 mM for inosine, L-homocysteine, and S-inosyl-L-homocysteine, respectively. No enzymatic activity was detected with S-adenosyl-L-homocysteine as the substrate in either the synthesis or hydrolysis direction. These results prompted us to redesignate the M. jannaschii enzyme an S-inosyl-L-homocysteine hydrolase (SIHH). Identification of SIHH demonstrates a modified pathway in this methanogen for the regeneration of SAM from S-adenosyl-L-homocysteine that uses the deamination of S-adenosyl-L-homocysteine to form S-inosyl-L-homocysteine. In strictly anaerobic methanogenic archaea, such as Methanocaldococcus jannaschii, canonical metabolic pathways are often not present, and instead, unique pathways that are deeply rooted on the phylogenetic tree are utilized by the organisms. Here, we discuss the recycling pathway for S-adenosyl-L-homocysteine, produced from S-adenosyl-L-methionine (SAM)-dependent methylation reactions, which uses a hydrolase specific for S-inosyl-L-homocysteine, an uncommon metabolite. Identification of the pathways and the enzymes involved in the unique pathways in the methanogens will provide insight into the

  7. Phenotypic assessment of THC discriminative stimulus properties in fatty acid amide hydrolase knockout and wildtype mice.

    Science.gov (United States)

    Walentiny, D Matthew; Vann, Robert E; Wiley, Jenny L

    2015-06-01

    A number of studies have examined the ability of the endogenous cannabinoid anandamide to elicit Δ(9)-tetrahydrocannabinol (THC)-like subjective effects, as modeled through the THC discrimination paradigm. In the present study, we compared transgenic mice lacking fatty acid amide hydrolase (FAAH), the enzyme primarily responsible for anandamide catabolism, to wildtype counterparts in a THC discrimination procedure. THC (5.6 mg/kg) served as a discriminative stimulus in both genotypes, with similar THC dose-response curves between groups. Anandamide fully substituted for THC in FAAH knockout, but not wildtype, mice. Conversely, the metabolically stable anandamide analog O-1812 fully substituted in both groups, but was more potent in knockouts. The CB1 receptor antagonist rimonabant dose-dependently attenuated THC generalization in both groups and anandamide substitution in FAAH knockouts. Pharmacological inhibition of monoacylglycerol lipase (MAGL), the primary catabolic enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG), with JZL184 resulted in full substitution for THC in FAAH knockout mice and nearly full substitution in wildtypes. Quantification of brain endocannabinoid levels revealed expected elevations in anandamide in FAAH knockout mice compared to wildtypes and equipotent dose-dependent elevations in 2-AG following JZL184 administration. Dual inhibition of FAAH and MAGL with JZL195 resulted in roughly equipotent increases in THC-appropriate responding in both groups. While the notable similarity in THC's discriminative stimulus effects across genotype suggests that the increased baseline brain anandamide levels (as seen in FAAH knockout mice) do not alter THC's subjective effects, FAAH knockout mice are more sensitive to the THC-like effects of pharmacologically induced increases in anandamide and MAGL inhibition (e.g., JZL184). Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Engineering and introduction of de novo disulphide bridges in organophosphorus hydrolase enzyme for thermostability improvement.

    Science.gov (United States)

    Farnoosh, Gholamreza; Khajeh, Khosro; Latifi, Ali Mohammad; Aghamollaei, Hossein

    2016-12-01

    The organophosphorus hydrolase (OPH) has been used to degrade organophosphorus chemicals, as one of the most frequently used decontamination methods. Under chemical and thermal denaturing conditions, the enzyme has been shown to unfold. To utilize this enzyme in various applications, the thermal stability is of importance. The engineering of de novo disulphide bridges has been explored as a means to increase the thermal stability of enzymes in the rational method of protein engineering. In this study, Disulphide by Design software, homology modelling and molecular dynamics simulations were used to select appropriate amino acid pairs for the introduction of disulphide bridge to improve protein thermostability. The thermostability of the wild-type and three selected mutant enzymes were evaluated by half-life, delta G inactivation (ΔGi) and structural studies (fluorescence and far-UV CD analysis). Data analysis showed that half-life of A204C/T234C and T128C/E153C mutants were increased up to 4 and 24 min, respectively; however, for the G74C/A78C mutant, the half-life was decreased up to 9 min. For the T128C/E124C mutant, both thermal stability and Catalytic efficiency (kcat) were also increased. The half-life and ΔGi results were correlated to the obtained information from structural studies by circular dichroism (CD) spectrometry and extrinsic fluorescence experiments; as rigidity increased in A204C/T2234C and T128C/E153C mutants, half-life and ΔGi also increased. For G74C/A78C mutant, these parameters decreased due to its higher flexibility. The results were submitted a strong evidence for the possibility to improve the thermostability of OPH enzyme by introducing a disulphide bridge after bioinformatics design, even though this design would not be always successful.

  9. Identification of oxidized protein hydrolase as a potential prodrug target in prostate cancer

    International Nuclear Information System (INIS)

    McGoldrick, Christopher A; Jiang, Yu-Lin; Paromov, Victor; Brannon, Marianne; Krishnan, Koyamangalath; Stone, William L

    2014-01-01

    Esterases are often overexpressed in cancer cells and can have chiral specificities different from that of the corresponding normal tissues. For this reason, ester prodrugs could be a promising approach in chemotherapy. In this study, we focused on the identification and characterization of differentially expressed esterases between non-tumorigenic and tumorigenic prostate epithelial cells. Cellular lysates from LNCaP, DU 145, and PC3 prostate cancer cell lines, tumorigenic RWPE-2 prostate epithelial cells, and non-tumorigenic RWPE-1 prostate epithelial cells were separated by native polyacrylamide gel electrophoresis (n-PAGE) and the esterase activity bands visualized using α-naphthyl acetate or α-naphthyl-N-acetylalaninate (ANAA) chiral esters and Fast Blue RR salt. The esterases were identified using nanospray LC/MS-MS tandem mass spectrometry and confirmed by Western blotting, native electroblotting, inhibition assays, and activity towards a known specific substrate. The serine protease/esterase oxidized protein hydrolase (OPH) was overexpressed in COS-7 cells to verify our results. The major esterase observed with the ANAA substrates within the n-PAGE activity bands was identified as OPH. OPH (EC 3.4.19.1) is a serine protease/esterase and a member of the prolyl oligopeptidase family. We found that LNCaP lysates contained approximately 40% more OPH compared to RWPE-1 lysates. RWPE-2, DU145 and PC3 cell lysates had similar levels of OPH activity. OPH within all of the cell lysates tested had a chiral preference for the S-isomer of ANAA. LNCaP cells were stained more intensely with ANAA substrates than RWPE-1 cells and COS-7 cells overexpressing OPH were found to have a higher activity towards the ANAA and AcApNA than parent COS-7 cells. These data suggest that prodrug derivatives of ANAA and AcApNA could have potential as chemotherapeutic agents for the treatment of prostate cancer tumors that overexpress OPH

  10. Fatty acid amide hydrolase inhibition heightens anandamide signaling without producing reinforcing effects in primates

    Science.gov (United States)

    Justinova, Zuzana; Mangieri, Regina A.; Bortolato, Marco; Chefer, Svetlana I.; Mukhin, Alexey G.; Clapper, Jason R.; King, Alvin R.; Redhi, Godfrey H.; Yasar, Sevil; Piomelli, Daniele; Goldberg, Steven R.

    2008-01-01

    Background CB1 cannabinoid receptors in the brain are known to participate in the regulation of reward-based behaviors, however, the contribution of each of the endocannabinoid transmitters, anandamide and 2-arachidonoylglycerol (2-AG), to these behaviors remains undefined. To address this question, we assessed the effects of URB597, a selective anandamide deactivation inhibitor, as a reinforcer of drug-seeking and drug-taking behavior in squirrel monkeys. Methods We investigated the reinforcing effects of the fatty acid amide hydrolase (FAAH) inhibitor URB597 in monkeys trained to intravenously self-administer Δ9-tetrahydrocannabinol (THC), anandamide or cocaine, and quantified brain endocannabinoid levels using liquid chromatography/mass spectrometry. We measured brain FAAH activity using an ex vivo enzyme assay. Results URB597 (0.3 mg/kg, intravenous) blocked FAAH activity and increased anandamide levels throughout the monkey brain. This effect was accompanied by a marked compensatory decrease in 2-AG levels. Monkeys did not self-administer URB597 and the drug did not promote reinstatement of extinguished drug-seeking behavior previously maintained by THC, anandamide, or cocaine. Pretreatment with URB597 did not modify self-administration of THC or cocaine even though, as expected, it significantly potentiated anandamide self-administration. Conclusions In the monkey brain, the FAAH inhibitor URB597 increases anandamide levels while causing a compensatory down-regulation in 2-AG levels. These effects are accompanied by a striking lack of reinforcing properties, which distinguishes URB597 from direct-acting cannabinoid agonists such as THC. Our results reveal an unexpected functional heterogeneity within the endocannabinoid signaling system, and suggest that FAAH inhibitors might be used therapeutically without risk of abuse or triggering of relapse to drug abuse. PMID:18814866

  11. Members of Glycosyl-Hydrolase Family 17 of A. fumigatus Differentially Affect Morphogenesis

    Directory of Open Access Journals (Sweden)

    Nicolas Millet

    2018-01-01

    Full Text Available Cell wall biosynthesis and remodeling are essential for fungal growth and development. In the fungal pathogen Aspergillus fumigatus, the β(1,3glucan is the major cell wall polysaccharide. This polymer is synthesized at the plasma membrane by a transmembrane complex, then released into the parietal space to be remodeled by enzymes, and finally incorporated into the pre-existing cell wall. In the Glycosyl-Hydrolases family 17 (GH17 of A. fumigatus, two β(1,3glucanosyltransferases, Bgt1p and Bgt2p, have been previously characterized. Disruption of BGT1 and BGT2 did not result in a phenotype, but sequence comparison and hydrophobic cluster analysis showed that three other genes in A. fumigatus belong to the GH17 family, SCW4, SCW11, and BGT3. In constrast to Δbgt1bgt2 mutants, single and multiple deletion of SCW4, SCW11, and BGT3 showed a decrease in conidiation associated with a higher conidial mortality and an abnormal conidial shape. Moreover, mycelium was also affected with a slower growth, stronger sensitivity to cell wall disturbing agents, and altered cell wall composition. Finally, the synthetic interactions between Bgt1p, Bgt2p, and the three other members, which support a functional cooperation in cell-wall assembly, were analyzed. Our data suggest that Scw4p, Scw11p, and Bgt3p are essential for cell wall integrity and might have antagonistic and distinct functions to Bgt1p and Bgt2p.

  12. Members of Glycosyl-Hydrolase Family 17 of A. fumigatus Differentially Affect Morphogenesis

    Science.gov (United States)

    Millet, Nicolas; Latgé, Jean-Paul; Mouyna, Isabelle

    2018-01-01

    Cell wall biosynthesis and remodeling are essential for fungal growth and development. In the fungal pathogen Aspergillus fumigatus, the β(1,3)glucan is the major cell wall polysaccharide. This polymer is synthesized at the plasma membrane by a transmembrane complex, then released into the parietal space to be remodeled by enzymes, and finally incorporated into the pre-existing cell wall. In the Glycosyl-Hydrolases family 17 (GH17) of A. fumigatus, two β(1,3)glucanosyltransferases, Bgt1p and Bgt2p, have been previously characterized. Disruption of BGT1 and BGT2 did not result in a phenotype, but sequence comparison and hydrophobic cluster analysis showed that three other genes in A. fumigatus belong to the GH17 family, SCW4, SCW11, and BGT3. In constrast to Δbgt1bgt2 mutants, single and multiple deletion of SCW4, SCW11, and BGT3 showed a decrease in conidiation associated with a higher conidial mortality and an abnormal conidial shape. Moreover, mycelium was also affected with a slower growth, stronger sensitivity to cell wall disturbing agents, and altered cell wall composition. Finally, the synthetic interactions between Bgt1p, Bgt2p, and the three other members, which support a functional cooperation in cell-wall assembly, were analyzed. Our data suggest that Scw4p, Scw11p, and Bgt3p are essential for cell wall integrity and might have antagonistic and distinct functions to Bgt1p and Bgt2p. PMID:29385695

  13. S-Adenosyl-L-Homocysteine Hydrolase Inhibition by a Synthetic Nicotinamide Cofactor Biomimetic

    Directory of Open Access Journals (Sweden)

    Lyn L. Kailing

    2018-03-01

    Full Text Available S-adenosyl-L-homocysteine (SAH hydrolases (SAHases are involved in the regulation of methylation reactions in many organisms and are thus crucial for numerous cellular functions. Consequently, their dysregulation is associated with severe health problems. The SAHase-catalyzed reaction is reversible and both directions depend on the redox activity of nicotinamide adenine dinucleotide (NAD+ as a cofactor. Therefore, nicotinamide cofactor biomimetics (NCB are a promising tool to modulate SAHase activity. In the present in vitro study, we investigated 10 synthetic truncated NAD+ analogs against a SAHase from the root-nodulating bacterium Bradyrhizobium elkanii. Among this set of analogs, one was identified to inhibit the SAHase in both directions. Isothermal titration calorimetry (ITC and crystallography experiments suggest that the inhibitory effect is not mediated by a direct interaction with the protein. Neither the apo-enzyme (i.e., deprived of the natural cofactor, nor the holo-enzyme (i.e., in the NAD+-bound state were found to bind the inhibitor. Yet, enzyme kinetics point to a non-competitive inhibition mechanism, where the inhibitor acts on both, the enzyme and enzyme-SAH complex. Based on our experimental results, we hypothesize that the NCB inhibits the enzyme via oxidation of the enzyme-bound NADH, which may be accessible through an open molecular gate, leaving the enzyme stalled in a configuration with oxidized cofactor, where the reaction intermediate can be neither converted nor released. Since the reaction mechanism of SAHase is quite uncommon, this kind of inhibition could be a viable pharmacological route, with a low risk of off-target effects. The NCB presented in this work could be used as a template for the development of more potent SAHase inhibitors.

  14. Evaluation of the organophosphorus hydrolase enzyme activity in creams and investigation of its stability

    Directory of Open Access Journals (Sweden)

    Mariye Rajaie

    2016-06-01

    Full Text Available The main purpose of this project is investigation of the organophosphorus hydrolase (OPH enzyme activity in water in oil (w/o and oil in water (o/w creams and investigation of the OPH enzyme stability in formulated creams. OPH enzyme was extracted and purified from strain flavobacterium. The w/o and o/w creams were prepared using different formulations. In order to achieve an emulsion with maximum stability, appropriate percentage of the cream components was selected by studying different formulations and the physical and chemical stability of the produced cream were considered. 5Uenzyme/90gcream enzyme was used for each formulation. To measure the enzyme activity in creams, extraction method was used and enzyme activity was determined based on parathion hydrolysis. The thermal stability of OPH in both types of w/o and o/w creams was studied at 4 and 30  °C for various time periods. The average enzyme activity was about 0.0065 U/gcream and 0.018 U/gcream for w/o and o/w creams respectivly. According to the results, the relative activity at 4 °C was reduced to 50% after 26 and 45 days in w/o and o/w creams, respectivly. The results showed that the OPH enzyme activity in o/w cream was 2.6 times more than that of w/o cream, because of the higher hydrophobicity of o/w cream compared to w/o. The OPH enzyme stability in o/w cream was greater in comparison to w/o cream. The OPH enzyme was active for nearly 2 months on o/w creams at 4 °C .

  15. Construction of a rice glycoside hydrolase phylogenomic database and identification of targets for biofuel research

    Directory of Open Access Journals (Sweden)

    Rita eSharma

    2013-08-01

    Full Text Available Glycoside hydrolases (GH catalyze the hydrolysis of glycosidic bonds in cell wall polymers and can have major effects on cell wall architecture. Taking advantage of the massive datasets available in public databases, we have constructed a rice phylogenomic database of GHs (http://ricephylogenomics.ucdavis.edu/cellwalls/gh/. This database integrates multiple data types including the structural features, orthologous relationships, mutant availability and gene expression patterns for each GH family in a phylogenomic context. The rice genome encodes 437 GH genes classified into 34 families. Based on pairwise comparison with eight dicot and four monocot genomes, we identified 138 GH genes that are highly diverged between monocots and dicots, 57 of which have diverged further in rice as compared with four monocot genomes scanned in this study. Chromosomal localization and expression analysis suggest a role for both whole-genome and localized gene duplications in expansion and diversification of GH families in rice. We examined the meta-profiles of expression patterns of GH genes in twenty different anatomical tissues of rice. Transcripts of 51 genes exhibit tissue or developmental stage-preferential expression, whereas, seventeen other genes preferentially accumulate in actively growing tissues. When queried in RiceNet, a probabilistic functional gene network that facilitates functional gene predictions, nine out of seventeen genes form a regulatory network with the well-characterized genes involved in biosynthesis of cell wall polymers including cellulose synthase and cellulose synthase-like genes of rice. Two-thirds of the GH genes in rice are up regulated in response to biotic and abiotic stress treatments indicating a role in stress adaptation. Our analyses identify potential GH targets for cell wall modification.

  16. Identification of a dithiol-dependent nucleoside triphosphate hydrolase in Sarcocystis neurona.

    Science.gov (United States)

    Zhang, Deqing; Gaji, Rajshekhar Y; Howe, Daniel K

    2006-09-01

    A putative nucleoside triphosphate hydrolase (NTPase) gene was identified in a database of expressed sequence tags (ESTs) from the apicomplexan parasite Sarcocystis neurona. Analysis of culture-derived S. neurona merozoites demonstrated a dithiol-dependent NTPase activity, consistent with the presence of a homologue to the TgNTPases of Toxoplasma gondii. A complete cDNA was obtained for the S. neurona gene and the predicted amino acid sequence shared 38% identity with the two TgNTPase isoforms from T. gondii. Based on the obvious homology, the S. neurona protein was designated SnNTP1. The SnNTP1 cDNA encodes a polypeptide of 714 amino acids with a predicted 22-residue signal peptide and an estimated mature molecular mass of 70kDa. Southern blot analysis of the SnNTP1 locus revealed that the gene exists as a single copy in the S. neurona genome, unlike the multiple gene copies that have been observed in T. gondii and Neospora caninum. Analyses of the SnNTP1 protein demonstrated that it is soluble and secreted into the culture medium by extracellular merozoites. Surprisingly, indirect immunofluorescence analysis of intracellular S. neurona revealed apical localisation of SnNTP1 and temporal expression characteristics that are comparable with the microneme protein SnMIC10. The absence of SnNTP1 during much of endopolygeny implies that this protein does not serve a function during intracellular growth and development of S. neurona schizonts. Instead, SnNTP1 may play a role in events that occur during or proximal to merozoite egress from and/or invasion into cells.

  17. Insight into Dominant Cellulolytic Bacteria from Two Biogas Digesters and Their Glycoside Hydrolase Genes

    Science.gov (United States)

    Zhang, Jun; Zhang, Lei; Geng, Alei; Liu, Fanghua; Zhao, Guoping; Wang, Shengyue; Zhou, Zhihua; Yan, Xing

    2015-01-01

    Diverse cellulolytic bacteria are essential for maintaining high lignocellulose degradation ability in biogas digesters. However, little was known about functional genes and gene clusters of dominant cellulolytic bacteria in biogas digesters. This is the foundation to understand lignocellulose degradation mechanisms of biogas digesters and apply these gene resource for optimizing biofuel production. A combination of metagenomic and 16S rRNA gene clone library methods was used to investigate the dominant cellulolytic bacteria and their glycoside hydrolase (GH) genes in two biogas digesters. The 16S rRNA gene analysis revealed that the dominant cellulolytic bacteria were strains closely related to Clostridium straminisolvens and an uncultured cellulolytic bacterium designated BG-1. To recover GH genes from cellulolytic bacteria in general, and BG-1 in particular, a refined assembly approach developed in this study was used to assemble GH genes from metagenomic reads; 163 GH-containing contigs ≥ 1 kb in length were obtained. Six recovered GH5 genes that were expressed in E. coli demonstrated multiple lignocellulase activities and one had high mannanase activity (1255 U/mg). Eleven fosmid clones harboring the recovered GH-containing contigs were sequenced and assembled into 10 fosmid contigs. The composition of GH genes in the 163 assembled metagenomic contigs and 10 fosmid contigs indicated that diverse GHs and lignocellulose degradation mechanisms were present in the biogas digesters. In particular, a small portion of BG-1 genome information was recovered by PhyloPythiaS analysis. The lignocellulase gene clusters in BG-1 suggested that it might use a possible novel lignocellulose degradation mechanism to efficiently degrade lignocellulose. Dominant cellulolytic bacteria of biogas digester possess diverse GH genes, not only in sequences but also in their functions, which may be applied for production of biofuel in the future. PMID:26070087

  18. Computational redesign reveals allosteric mutation hotspots of organophosphate hydrolase that enhance organophosphate hydrolysis

    Energy Technology Data Exchange (ETDEWEB)

    Jacob, Reed B. [Univ. of North Carolina, Chapel Hill, NC (United States); Ding, Feng [Clemson Univ., SC (United States); Ye, Dongmei [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Ackerman, Eric [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Dokholyan, Nikolay V. [Univ. of North Carolina, Chapel Hill, NC (United States)

    2015-04-01

    Organophosphates are widely used for peaceful (agriculture) and military purposes (chemical warfare agents). The extraordinary toxicity of organophosphates and the risk of deployment, make it critical to develop means for their rapid and efficient deactivation. Organophosphate hydrolase (OPH) already plays an important role in organophosphate remediation, but is insufficient for therapeutic or prophylactic purposes primarily due to low substrate affinity. Current efforts focus on directly modifying the active site to differentiate substrate specificity and increase catalytic activity. Here, we present a novel strategy for enhancing the general catalytic efficiency of OPH through computational redesign of the residues that are allosterically coupled to the active site and validated our design by mutagenesis. Specifically, we identify five such hot-spot residues for allosteric regulation and assay these mutants for hydrolysis activity against paraoxon, a chemical-weapons simulant. A high percentage of the predicted mutants exhibit enhanced activity over wild-type (kcat =16.63 s-1), such as T199I/T54I (899.5 s-1) and C227V/T199I/T54I (848 s-1), while the Km remains relatively unchanged in our high-throughput cell-free expression system. Further computational studies of protein dynamics reveal four distinct distal regions coupled to the active site that display significant changes in conformation dynamics upon these identified mutations. These results validate a computational design method that is both efficient and easily adapted as a general procedure for enzymatic enhancement.

  19. Structural and biochemical characterization of novel bacterial α-galactosidases belonging to glycoside hydrolase family 31.

    Science.gov (United States)

    Miyazaki, Takatsugu; Ishizaki, Yuichi; Ichikawa, Megumi; Nishikawa, Atsushi; Tonozuka, Takashi

    2015-07-01

    Glycoside hydrolase family 31 (GH31) proteins have been reportedly identified as exo-α-glycosidases with activity for α-glucosides and α-xylosides. We focused on a GH31 subfamily, which contains proteins with low sequence identity (Pedobacter heparinus and Pedobacter saltans. The enzymes unexpectedly exhibited α-galactosidase activity, but were not active on α-glucosides and α-xylosides. The crystal structures of one of the enzymes, PsGal31A, in unliganded form and in complexes with D-galactose or L-fucose and the catalytic nucleophile mutant in unliganded form and in complex with p-nitrophenyl-α-D-galactopyranoside, were determined at 1.85-2.30 Å (1 Å=0.1 nm) resolution. The overall structure of PsGal31A contains four domains and the catalytic domain adopts a (β/α)8-barrel fold that resembles the structures of other GH31 enzymes. Two catalytic aspartic acid residues are structurally conserved in the enzymes, whereas most residues forming the active site differ from those of GH31 α-glucosidases and α-xylosidases. PsGal31A forms a dimer via a unique loop that is not conserved in other reported GH31 enzymes; this loop is involved in its aglycone specificity and in binding L-fucose. Considering potential genes for α-L-fucosidases and carbohydrate-related proteins within the vicinity of Pedobacter Gal31, the identified Gal31 enzymes are likely to function in a novel sugar degradation system. This is the first report of α-galactosidases which belong to GH31 family. © 2015 Authors; published by Portland Press Limited.

  20. Prunasin Hydrolases during Fruit Development in Sweet and Bitter Almonds1[C][W][OA

    Science.gov (United States)

    Sánchez-Pérez, Raquel; Belmonte, Fara Sáez; Borch, Jonas; Dicenta, Federico; Møller, Birger Lindberg; Jørgensen, Kirsten

    2012-01-01

    Amygdalin is a cyanogenic diglucoside and constitutes the bitter component in bitter almond (Prunus dulcis). Amygdalin concentration increases in the course of fruit formation. The monoglucoside prunasin is the precursor of amygdalin. Prunasin may be degraded to hydrogen cyanide, glucose, and benzaldehyde by the action of the β-glucosidase prunasin hydrolase (PH) and mandelonitirile lyase or be glucosylated to form amygdalin. The tissue and cellular localization of PHs was determined during fruit development in two sweet and two bitter almond cultivars using a specific antibody toward PHs. Confocal studies on sections of tegument, nucellus, endosperm, and embryo showed that the localization of the PH proteins is dependent on the stage of fruit development, shifting between apoplast and symplast in opposite patterns in sweet and bitter cultivars. Two different PH genes, Ph691 and Ph692, have been identified in a sweet and a bitter almond cultivar. Both cDNAs are 86% identical on the nucleotide level, and their encoded proteins are 79% identical to each other. In addition, Ph691 and Ph692 display 92% and 86% nucleotide identity to Ph1 from black cherry (Prunus serotina). Both proteins were predicted to contain an amino-terminal signal peptide, with the size of 26 amino acid residues for PH691 and 22 residues for PH692. The PH activity and the localization of the respective proteins in vivo differ between cultivars. This implies that there might be different concentrations of prunasin available in the seed for amygdalin synthesis and that these differences may determine whether the mature almond develops into bitter or sweet. PMID:22353576

  1. Meta-analysis of microsomal epoxide hydrolase gene polymorphism and risk of hepatocellular carcinoma.

    Directory of Open Access Journals (Sweden)

    Jian-Hong Zhong

    Full Text Available BACKGROUND: Hepatocarcinogenesis is a complex process that may be influenced by many factors, including polymorphism in microsomal epoxide hydrolase (mEH. Previous work suggests an association between the Tyr113His and His139Arg mEH polymorphisms and susceptibility to hepatocellular carcinoma (HCC, but the results have been inconsistent. METHODS: PubMed, EMBASE, Google Scholar and the Chinese National Knowledge Infrastructure databases were systematically searched to identify relevant studies. A meta-analysis was performed to examine the association between Tyr113His and His139Arg mEH polymorphism and susceptibility to HCC. Odds ratios (ORs and 95% confidence intervals (95% CIs were calculated. RESULTS: Eleven studies were included in the meta-analysis, involving 1,696 HCC cases and 3,600 controls. The 113His- mEH allele was significantly associated with increased risk of HCC based on allelic contrast (OR = 1.35, 95% CI = 1.04-1.75, p = 0.02, homozygote comparison (OR = 1.65, 95% CI = 1.07-2.54, p = 0.02 and a recessive genetic model (OR = 1.54, 95% CI = 1.21-1.96, p<0.001, while individuals carrying the Arg139Arg mEH genotype had no association with increased or decreased risk of HCC. CONCLUSION: The 113His- allele polymorphism in mEH may be a risk factor for hepatocarcinogenesis, while the mEH 139Arg- allele may not be a risk or protective factor. There is substantial evidence that mEH polymorphisms interact synergistically with other genes and the environment to modulate risk of HCC. Further large and well-designed studies are needed to confirm these conclusions.

  2. Inhibition of the soluble epoxide hydrolase promotes albuminuria in mice with progressive renal disease.

    Directory of Open Access Journals (Sweden)

    Oliver Jung

    2010-08-01

    Full Text Available Epoxyeicotrienoic acids (EETs are cytochrome P450-dependent anti-hypertensive and anti-inflammatory derivatives of arachidonic acid, which are highly abundant in the kidney and considered reno-protective. EETs are degraded by the enzyme soluble epoxide hydrolase (sEH and sEH inhibitors are considered treatment for chronic renal failure (CRF. We determined whether sEH inhibition attenuates the progression of CRF in the 5/6-nephrectomy model (5/6-Nx in mice. 5/6-Nx mice were treated with a placebo, an ACE-inhibitor (Ramipril, 40 mg/kg, the sEH-inhibitor cAUCB or the CYP-inhibitor fenbendazole for 8 weeks. 5/6-Nx induced hypertension, albuminuria, glomerulosclerosis and tubulo-interstitial damage and these effects were attenuated by Ramipril. In contrast, cAUCB failed to lower the blood pressure and albuminuria was more severe as compared to placebo. Plasma EET-levels were doubled in 5/6 Nx-mice as compared to sham mice receiving placebo. Renal sEH expression was attenuated in 5/6-Nx mice but cAUCB in these animals still further increased the EET-level. cAUCB also increased 5-HETE and 15-HETE, which derive from peroxidation or lipoxygenases. Similar to cAUCB, CYP450 inhibition increased HETEs and promoted albuminuria. Thus, sEH-inhibition failed to elicit protective effects in the 5/6-Nx model and showed a tendency to aggravate the disease. These effects might be consequence of a shift of arachidonic acid metabolism into the lipoxygenase pathway.

  3. Altered soluble epoxide hydrolase-derived oxylipins in patients with seasonal major depression: An exploratory study.

    Science.gov (United States)

    Hennebelle, Marie; Otoki, Yurika; Yang, Jun; Hammock, Bruce D; Levitt, Anthony J; Taha, Ameer Y; Swardfager, Walter

    2017-06-01

    Many cytochrome p450-derived lipids promote resolution of inflammation, in contrast to their soluble epoxide hydrolase(sEH)-derived oxylipin breakdown products. Here we compare plasma oxylipins and precursor fatty acids between seasons in participants with major depressive disorder with seasonal pattern (MDD-s). Euthymic participants with a history of MDD-s recruited in summer-fall were followed-up in winter. At both visits, a structured clinical interview (DSM-5 criteria) and the Beck Depression Inventory II (BDI-II) were administered. Unesterified and total oxylipin pools were assayed by liquid chromatography tandem mass-spectrometry (LC-MS/MS). Precursor fatty acids were measured by gas chromatography. In nine unmedicated participants euthymic at baseline who met depression criteria in winter, BDI-II scores increased from 4.9±4.4 to 19.9±7.7. Four sEH-derived oxylipins increased in winter compared to summer-fall with moderate to large effect sizes. An auto-oxidation product (unesterified epoxyketooctadecadienoic acid) and lipoxygenase-derived 13-hydroxyoctadecadienoic acid also increased in winter. The cytochrome p450-derived 20-COOH-leukotriene B4 (unesterified) and total 14(15)-epoxyeicosatetraenoic acid, and the sEH-derived 14,15-dihydroxyeicostrienoic acid (unesterified), decreased in winter. We conclude that winter depression was associated with changes in cytochrome p450- and sEH-derived oxylipins, suggesting that seasonal shifts in omega-6 and omega-3 fatty acid metabolism mediated by sEH may underlie inflammatory states in symptomatic MDD-s. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

  4. Nasopharyngeal colonization and invasive disease are enhanced by the cell wall hydrolases LytB and LytC of Streptococcus pneumoniae.

    Directory of Open Access Journals (Sweden)

    Elisa Ramos-Sevillano

    Full Text Available BACKGROUND: Streptococcus pneumoniae is a common colonizer of the human nasopharynx and one of the major pathogens causing invasive disease worldwide. Dissection of the molecular pathways responsible for colonization, invasion, and evasion of the immune system will provide new targets for antimicrobial or vaccine therapies for this common pathogen. METHODOLOGY/PRINCIPAL FINDINGS: We have constructed mutants lacking the pneumococcal cell wall hydrolases (CWHs LytB and LytC to investigate the role of these proteins in different phases of the pneumococcal pathogenesis. Our results show that LytB and LytC are involved in the attachment of S. pneumoniae to human nasopharyngeal cells both in vitro and in vivo. The interaction of both proteins with phagocytic cells demonstrated that LytB and LytC act in concert avoiding pneumococcal phagocytosis mediated by neutrophils and alveolar macrophages. Furthermore, C3b deposition was increased on the lytC mutant confirming that LytC is involved in complement evasion. As a result, the lytC mutant showed a reduced ability to successfully cause pneumococcal pneumonia and sepsis. Bacterial mutants lacking both LytB and LytC showed a dramatically impaired attachment to nasopharyngeal cells as well as a marked degree of attenuation in a mouse model of colonization. In addition, C3b deposition and phagocytosis was more efficient for the double lytB lytC mutant and its virulence was greatly impaired in both systemic and pulmonary models of infection. CONCLUSIONS/SIGNIFICANCE: This study confirms that the CWHs LytB and LytC of S. pneumoniae are essential virulence factors involved in the colonization of the nasopharynx and in the progress of invasive disease by avoiding host immunity.

  5. Bile-Salt-Hydrolases from the Probiotic Strain Lactobacillus johnsonii La1 Mediate Anti-giardial Activity in Vitro and in Vivo

    Directory of Open Access Journals (Sweden)

    Thibault Allain

    2018-01-01

    Full Text Available Giardia duodenalis (syn. G. lamblia, G. intestinalis is the protozoan parasite responsible for giardiasis, the most common and widely spread intestinal parasitic disease worldwide, affecting both humans and animals. After cysts ingestion (through either contaminated food or water, Giardia excysts in the upper intestinal tract to release replicating trophozoites that are responsible for the production of symptoms. In the gut, Giardia cohabits with the host's microbiota, and several studies have revealed the importance of this gut ecosystem and/or some probiotic bacteria in providing protection against G. duodenalis infection through mechanisms that remain incompletely understood. Recent findings suggest that Bile-Salt-Hydrolase (BSH-like activities from the probiotic strain of Lactobacillus johnsonii La1 may contribute to the anti-giardial activity displayed by this strain. Here, we cloned and expressed each of the three bsh genes present in the L. johnsonii La1 genome to study their enzymatic and biological properties. While BSH47 and BSH56 were expressed as recombinant active enzymes, no significant enzymatic activity was detected with BSH12. In vitro assays allowed determining the substrate specificities of both BSH47 and BSH56, which were different. Modeling of these BSHs indicated a strong conservation of their 3-D structures despite low conservation of their primary structures. Both recombinant enzymes were able to mediate anti-giardial biological activity against Giardia trophozoites in vitro. Moreover, BSH47 exerted significant anti-giardial effects when tested in a murine model of giardiasis. These results shed new light on the mechanism, whereby active BSH derived from the probiotic strain Lactobacillus johnsonii La1 may yield anti-giardial effects in vitro and in vivo. These findings pave the way toward novel approaches for the treatment of this widely spread but neglected infectious disease, both in human and in veterinary medicine.

  6. Draft genome sequence of Streptomyces sp. strain F1, a potential source for glycoside hydrolases isolated from Brazilian soil

    Directory of Open Access Journals (Sweden)

    Ricardo Rodrigues de Melo

    Full Text Available ABSTRACT Here, we show the draft genome sequence of Streptomyces sp. F1, a strain isolated from soil with great potential for secretion of hydrolytic enzymes used to deconstruct cellulosic biomass. The draft genome assembly of Streptomyces sp. strain F1 has 69 contigs with a total genome size of 8,142,296 bp and G + C 72.65%. Preliminary genome analysis identified 175 proteins as Carbohydrate-Active Enzymes, being 85 glycoside hydrolases organized in 33 distinct families. This draft genome information provides new insights on the key genes encoding hydrolytic enzymes involved in biomass deconstruction employed by soil bacteria.

  7. Glycoside Hydrolase (GH) 45 and 5 Candidate Cellulases in Aphelenchoides besseyi Isolated from Bird?s-Nest Fern

    OpenAIRE

    Wu, Guan-Long; Kuo, Tzu-Hao; Tsay, Tung-Tsuan; Tsai, Isheng J.; Chen, Peichen J.

    2016-01-01

    Five Aphelenchoides besseyi isolates collected from bird's-nest ferns or rice possess different parasitic capacities in bird's-nest fern. Two different glycoside hydrolase (GH) 45 genes were identified in the fern isolates, and only one was found in the rice isolates. A Abe GH5-1 gene containing an SCP-like family domain was found only in the fern isolates. Abe GH5-1 gene has five introns suggesting a eukaryotic origin. A maximum likelihood phylogeny revealed that Abe GH5-1 is part of the nem...

  8. Enhanced staphylolytic activity of the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88 HydH5 virion associated peptidoglycan hydrolase: fusions, deletions and synergy with LysH5

    Science.gov (United States)

    Virion-associated peptidoglycan hydrolases have a potential as antimicrobial agents due to their ability to lyse Gram positive bacteria on contact. In this work, our aim was to improve the lytic activity of HydH5, a virion associated peptidoglycan hydrolase from the Staphylococcus aureus bacteriopha...

  9. The impact of nonpolar lipids on the regulation of the steryl ester hydrolases Tgl1p and Yeh1p in the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Klein, Isabella; Korber, Martina; Athenstaedt, Karin; Daum, Günther

    2017-12-01

    In the yeast Saccharomyces cerevisiae degradation of steryl esters is catalyzed by the steryl ester hydrolases Tgl1p, Yeh1p and Yeh2p. The two steryl ester hydrolases Tgl1p and Yeh1p localize to lipid droplets, a cell compartment storing steryl esters and triacylglycerols. In the present study we investigated regulatory aspects of these two hydrolytic enzymes, namely the gene expression level, protein amount, stability and enzyme activity of Tgl1p and Yeh1p in strains lacking both or only one of the two major nonpolar lipids, steryl esters and triacylglycerols. In a strain lacking both nonpolar lipids and consequently lipid droplets, Tgl1p as well as Yeh1p were present at low amount, became highly unstable compared to wild-type cells, and lost their enzymatic activity. Under these conditions both steryl ester hydrolases were retained in the endoplasmic reticulum. The lack of steryl esters alone was not sufficient to cause an altered intracellular localization of Tgl1p and Yeh1p. Surprisingly, the stability of Tgl1p and Yeh1p was markedly reduced in a strain lacking triacylglycerols, but their capacity to mobilize steryl esters remained unaffected. We also tested a possible cross-regulation of Tgl1p and Yeh1p by analyzing the behavior of each hydrolase in the absence of its counterpart steryl ester hydrolases. In summary, this study demonstrates a strong regulation of the two lipid droplet associated steryl ester hydrolases Tgl1p and Yeh1p due to the presence/absence of their host organelle. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Production and characterisation of glycoside hydrolases from GH3, GH5, GH10, GH11 and GH61 for chemo-enzymatic synthesis of xylo- and mannooligosaccharides

    DEFF Research Database (Denmark)

    Dilokpimol, Adiphol

    Produktion og karakterisering af glykosid hydrolaser fro GH3, GH5, GH10, GH11 og GH61 til chemo-enzymatisk syntese af xylo- og mannooligosakkarider Biprodukter fra hydrolyse af plantecellevægge er kilder til oligosakkarider, som potentielt kan fungere som prebiotika ved at stimulere væksten af...... omfatter karakterisering af de producerede enzymer samt cDNA kloning af formodet GH61 endo Produktion og karakterisering af glykosid hydrolaser fro GH3, GH5, GH10, GH11 og GH61 til chemo-enzymatisk syntese af xylo- og mannooligosakkarider Biprodukter fra hydrolyse af plantecellevægge er kilder til...

  11. Effect of inhibition of fatty acid amide hydrolase on MPTP-induced dopaminergic neuronal damage.

    Science.gov (United States)

    Viveros-Paredes, J M; Gonzalez-Castañeda, R E; Escalante-Castañeda, A; Tejeda-Martínez, A R; Castañeda-Achutiguí, F; Flores-Soto, M E

    2017-01-16

    Parkinson's disease (PD) is a neurodegenerative disorder characterised by balance problems, muscle rigidity, and slow movement due to low dopamine levels and loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). The endocannabinoid system is known to modulate the nigrostriatal pathway through endogenous ligands such as anandamide (AEA), which is hydrolysed by fatty acid amide hydrolase (FAAH). The purpose of this study was to increase AEA levels using FAAH inhibitor URB597 to evaluate the modulatory effect of AEA on dopaminergic neuronal death induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Our study included 4 experimental groups (n = 6 mice per group): a control group receiving no treatment, a group receiving URB597 (0.2mg/kg) every 3 days for 30 days, a group treated with MPTP (30mg/kg) for 5 days, and a group receiving URB597 and subsequently MPTP injections. Three days after the last dose, we conducted a series of behavioural tests (beam test, pole test, and stride length test) to compare motor coordination between groups. We subsequently analysed immunoreactivity of dopaminergic cells and microglia in the SNpc and striatum. Mice treated with URB597 plus MPTP were found to perform better on behavioural tests than mice receiving MPTP only. According to the immunohistochemistry study, mice receiving MPTP showed fewer dopaminergic cells and fibres in the SNpc and striatum. Animals treated with URB597 plus MPTP displayed increased tyrosine hydroxylase immunoreactivity compared to those treated with MPTP only. Regarding microglial immunoreactivity, the group receiving MPTP showed higher Iba1 immunoreactivity in the striatum and SNpc than did the group treated with URB597 plus MPTP. Our results show that URB597 exerts a protective effect since it inhibits dopaminergic neuronal death, decreases microglial immunoreactivity, and improves MPTP-induced motor alterations. Copyright © 2016 Sociedad Española de Neurología. Publicado

  12. Genomic and expression analysis of the flax (Linum usitatissimum) family of glycosyl hydrolase 35 genes.

    Science.gov (United States)

    Hobson, Neil; Deyholos, Michael K

    2013-05-23

    Several β-galactosidases of the Glycosyl Hydrolase 35 (GH35) family have been characterized, and many of these modify cell wall components, including pectins, xyloglucans, and arabinogalactan proteins. The phloem fibres of flax (Linum usitatissimum) have gelatinous-type cell walls that are rich in crystalline cellulose and depend on β-galactosidase activity for their normal development. In this study, we investigate the transcript expression patterns and inferred evolutionary relationships of the complete set of flax GH35 genes, to better understand the functions of these genes in flax and other species. Using the recently published flax genome assembly, we identified 43 β-galactosidase-like (BGAL) genes, based on the presence of a GH35 domain. Phylogenetic analyses of their protein sequences clustered them into eight sub-families. Sub-family B, whose members in other species were known to be expressed in developing flowers and pollen, was greatly under represented in flax (p-value < 0.01). Sub-family A5, whose sole member from arabidopsis has been described as its primary xyloglucan BGAL, was greatly expanded in flax (p-value < 0.01). A number of flax BGALs were also observed to contain non-consensus GH35 active sites. Expression patterns of the flax BGALs were investigated using qRT-PCR and publicly available microarray data. All predicted flax BGALs showed evidence of expression in at least one tissue. Flax has a large number of BGAL genes, which display a distinct distribution among the BGAL sub-families, in comparison to other closely related species with available whole genome assemblies. Almost every flax BGAL was expressed in fibres, the majority of which expressed predominately in fibres as compared to other tissues, suggesting an important role for the expansion of this gene family in the development of this species as a fibre crop. Variations displayed in the canonical GH35 active site suggest a variety of roles unique to flax, which will require

  13. Insight into glycoside hydrolases for debranched xylan degradation from extremely thermophilic bacterium Caldicellulosiruptor lactoaceticus.

    Directory of Open Access Journals (Sweden)

    Xiaojing Jia

    Full Text Available Caldicellulosiruptor lactoaceticus 6A, an anaerobic and extremely thermophilic bacterium, uses natural xylan as carbon source. The encoded genes of C. lactoaceticus 6A for glycoside hydrolase (GH provide a platform for xylan degradation. The GH family 10 xylanase (Xyn10A and GH67 α-glucuronidase (Agu67A from C. lactoaceticus 6A were heterologously expressed, purified and characterized. Both Xyn10A and Agu67A are predicted as intracellular enzymes as no signal peptides identified. Xyn10A and Agu67A had molecular weight of 47.0 kDa and 80.0 kDa respectively as determined by SDS-PAGE, while both appeared as homodimer when analyzed by gel filtration. Xyn10A displayed the highest activity at 80 °C and pH 6.5, as 75 °C and pH 6.5 for Agu67A. Xyn10A had good stability at 75 °C, 80 °C, and pH 4.5-8.5, respectively, and was sensitive to various metal ions and reagents. Xyn10A possessed hydrolytic activity towards xylo-oligosaccharides (XOs and beechwood xylan. At optimum conditions, the specific activity of Xyn10A was 44.6 IU/mg with beechwood xylan as substrate, and liberated branched XOs, xylobiose, and xylose. Agu67A was active on branched XOs with methyl-glucuronic acids (MeGlcA sub-chains, and primarily generated XOs equivalents and MeGlcA. The specific activity of Agu67A was 1.3 IU/mg with aldobiouronic acid as substrate. The synergistic action of Xyn10A and Agu67A was observed with MeGlcA branched XOs and xylan as substrates, both backbone and branched chain of substrates were degraded, and liberated xylose, xylobiose, and MeGlcA. The synergism of Xyn10A and Agu67A provided not only a thermophilic method for natural xylan degradation, but also insight into the mechanisms for xylan utilization of C. lactoaceticus.

  14. Soluble epoxide hydrolase contamination of specific catalase preparations inhibits epoxyeicosatrienoic acid vasodilation of rat renal arterioles

    Science.gov (United States)

    Olson, Lauren; Harder, Adam; Isbell, Marilyn; Imig, John D.; Gutterman, David D.; Falck, J. R.; Campbell, William B.

    2011-01-01

    Cytochrome P-450 metabolites of arachidonic acid, the epoxyeicosatrienoic acids (EETs) and hydrogen peroxide (H2O2), are important signaling molecules in the kidney. In renal arteries, EETs cause vasodilation whereas H2O2 causes vasoconstriction. To determine the physiological contribution of H2O2, catalase is used to inactivate H2O2. However, the consequence of catalase action on EET vascular activity has not been determined. In rat renal afferent arterioles, 14,15-EET caused concentration-related dilations that were inhibited by Sigma bovine liver (SBL) catalase (1,000 U/ml) but not Calbiochem bovine liver (CBL) catalase (1,000 U/ml). SBL catalase inhibition was reversed by the soluble epoxide hydrolase (sEH) inhibitor tAUCB (1 μM). In 14,15-EET incubations, SBL catalase caused a concentration-related increase in a polar metabolite. Using mass spectrometry, the metabolite was identified as 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), the inactive sEH metabolite. 14,15-EET hydrolysis was not altered by the catalase inhibitor 3-amino-1,2,4-triazole (3-ATZ; 10–50 mM), but was abolished by the sEH inhibitor BIRD-0826 (1–10 μM). SBL catalase EET hydrolysis showed a regioisomer preference with greatest hydrolysis of 14,15-EET followed by 11,12-, 8,9- and 5,6-EET (Vmax = 0.54 ± 0.07, 0.23 ± 0.06, 0.18 ± 0.01 and 0.08 ± 0.02 ng DHET·U catalase−1·min−1, respectively). Of five different catalase preparations assayed, EET hydrolysis was observed with two Sigma liver catalases. These preparations had low specific catalase activity and positive sEH expression. Mass spectrometric analysis of the SBL catalase identified peptide fragments matching bovine sEH. Collectively, these data indicate that catalase does not affect EET-mediated dilation of renal arterioles. However, some commercial catalase preparations are contaminated with sEH, and these contaminated preparations diminish the biological activity of H2O2 and EETs. PMID:21753077

  15. Identification of key peptidoglycan hydrolases for morphogenesis, autolysis, and peptidoglycan composition of Lactobacillus plantarum WCFS1

    Directory of Open Access Journals (Sweden)

    Rolain Thomas

    2012-10-01

    Full Text Available Abstract Background Lactobacillus plantarum is commonly used in industrial fermentation processes. Selected strains are also marketed as probiotics for their health beneficial effects. Although the functional role of peptidoglycan-degrading enzymes is increasingly documented to be important for a range of bacterial processes and host-microbe interactions, little is known about their functional roles in lactobacilli. This knowledge holds important potential for developing more robust strains resistant to autolysis under stress conditions as well as peptidoglycan engineering for a better understanding of the contribution of released muramyl-peptides as probiotic immunomodulators. Results Here, we explored the functional role of the predicted peptidoglycan hydrolase (PGH complement encoded in the genome of L. plantarum by systematic gene deletion. From twelve predicted PGH-encoding genes, nine could be individually inactivated and their corresponding mutant strains were characterized regarding their cell morphology, growth, and autolysis under various conditions. From this analysis, we identified two PGHs, the predicted N-acetylglucosaminidase Acm2 and NplC/P60 D,L-endopeptidase LytA, as key determinants in the morphology of L. plantarum. Acm2 was demonstrated to be required for the ultimate step of cell separation of daughter cells, whereas LytA appeared to be required for cell shape maintenance and cell-wall integrity. We also showed by autolysis experiments that both PGHs are involved in the global autolytic process with a dominant role for Acm2 in all tested conditions, identifying Acm2 as the major autolysin of L. plantarum WCFS1. In addition, Acm2 and the putative N-acetylmuramidase Lys2 were shown to play redundant roles in both cell separation and autolysis under stress conditions. Finally, the analysis of the peptidoglycan composition of Acm2- and LytA-deficient derivatives revealed their potential hydrolytic activities by the

  16. Coexpression of bile salt hydrolase gene and catalase gene remarkably improves oxidative stress and bile salt resistance in Lactobacillus casei.

    Science.gov (United States)

    Wang, Guohong; Yin, Sheng; An, Haoran; Chen, Shangwu; Hao, Yanling

    2011-08-01

    Lactic acid bacteria (LAB) encounter various types of stress during industrial processes and gastrointestinal transit. Catalase (CAT) and bile salt hydrolase (BSH) can protect bacteria from oxidative stress or damage caused by bile salts by decomposing hydrogen peroxide (H(2)O(2)) or deconjugating the bile salts, respectively. Lactobacillus casei is a valuable probiotic strain and is often deficient in both CAT and BSH. In order to improve the resistance of L. casei to both oxidative and bile salts stress, the catalase gene katA from L. sakei and the bile salt hydrolase gene bsh1 from L. plantarum were coexpressed in L. casei HX01. The enzyme activities of CAT and BSH were 2.41 μmol H(2)O(2)/min/10(8) colony-forming units (CFU) and 2.11 μmol glycine/min/ml in the recombinant L. casei CB, respectively. After incubation with 8 mM H(2)O(2), survival ratio of L. casei CB was 40-fold higher than that of L. casei CK. Treatment of L. casei CB with various concentrations of sodium glycodeoxycholate (GDCA) showed that ~10(5) CFU/ml cells survived after incubation with 0.5% GDCA, whereas almost all the L. casei CK cells were killed when treaded with 0.4% GDCA. These results indicate that the coexpression of CAT and BSH confers high-level resistance to both oxidative and bile salts stress conditions in L. casei HX01.

  17. [Soil hydrolase characteristics in late soil-thawing period in subalpine/alpine forests of west Sichuan].

    Science.gov (United States)

    Tan, Bo; Wu, Fu-Zhong; Yang, Wan-Qin; Yu, Sheng; Yang, Yu-Lian; Wang, Ao

    2011-05-01

    Late soil-thawing period is a critical stage connecting winter and growth season. The significant temperature fluctuation at this stage might have strong effects on soil ecological processes. In order to understand the soil biochemical processes at this stage in the subalpine/alpine forests of west Sichuan, soil samples were collected from the representative forests including primary fir forest, fir and birch mixed forest, and secondary fir forest in March 5-April 25, 2009, with the activities of soil invertase, urease, and phosphatase (neutral, acid and alkaline phosphatases) measured. In soil frozen period, the activities of the three enzymes in test forests still kept relatively higher. With the increase of soil temperature, the activities of hydrolases at the early stage of soil-thawing decreased rapidly after a sharp increase, except for neutral phosphatease. Thereafter, there was an increase in the activities of urease and phosphatase. Relative to soil mineral layer, soil organic layer had higher hydrolase activity in late soil-thawing period, and showed more obvious responses to the variation of soil temperature.

  18. First glycoside hydrolase family 2 enzymes from Thermus antranikianii and Thermus brockianus with β-glucosidase activity

    Directory of Open Access Journals (Sweden)

    Carola eSchröder

    2015-06-01

    Full Text Available Two genes tagh2 and tbgh2 coding for enzymes with hydrolytic activity towards esculin were identified from the extreme thermophilic, aerobic bacteria Thermus antranikianii (Ta and T. brockianus (Tb. Shortened conserved domains predicted a membership of the enzymes of glycoside hydrolase (GH family 2. At present, β-galactosidase activity is found frequently in GH family 2 but β-glucosidase activity has not been reported in this family before. The enzymes TaGH2 and TbGH2 preferred hydrolysis of nitrophenol-linked β-D-glucopyranosides with specific activities of 3,966 U/mg and 660 U/mg, respectively. Residual activities of 40 % (TaGH2 and 51 % (TbGH2 towards 4-NP-β-D-galactopyranoside were observed. Furthermore, TaGH2 hydrolyzed cellobiose. TbGH2, however, showed no activity on cellobiose or lactose. The enzymes exhibited highest activity at 95 °C (TaGH2 and 90 °C (TbGH2 at pH 6.5. Both enzymes were extremely thermostable and thermal activation up to 250 % was observed at temperatures between 50 and 60 °C. Accordingly, the first thermoactive glycoside hydrolase family 2 enzymes with β glucosidase activity have been identified and characterized. The hydrolysis of cellobiose is a unique property of TaGH2 when compared to the enzymes of GH family 2.

  19. GH97 is a new family of glycoside hydrolases, which is related to the α-galactosidase superfamily

    Directory of Open Access Journals (Sweden)

    Naumoff Daniil G

    2005-08-01

    Full Text Available Abstract Background As a rule, about 1% of genes in a given genome encode glycoside hydrolases and their homologues. On the basis of sequence similarity they have been grouped into more than ninety GH families during the last 15 years. The GH97 family has been established very recently and initially included only 18 bacterial proteins. However, the evolutionary relationship of the genes encoding proteins of this family remains unclear, as well as their distribution among main groups of the living organisms. Results The extensive search of the current databases allowed us to double the number of GH97 family proteins. Five subfamilies were distinguished on the basis of pairwise sequence comparison and phylogenetic analysis. Iterative sequence analysis revealed the relationship of the GH97 family with the GH27, GH31, and GH36 families of glycosidases, which belong to the α-galactosidase superfamily, as well as a more distant relationship with some other glycosidase families (GH13 and GH20. Conclusion The results of this study show an unexpected sequence similarity of GH97 family proteins with glycoside hydrolases from several other families, that have (β/α8-barrel fold of the catalytic domain and a retaining mechanism of the glycoside bond hydrolysis. These data suggest a common evolutionary origin of glycosidases representing different families and clans.

  20. Serum concentration of ubiquitin c-terminal hydrolase-L1 in detecting severity of traumatic brain injury

    Science.gov (United States)

    Siahaan, A. M. P.; Japardi, I.; Hakim, A. A.

    2018-03-01

    One of the main problems with ahead injury is assessing the severity. While physical examination and imaging had limitations, neuronal damage markers, ubiquitin C-terminal hydrolase-L1 (UCH-L1), released in theblood may provide valuable information about diagnosis the traumatic brain injury (TBI).Analyzing the concentrations of serum ubiquitin C-terminal hydrolase-L1 (UCH-L1), there must have a neuronal injury biomarker, in theTBI patients serum and their association with clinical characteristics and outcome. There were 80 TBI subjects, and there are mild, moderate, and severe involved in this study of case- control. By using ELISA, we studied the profile of serum UCH-L1 levels for TBI patients. TheUCH-L1 serum level of moderate and severe head injury is higher than in mild head injury (pinjury patients. There is no particular correlation found between serum UCH-L1 level and outcome. Serum levels of UCH-L1 appear to have potential clinical utility in diagnosing TBI but do not correlate with outcome.

  1. Potassium biphthalate buffer for pH control to optimize glycosyl hydrolase production in shake flasks using filamentous fungi

    Directory of Open Access Journals (Sweden)

    Patrícia dos Santos Costa

    Full Text Available Abstract The optimization of culture medium with statistical methods is widely used in filamentous fungi glycosyl hydrolase production. The implementation of such methodology in bioreactors is very expensive as it requires several pH-controlled systems operating in parallel in order to test a large number of culture media components. The objective of this study was to evaluate potassium biphthalate buffer for pH control, which allows the optimization studies to be performed in shake flasks.The results have shown that buffering the culture medium with 0.1 M potassium biphthalate allowed pH control, resulting in a decrease of the standard deviation of triplicates for pH and activities of glycosyl hydrolase measurements. The use of this buffer allowed shake flask culture media optimization of enzyme production by Trichoderma harzianum, increasing the cellulase activity by more than 2 times compared to standard unbuffered culture medium. The same buffer can be used for culture media optimization of other fungi, such as Penicillium echinulatum.

  2. Gene-centric metagenomics of the fiber-adherent bovine rumen microbiome reveals forage specific glycoside hydrolases.

    Science.gov (United States)

    Brulc, Jennifer M; Antonopoulos, Dionysios A; Miller, Margret E Berg; Wilson, Melissa K; Yannarell, Anthony C; Dinsdale, Elizabeth A; Edwards, Robert E; Frank, Edward D; Emerson, Joanne B; Wacklin, Pirjo; Coutinho, Pedro M; Henrissat, Bernard; Nelson, Karen E; White, Bryan A

    2009-02-10

    The complex microbiome of the rumen functions as an effective system for the conversion of plant cell wall biomass to microbial protein, short chain fatty acids, and gases. As such, it provides a unique genetic resource for plant cell wall degrading microbial enzymes that could be used in the production of biofuels. The rumen and gastrointestinal tract harbor a dense and complex microbiome. To gain a greater understanding of the ecology and metabolic potential of this microbiome, we used comparative metagenomics (phylotype analysis and SEED subsystems-based annotations) to examine randomly sampled pyrosequence data from 3 fiber-adherent microbiomes and 1 pooled liquid sample (a mixture of the liquid microbiome fractions from the same bovine rumens). Even though the 3 animals were fed the same diet, the community structure, predicted phylotype, and metabolic potentials in the rumen were markedly different with respect to nutrient utilization. A comparison of the glycoside hydrolase and cellulosome functional genes revealed that in the rumen microbiome, initial colonization of fiber appears to be by organisms possessing enzymes that attack the easily available side chains of complex plant polysaccharides and not the more recalcitrant main chains, especially cellulose. Furthermore, when compared with the termite hindgut microbiome, there are fundamental differences in the glycoside hydrolase content that appear to be diet driven for either the bovine rumen (forages and legumes) or the termite hindgut (wood).

  3. Glycoside hydrolase family 13 α-glucosidases encoded by Bifidobacterium breve UCC2003; A comparative analysis of function, structure and phylogeny.

    Science.gov (United States)

    Kelly, Emer D; Bottacini, Francesca; O'Callaghan, John; Motherway, Mary O'Connell; O'Connell, Kerry Joan; Stanton, Catherine; van Sinderen, Douwe

    2016-05-02

    Bifidobacterium breve is a noted inhabitant and one of the first colonizers of the human gastro intestinal tract (GIT). The ability of this bacterium to persist in the GIT is reflected by the abundance of carbohydrate-active enzymes that are encoded by its genome. One such family of enzymes is represented by the α-glucosidases, of which three, Agl1, Agl2 and MelD, have previously been identified and characterized in the prototype B. breve strain UCC2003. In this report, we describe an additional B. breve UCC2003-encoded α-glucosidase, along with a B. breve UCC2003-encoded α-glucosidase-like protein, designated here as Agl3 and Agl4, respectively, which together with the three previously described enzymes belong to glycoside hydrolase (GH) family 13. Agl3 was shown to exhibit hydrolytic specificity towards the α-(1→6) linkage present in palatinose; the α-(1→3) linkage present in turanose; the α-(1→4) linkages found in maltotriose and maltose; and to a lesser degree, the α-(1→2) linkage found in sucrose and kojibiose; and the α-(1→5) linkage found in leucrose. Surprisingly, based on the substrates analyzed, Agl4 did not exhibit biologically relevant α-glucosidic activity. With the presence of four functionally active GH13 α-glucosidases, B. breve UCC2003 is capable of hydrolyzing all α-glucosidic linkages that can be expected in glycan substrates in the lower GIT. This abundance of α-glucosidases provides B. breve UCC2003 with an adaptive ability and metabolic versatility befitting the transient nature of growth substrates in the GIT. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Glycosyl hydrolases from Bifidobacterium adolescentis DSM20083 : Their role in the metabolism and synthesis of oligosaccharides.

    NARCIS (Netherlands)

    Broek, van den L.A.M.

    2005-01-01

    Nowadays, there is an increasing interest for food, which is health beneficial for humans. Prebiotics e.g. is used to stimulate the growth of bacteria in the gut that have a positive effect on human health. It is claimed that bifidobacteria in the colon improve health and well being of humans. The

  5. Molecular Cloning and Nucleotide Sequence of the Gene Encoding the Major Peptidoglycan Hydrolase of Lactococcus lactis, a Muramidase Needed for Cell Separation

    NARCIS (Netherlands)

    Buist, Girbe; Kok, Jan; Leenhouts, Kees J.; Dabrowska, Magdalena; Venema, Gerhardus; Haandrikman, Alfred J.

    A gene of Lactococcus lactis subsp, cremoris MG1363 encoding a peptidoglycan hydrolase was identified in a genomic library of the strain in pUC19 by screening Escherichia coli transformants for cell wall lysis activity on a medium containing autoclaved, lyophilized Micrococcus lysodeikticus cells,

  6. Transient changes of enzyme activity of five acid hydrolases in the supernatants of homogenates of hearts of mice due to ultraviolet irradiation

    International Nuclear Information System (INIS)

    Droba, B.; Jagiellonian Univ., Krakow

    1977-01-01

    Enzymatic activity of five lysosomal hydrolases: acid p-nitrophenyl phosphatase (EC 3.1.3.2), acid β-glycerophosphatase (EC 3.1.3.2), arylsulphatase (EC 3.1.6.1), β-galactosidase (EC 3.2.1.23) and β-N-acetylhexoaminidase (EC 3.2.1.30) was studied in the supernatants of homogenates of hearts of unirradiated mice, serving as controls, and a group of UV-irradiated mice. In the control group, determinations made at 6-hr intervals showed rhythmic diurnal changes in activities of three acid hydrolases. These changes were statistically significant in the case of acid p-nitrophenyl phosphatase, acid β-glycerophosphatase, and β-N-acetylhexosaminidase. The effect of UV-irradiation was manifested mainly by depression of enzyme activities of the acid hydrolases during the first few hours after exposure. Depression of activities of arylsulphatase and β-N-acetylhexosaminidase by UV light was statistically significant. Presumably, the fall in enzyme activities of the acid hydrolases was due to chemical mediators formed in the skin under the influence of UV-radiation and adrenal corticoids secreted into the blood

  7. Key aromatic residues at subsites +2 and +3 of glycoside hydrolase family 31 α-glucosidase contribute to recognition of long-chain substrates

    DEFF Research Database (Denmark)

    Tagami, Takayoshi; Okuyama, Masayuki; Nakai, Hiroyuki

    2013-01-01

    Glycoside hydrolase family 31 α-glucosidases (31AGs) show various specificities for maltooligosaccharides according to chain length. Aspergillus niger α-glucosidase (ANG) is specific for short-chain substrates with the highest kcat/Km for maltotriose, while sugar beet α-glucosidase (SBG) prefers...

  8. Enzymatic synthesis of β-xylosyl-oligosaccharides by transxylosylation using two beta-xylosidases of glycoside hydrolase family 3 from Aspergillus nidulans FGSC A4

    DEFF Research Database (Denmark)

    Dilokpimol, Adiphol; Nakai, Hiroyuki; Gotfredsen, Charlotte Held

    2011-01-01

    Two beta-xylosidases of glycoside hydrolase family 3 (GH 3) from Aspergillus nidulans FGSC A4, BxlA and BxlB were produced recombinantly in Pichia pastoris and secreted to the culture supernatants in yields of 16 and 118 mg/L, respectively. BxlA showed about sixfold higher catalytic efficiency (k...

  9. Ethanol production with a flocculating mutant of Zymomonas mobilis and immobilized glycoside hydrolases. Ethanolgewinnung mit einer flockenden Mutante von Zymomonas mobilis und immobilisierten Glycosidhydrolasen

    Energy Technology Data Exchange (ETDEWEB)

    Tramm-Werner, S.

    1987-05-25

    A method to extend the substrate spectrum of Z. mobilis was developed. Higher ethanol yields were achieved by simultaneous use of hydrolases cross-linked with glutar aldehyde together with the flocculating Zymomonas cells (TW 602). Apart from the high product yields, the method is characterized by low susceptibility to infections.

  10. Efficacy of S-adenosylhomocysteine hydrolase inhibitors, D-eritadenine and (S)-DHPA, against the growth of Cryptosporidium parvum in vitro

    Czech Academy of Sciences Publication Activity Database

    Čtrnáctá, Vlasta; Fritzler, J. M.; Šurínová, M.; Hrdý, I.; Zhu, G.; Stejskal, F.

    2010-01-01

    Roč. 126, č. 2 (2010), s. 113-116 ISSN 0014-4894 Institutional research plan: CEZ:AV0Z50520701 Keywords : S-adenosylhomocysteine hydrolase * D-eritadenine * (S)-DHPA Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.869, year: 2010

  11. Cloning, characterization and heterologous expression of epoxide hydrolase-encoding cDNA sequences from yeasts belonging to the genera Rhodotorula and Rhodosporidium

    NARCIS (Netherlands)

    Visser, H.; Weijers, C.A.G.M.; Ooyen, van A.J.J.; Verdoes, J.C.

    2002-01-01

    Epoxide hydrolase-encoding cDNA sequences were isolated from the basidiomycetous yeast species Rhodosporidium toruloides CBS 349, Rhodosporidium toruloides CBS 14 and Rhodotorula araucariae CBS 6031 in order to evaluate the molecular data and potential application of this type of enzymes. The

  12. Stereoselectivity and substrate specificity in the kinetic resolution of methyl-substituted 1-oxaspiro[2.5]octanes by Rhodotorula glutinis epoxide hydrolase

    NARCIS (Netherlands)

    Weijers, C.A.G.M.; Meeuwse, P.; Herpers, R.L.J.M.; Franssen, M.C.R.; Sudhölter, E.J.R.

    2005-01-01

    [GRAPHICS] The kinetic resolution of a range of methyl-substituted 1-oxaspiro[2.5]octanes by yeast epoxide hydrolase (YEH) from Rhodotorula glutinis has been investigated. The structural determinants of substrate specificity and stereoselectivity of YEH toward these substrates appeared to be the

  13. Mining novel starch-converting Glycoside Hydrolase 70 enzymes from the Nestlé Culture Collection genome database : The Lactobacillus reuteri NCC 2613 GtfB

    NARCIS (Netherlands)

    Gangoiti, Joana; van Leeuwen, Sander S.; Meng, Xiangfeng; Duboux, Stéphane; Vafiadi, Christina; Pijning, Tjaard; Dijkhuizen, Lubbert

    2017-01-01

    The Glycoside hydrolase (GH) family 70 originally was established for glucansucrases of lactic acid bacteria (LAB) converting sucrose into α-glucan polymers. In recent years we have identified 3 subfamilies of GH70 enzymes (designated GtfB, GtfC and GtfD) as 4,6-α-glucanotransferases, cleaving

  14. 4-alkyl-L-(Dehydro)proline biosynthesis in actinobacteria involves N-terminal nucleophile-hydrolase activity of γ-glutamyltranspeptidase homolog for C-C bond cleavage

    Science.gov (United States)

    Zhong, Guannan; Zhao, Qunfei; Zhang, Qinglin; Liu, Wen

    2017-07-01

    γ-Glutamyltranspeptidases (γ-GTs), ubiquitous in glutathione metabolism for γ-glutamyl transfer/hydrolysis, are N-terminal nucleophile (Ntn)-hydrolase fold proteins that share an autoproteolytic process for self-activation. γ-GT homologues are widely present in Gram-positive actinobacteria where their Ntn-hydrolase activities, however, are not involved in glutathione metabolism. Herein, we demonstrate that the formation of 4-Alkyl-L-(dehydro)proline (ALDP) residues, the non-proteinogenic α-amino acids that serve as vital components of many bioactive metabolites found in actinobacteria, involves unprecedented Ntn-hydrolase activity of γ-GT homologue for C-C bond cleavage. The related enzymes share a key Thr residue, which acts as an internal nucleophile for protein hydrolysis and then as a newly released N-terminal nucleophile for carboxylate side-chain processing likely through the generation of an oxalyl-Thr enzyme intermediate. These findings provide mechanistic insights into the biosynthesis of various ALDP residues/associated natural products, highlight the versatile functions of Ntn-hydrolase fold proteins, and particularly generate interest in thus far less-appreciated γ-GT homologues in actinobacteria.

  15. The phosducin-like protein PhLP1 impacts regulation of glycoside hydrolases and light response in Trichoderma reesei

    Directory of Open Access Journals (Sweden)

    Tisch Doris

    2011-12-01

    Full Text Available Abstract Background In the biotechnological workhorse Trichoderma reesei (Hypocrea jecorina transcription of cellulase genes as well as efficiency of the secreted cellulase mixture are modulated by light. Components of the heterotrimeric G-protein pathway interact with light-dependent signals, rendering this pathway a key regulator of cellulase gene expression. Results As regulators of heterotrimeric G-protein signaling, class I phosducin-like proteins, are assumed to act as co-chaperones for G-protein beta-gamma folding and exert their function in response to light in higher eukaryotes. Our results revealed light responsive transcription of the T. reesei class I phosducin-like protein gene phlp1 and indicate a light dependent function of PhLP1 also in fungi. We showed the functions of PhLP1, GNB1 and GNG1 in the same pathway, with one major output being the regulation of transcription of glycoside hydrolase genes including cellulase genes in T. reesei. We found no direct correlation between the growth rate and global regulation of glycoside hydrolases, which suggests that regulation of growth does not occur only at the level of substrate degradation efficiency. Additionally, PhLP1, GNB1 and GNG1 are all important for proper regulation of light responsiveness during long term exposure. In their absence, the amount of light regulated genes increased from 2.7% in wild type to 14% in Δphlp1. Besides from the regulation of degradative enzymes, PhLP1 was also found to impact on the transcription of genes involved in sexual development, which was in accordance with decreased efficiency of fruiting body formation in Δphlp1. The lack of GNB1 drastically diminished ascospore discharge in T. reesei. Conclusions The heterotrimeric G-protein pathway is crucial for the interconnection of nutrient signaling and light response of T. reesei, with the class I phosducin-like protein PhLP1, GNB1 and GNG1 acting as important nodes, which influence light

  16. Comparative Community Proteomics Demonstrates the Unexpected Importance of Actinobacterial Glycoside Hydrolase Family 12 Protein for Crystalline Cellulose Hydrolysis

    Energy Technology Data Exchange (ETDEWEB)

    Hiras, Jennifer; Wu, Yu-Wei; Deng, Kai; Nicora, Carrie D.; Aldrich, Joshua T.; Frey, Dario; Kolinko, Sebastian; Robinson, Errol W.; Jacobs, Jon M.; Adams, Paul D.; Northen, Trent R.; Simmons, Blake A.; Singer, Steven W.

    2016-08-23

    ABSTRACT

    Glycoside hydrolases (GHs) are key enzymes in the depolymerization of plant-derived cellulose, a process central to the global carbon cycle and the conversion of plant biomass to fuels and chemicals. A limited number of GH families hydrolyze crystalline cellulose, often by a processive mechanism along the cellulose chain. During cultivation of thermophilic cellulolytic microbial communities, substantial differences were observed in the crystalline cellulose saccharification activities of supernatants recovered from divergent lineages. Comparative community proteomics identified a set of cellulases from a population closely related to actinobacteriumThermobispora bisporathat were highly abundant in the most active consortium. Among the cellulases fromT. bispora, the abundance of a GH family 12 (GH12) protein correlated most closely with the changes in crystalline cellulose hydrolysis activity. This result was surprising since GH12 proteins have been predominantly characterized as enzymes active on soluble polysaccharide substrates. Heterologous expression and biochemical characterization of the suite ofT. bisporahydrolytic cellulases confirmed that the GH12 protein possessed the highest activity on multiple crystalline cellulose substrates and demonstrated that it hydrolyzes cellulose chains by a predominantly random mechanism. This work suggests that the role of GH12 proteins in crystalline cellulose hydrolysis by cellulolytic microbes should be reconsidered.

    IMPORTANCECellulose is the most abundant organic polymer on earth, and its enzymatic hydrolysis is a key reaction in the global carbon cycle and the conversion of plant biomass to biofuels. The glycoside hydrolases that depolymerize crystalline cellulose have been primarily characterized from isolates. In this study, we demonstrate that adapting microbial consortia from compost to grow on crystalline cellulose

  17. Crystal Structure of Homoserine Transacetylase from Haemophilus Influenzae Reveals a New Family of alpha/beta-Hydrolases

    Energy Technology Data Exchange (ETDEWEB)

    Mirza,I.; Nazi, I.; Korczynska, M.; Wright, G.; Berghuis, A.

    2005-01-01

    Homoserine transacetylase catalyzes one of the required steps in the biosynthesis of methionine in fungi and several bacteria. We have determined the crystal structure of homoserine transacetylase from Haemophilus influenzae to a resolution of 1.65 A. The structure identifies this enzyme to be a member of the alpha/beta-hydrolase structural superfamily. The active site of the enzyme is located near the end of a deep tunnel formed by the juxtaposition of two domains and incorporates a catalytic triad involving Ser143, His337, and Asp304. A structural basis is given for the observed double displacement kinetic mechanism of homoserine transacetylase. Furthermore, the properties of the tunnel provide a rationale for how homoserine transacetylase catalyzes a transferase reaction vs. hydrolysis, despite extensive similarity in active site architecture to hydrolytic enzymes.

  18. Two sides of the same coin: Xyloglucan endotransglucosylases/hydrolases in host infection by the parasitic plant Cuscuta.

    Science.gov (United States)

    Olsen, Stian; Popper, Zoë A; Krause, Kirsten

    2016-01-01

    The holoparasitic angiosperm Cuscuta develops haustoria that enable it to feed on other plants. Recent findings corroborate the long-standing theory that cell wall modifications are required in order for the parasite to successfully infect a host, and further suggest that changes to xyloglucan through the activity of xyloglucan endotransglucosylases/hydrolases (XTHs) are essential. On the other hand, XTH expression was also detected in resistant tomato upon an attack by Cuscuta, which suggests that both host and parasite use these enzymes in their "arms race." Here, we summarize existing data on the cell wall-modifying activities of XTHs during parasitization and present a model suggesting how XTHs might function to make the host's resources accessible to Cuscuta.

  19. Discovery of potent inhibitors of soluble epoxide hydrolase by combinatorial library design and structure-based virtual screening.

    Science.gov (United States)

    Xing, Li; McDonald, Joseph J; Kolodziej, Steve A; Kurumbail, Ravi G; Williams, Jennifer M; Warren, Chad J; O'Neal, Janet M; Skepner, Jill E; Roberds, Steven L

    2011-03-10

    Structure-based virtual screening was applied to design combinatorial libraries to discover novel and potent soluble epoxide hydrolase (sEH) inhibitors. X-ray crystal structures revealed unique interactions for a benzoxazole template in addition to the conserved hydrogen bonds with the catalytic machinery of sEH. By exploitation of the favorable binding elements, two iterations of library design based on amide coupling were employed, guided principally by the docking results of the enumerated virtual products. Biological screening of the libraries demonstrated as high as 90% hit rate, of which over two dozen compounds were single digit nanomolar sEH inhibitors by IC(50) determination. In total the library design and synthesis produced more than 300 submicromolar sEH inhibitors. In cellular systems consistent activities were demonstrated with biochemical measurements. The SAR understanding of the benzoxazole template provides valuable insights into discovery of novel sEH inhibitors as therapeutic agents.

  20. Draft genome sequence of Streptomyces sp. strain F1, a potential source for glycoside hydrolases isolated from Brazilian soil.

    Science.gov (United States)

    Melo, Ricardo Rodrigues de; Persinoti, Gabriela Felix; Paixão, Douglas Antonio Alvaredo; Squina, Fábio Márcio; Ruller, Roberto; Sato, Helia Harumi

    Here, we show the draft genome sequence of Streptomyces sp. F1, a strain isolated from soil with great potential for secretion of hydrolytic enzymes used to deconstruct cellulosic biomass. The draft genome assembly of Streptomyces sp. strain F1 has 69 contigs with a total genome size of 8,142,296bp and G+C 72.65%. Preliminary genome analysis identified 175 proteins as Carbohydrate-Active Enzymes, being 85 glycoside hydrolases organized in 33 distinct families. This draft genome information provides new insights on the key genes encoding hydrolytic enzymes involved in biomass deconstruction employed by soil bacteria. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

  1. Pain and beyond: fatty acid amides and fatty acid amide hydrolase inhibitors in cardiovascular and metabolic diseases.

    Science.gov (United States)

    Pillarisetti, Sivaram; Alexander, Christopher W; Khanna, Ish

    2009-12-01

    Fatty acid amide hydrolase (FAAH) is responsible for the hydrolysis of several important endogenous fatty acid amides (FAAs), including anandamide, oleoylethanolamide and palmitoylethanolamide. Because specific FAAs interact with cannabinoid and vanilloid receptors, they are often referred to as 'endocannabinoids' or 'endovanilloids'. Initial interest in this area, therefore, has focused on developing FAAH inhibitors to augment the actions of FAAs and reduce pain. However, recent literature has shown that these FAAs - through interactions with unique receptors (extracellular and intracellular) - can induce a diverse array of effects that include appetite suppression, modulation of lipid and glucose metabolism, vasodilation, cardiac function and inflammation. This review gives an overview of FAAs and diverse FAAH inhibitors and their potential therapeutic utility in pain and non-pain indications.

  2. Purification, crystallization and preliminary X-ray crystallographic analysis of human ppGpp hydrolase, Mesh1

    Directory of Open Access Journals (Sweden)

    Dawei Sun

    2010-09-01

    Full Text Available Bacterial SpoT is a Mn2+-dependent pyrophosphohydrolase to hydrolyze guanosine 3’-diphosphate-5’-diphosphate to guanosine diphosphate and pyrophosphate. In this study, SpoT ortholog from Homo sapiens (hMesh1, was over-expressed in Escherichia coli, purified and crystallized using hanging-drop vapour-diffusion method with polyethylene glycol and sodium citrate. The native crystal of hMesh1 was diffracted to 2.1 Å using a synchrotron-radiation source and belonged to the monoclinic space group P21 with cell dimensions of a = 53.27 Å, b = 62.61 Å, c = 52.45 Å and β= 94.96˚. The crystal contains two molecules in the asymmetric unit, with a solvent content of 44% and a Matthews coefficient VM value of 2.18 Å3/Da.

  3. Chlorophyll Degradation: The Tocopherol Biosynthesis-Related Phytol Hydrolase in Arabidopsis Seeds Is Still Missing1[C][W][OPEN

    Science.gov (United States)

    Zhang, Wei; Liu, Tianqi; Ren, Guodong; Hörtensteiner, Stefan; Zhou, Yongming; Cahoon, Edgar B.; Zhang, Chunyu

    2014-01-01

    Phytyl diphosphate (PDP) is the prenyl precursor for tocopherol biosynthesis. Based on recent genetic evidence, PDP is supplied to the tocopherol biosynthetic pathway primarily by chlorophyll degradation and sequential phytol phosphorylation. Three enzymes of Arabidopsis (Arabidopsis thaliana) are known to be capable of removing the phytol chain from chlorophyll in vitro: chlorophyllase1 (CLH1), CLH2, and pheophytin pheophorbide hydrolase (PPH), which specifically hydrolyzes pheophytin. While PPH, but not chlorophyllases, is required for in vivo chlorophyll breakdown during Arabidopsis leaf senescence, little is known about the involvement of these phytol-releasing enzymes in tocopherol biosynthesis. To explore the origin of PDP for tocopherol synthesis, seed tocopherol concentrations were determined in Arabidopsis lines engineered for seed-specific overexpression of PPH and in single and multiple mutants in the three genes encoding known dephytylating enzymes. Except for modestly increasing tocopherol content observed in the PPH overexpressor, none of the remaining lines exhibited significantly reduced tocopherol concentrations, suggesting that the known chlorophyll-derived phytol-releasing enzymes do not play major roles in tocopherol biosynthesis. Tocopherol content of seeds from double mutants in NONYELLOWING1 (NYE1) and NYE2, regulators of chlorophyll degradation, had modest reduction compared with wild-type seeds, although mature seeds of the double mutant retained significantly higher chlorophyll levels. These findings suggest that NYEs may play limited roles in regulating an unknown tocopherol biosynthesis-related phytol hydrolase. Meanwhile, seeds of wild-type over-expressing NYE1 had lower tocopherol levels, suggesting that phytol derived from NYE1-dependent chlorophyll degradation probably doesn’t enter tocopherol biosynthesis. Potential routes of chlorophyll degradation are discussed in relation to tocopherol biosynthesis. PMID:25059706

  4. Omeprazole increases the efficacy of a soluble epoxide hydrolase inhibitor in a PGE2 induced pain model

    International Nuclear Information System (INIS)

    Goswami, Sumanta Kumar; Inceoglu, Bora; Yang, Jun; Wan, Debin; Kodani, Sean D.; Trindade da Silva, Carlos Antonio; Morisseau, Christophe; Hammock, Bruce D.

    2015-01-01

    Epoxyeicosatrienoic acids (EETs) are potent endogenous analgesic metabolites produced from arachidonic acid by cytochrome P450s (P450s). Metabolism of EETs by soluble epoxide hydrolase (sEH) reduces their activity, while their stabilization by sEH inhibition decreases both inflammatory and neuropathic pain. Here, we tested the complementary hypothesis that increasing the level of EETs through induction of P450s by omeprazole (OME), can influence pain related signaling by itself, and potentiate the anti-hyperalgesic effect of sEH inhibitor. Rats were treated with OME (100 mg/kg/day, p.o., 7 days), sEH inhibitor TPPU (3 mg/kg/day, p.o.) and OME (100 mg/kg/day, p.o., 7 days) + TPPU (3 mg/kg/day, p.o., last 3 days of OME dose) dissolved in vehicle PEG400, and their effect on hyperalgesia (increased sensitivity to pain) induced by PGE 2 was monitored. While OME treatment by itself exhibited variable effects on PGE 2 induced hyperalgesia, it strongly potentiated the effect of TPPU in the same assay. The significant decrease in pain with OME + TPPU treatment correlated with the increased levels of EETs in plasma and increased activities of P450 1A1 and P450 1A2 in liver microsomes. The results show that reducing catabolism of EETs with a sEH inhibitor yielded a stronger analgesic effect than increasing generation of EETs by OME, and combination of both yielded the strongest pain reducing effect under the condition of this study. - Highlights: • The soluble epoxide hydrolase (sEH) inhibitor TPPU is anti-hyperalgesic. • Omeprazole potentiates the anti-hyperalgesic actions of TPPU. • This potentiation is associated with increased P450 activity. • The potentiation is associated with an increase in fatty acid epoxide/diol ratio. • Joint use of sEH inhibitors and P450 inducers could result in drug–drug interactions.

  5. Lipopolysaccharide-induced pulmonary inflammation is not accompanied by a release of anandamide into the lavage fluid or a down-regulation of the activity of fatty acid amide hydrolase

    DEFF Research Database (Denmark)

    Holt, S.; J. Fowler, C.; Rocksén, D.

    2004-01-01

    The effect of lipopolysaccharide inhalation upon lung anandamide levels, anandamide synthetic enzymes and fatty acid amide hydrolase has been investigated. Lipopolysaccharide exposure produced a dramatic extravasation of neutrophils and release of tumour necrosis factor a into the bronchoalveolar......-acyltransferase and N-acylphosphatidylethanolamine phospholipase D and the activity of fatty acid amide hydrolase in lung membrane fractions did not change significantly following the exposure to lipopolysaccharide. The non-selective fatty acid amide hydrolase inhibitor phenylmethylsulfonyl fluoride was a less potent...... inhibitor of lung fatty acid amide hydrolase than expected from the literature, and a dose of 30 mg/kg i.p. of this compound, which produced a complete inhibition of brain anandamide metabolism, only partially inhibited the lung metabolic activity....

  6. The ubiquitin C-terminal hydrolase UCH-L1 promotes bacterial invasion by altering the dynamics of the actin cytoskeleton

    DEFF Research Database (Denmark)

    Basseres, Eugene; Coppotelli, Giuseppe; Pfirrmann, Thorsten

    2010-01-01

    Invasion of eukaryotic target cells by pathogenic bacteria requires extensive remodelling of the membrane and actin cytoskeleton. Here we show that the remodelling process is regulated by the ubiquitin C-terminal hydrolase UCH-L1 that promotes the invasion of epithelial cells by Listeria monocyto......Invasion of eukaryotic target cells by pathogenic bacteria requires extensive remodelling of the membrane and actin cytoskeleton. Here we show that the remodelling process is regulated by the ubiquitin C-terminal hydrolase UCH-L1 that promotes the invasion of epithelial cells by Listeria...... of downstream ERK1/2- and AKT-dependent signalling in response to the natural ligand Hepatocyte Growth Factor (HGF). The regulation of cytoskeleton dynamics was further confirmed by the induction of actin stress fibres in HeLa expressing the active enzyme but not the catalytic mutant UCH-L1(C90S...

  7. Impact of the Staphylococcus epidermidis LytSR two-component regulatory system on murein hydrolase activity, pyruvate utilization and global transcriptional profile

    Directory of Open Access Journals (Sweden)

    Yu Fangyou

    2010-11-01

    Full Text Available Abstract Background Staphylococcus epidermidis has emerged as one of the most important nosocomial pathogens, mainly because of its ability to colonize implanted biomaterials by forming a biofilm. Extensive studies are focused on the molecular mechanisms involved in biofilm formation. The LytSR two-component regulatory system regulates autolysis and biofilm formation in Staphylococcus aureus. However, the role of LytSR played in S. epidermidis remained unknown. Results In the present study, we demonstrated that lytSR knock-out in S. epidermidis did not alter susceptibility to Triton X-100 induced autolysis. Quantitative murein hydrolase assay indicated that disruption of lytSR in S. epidermidis resulted in decreased activities of extracellular murein hydrolases, although zymogram showed no apparent differences in murein hydrolase patterns between S. epidermidis strain 1457 and its lytSR mutant. Compared to the wild-type counterpart, 1457ΔlytSR produced slightly more biofilm, with significantly decreased dead cells inside. Microarray analysis showed that lytSR mutation affected the transcription of 164 genes (123 genes were upregulated and 41 genes were downregulated. Specifically, genes encoding proteins responsible for protein synthesis, energy metabolism were downregulated, while genes involved in amino acid and nucleotide biosynthesis, amino acid transporters were upregulated. Impaired ability to utilize pyruvate and reduced activity of arginine deiminase was observed in 1457ΔlytSR, which is consistent with the microarray data. Conclusions The preliminary results suggest that in S. epidermidis LytSR two-component system regulates extracellular murein hydrolase activity, bacterial cell death and pyruvate utilization. Based on the microarray data, it appears that lytSR inactivation induces a stringent response. In addition, LytSR may indirectly enhance biofilm formation by altering the metabolic status of the bacteria.

  8. Evaluation of the precision-cut liver and lung slice systems for the study of induction of CYP1, epoxide hydrolase and glutathione S-transferase activities.

    Science.gov (United States)

    Pushparajah, Daphnee S; Umachandran, Meera; Plant, Kathryn E; Plant, Nick; Ioannides, Costas

    2007-02-28

    The principal objective was to ascertain whether precision-cut tissue slices can be used to evaluate the potential of chemicals to induce CYP1, epoxide hydrolase and glutathione S-transferase activities, all being important enzymes involved in the metabolism of polycyclic aromatic hydrocarbons. Precision-cut rat liver and lung slices were incubated with a range of benzo[a]pyrene concentrations for various time periods. A rise in the O-deethylation of ethoxyresorufin was seen in both liver and lung slices exposed to benzo[a]pyrene, which was accompanied by increased CYP1A apoprotein levels. Pulmonary CYP1B1 apoprotein levels and hepatic mRNA levels were similarly enhanced. Elevated epoxide hydrolase and glutathione S-transferase activities were also observed in liver slices following incubation for 24h; similarly, a rise in apoprotein levels of both enzymes was evident, peak levels occurring at the same time point. When mRNA levels were monitored, a rise in the levels of both enzymes was seen as early as 4h after incubation, but maximum levels were attained at 24 h. In lung slices, induction of epoxide hydrolase by benzo[a]pyrene was observed after a 24-h incubation, and at a concentration of 1 microM; a rise in apoprotein levels was seen at this time point. Glutathione S-transferase activity was not inducible in lung slices by benzo[a]pyrene but a modest increase was observed in hepatic slices. Collectively, these studies confirmed CYP1A induction in rat liver slices and established that CYP1B1 expression, and epoxide hydrolase and glutathione S-transferase activities are inducible in precision-cut tissue slices.

  9. Response of a diuron-degrading community to diuron exposure assessed by real-time quantitative PCR monitoring of phenylurea hydrolase A and B encoding genes

    OpenAIRE

    Pesce , S.; Beguet , J.; Rouard , N.; Devers Lamrani , M.; Martin Laurent , F.

    2013-01-01

    A real-time quantitative PCR method was developed to detect and quantify phenlylurea hydrolase genes' (puhA and puhB) sequences from environmental DNA samples to assess diuron-degrading genetic potential in some soil and sediment microbial communities. In the soil communities, mineralization rates (determined with [ring-14C]-labeled diuron) were linked to diuron-degrading genetic potentials estimated from puhB number copies, which increased following repeated diuron treatments. In the sedimen...

  10. Cloning, expression and characterization of a mammalian Nudix hydrolase-like enzyme that cleaves the pyrophosphate bond of UDP-glucose.

    OpenAIRE

    Yagi, Toshihiro; Baroja-Fernández, Edurne; Yamamoto, Ryuji; Muñoz, Francisco José; Akazawa, Takashi; Hong, Kyoung Su; Pozueta-Romero, Javier

    2003-01-01

    A distinct UDP-glucose (UDPG) pyrophosphatase (UGPPase, EC 3.6.1.45) has been characterized using pig kidney ( Sus scrofa ). This enzyme hydrolyses UDPG, the precursor molecule of numerous glycosylation reactions in animals, to produce glucose 1-phosphate (G1P) and UMP. Sequence analyses of the purified enzyme revealed that, similar to the case of a nucleotide-sugar hydrolase controlling the intracellular levels of ADP-glucose linked to glycogen biosynthesis in Escherichia coli [Moreno-Bruna,...

  11. Ubiquitin C-Terminal Hydrolase-Activity Is Involved in Sperm Acrosomal Function and Anti-polyspermy Defense During Porcine Fertilization

    Czech Academy of Sciences Publication Activity Database

    Yi, Y. J.; Manandhar, G.; Sutovsky, M.; Rongfeng, L.; Jonáková, Věra; Oko, R.; Park, C. S.; Prather, R.S.; Sutovsky, P.

    2007-01-01

    Roč. 77, č. 5 (2007), s. 780-793 ISSN 0006-3363 R&D Projects: GA ČR GA303/06/0895; GA MŠk 1M06011 Institutional research plan: CEZ:AV0Z50520514; CEZ:AV0Z50520701 Keywords : Ubiquitin * proteasome * hydrolase * spermadhesin Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.670, year: 2007

  12. 1,3-Disubstituted Ureas Functionalized with Ether Groups are Potent Inhibitors of the Soluble Epoxide Hydrolase with Improved Pharmacokinetic Properties

    OpenAIRE

    Kim, In-Hae; Tsai, Hsing-Ju; Nishi, Kosuke; Kasagami, Takeo; Morisseau, Christophe; Hammock, Bruce D.

    2007-01-01

    Soluble epoxide hydrolase (sEH) is a therapeutic target for treating hypertension and inflammation. 1,3-Disubstituted ureas functionalized with an ether group are potent sEH inhibitors. However, their relatively low metabolic stability leads to poor pharmacokinetic properties. To improve their bioavailability, we investigated the effect of incorporating various polar groups on the ether function on the inhibition potencies, physical properties, in vitro metabolic stability, and pharmacokineti...

  13. Mechanism-based fluorescent labeling of beta-galactosidases. An efficient method in proteomics for glycoside hydrolases.

    Science.gov (United States)

    Kurogochi, Masaki; Nishimura, Shin-Ichiro; Lee, Yuan Chuan

    2004-10-22

    (4-N-5-Dimethylaminonaphthalene-1-sulfonyl-2-difluoromethylphenyl)-beta-d-galactopyranoside was synthesized and successfully tested on beta-galactosidases from Xanthomonas manihotis (Wong-Madden, S. T., and Landry, D. Glycobiology (1995) 5, 19-28 and Taron, C. H., Benner, J. S., Hornstra, L. J., and Guthrie, E. P. (1995) Glycobiology 5, 603-610), Escherichia coli (Jacobson, R. H., Zhang, X. J., DuBose, R. F., and Matthews, B. W. (1994) Nature 369, 761-766), and Bacillus circulans (Fujimoto, H., Miyasato, M., Ito, Y., Sasaki, T., and Ajisaka, K. (1988) Glycoconj. J. 15, 155-160) for the rapid identification of the catalytic site. Reaction of the irreversible inhibitor with enzymes proceeded to afford a fluorescence-labeled protein suitable for further high throughput characterization by using antidansyl antibody and matrix-assisted laser desorption ionization time-of-flight/time-of-flight (MALDI-TOF/TOF). Specific probing by a fluorescent aglycon greatly facilitated identification of the labeled peptide fragments from beta-galactosidases. It was demonstrated by using X. manihotis beta-galactosidase that the Arg-58 residue, which is located within a sequence of 56IPRAYWKD63, was labeled by nucleophilic attack of the guanidinyl group. This sequence including Arg-58 (Leu-46 to Tyr-194) was similar to that (Met-1 to Tyr-151) of Thermus thermophilus A4, which is the first known structure of glycoside hydrolases family 42 (Hidaka, M., Fushinobu, S., Ohtsu, N., Motoshima, H., Matsuzawa, H., Shoun, H., and Wakagi, T. (2002) J. Mol. Biol. 322, 79-91). A catalytic glutamic acid (Glu-537) of E. coli beta-galactosidase was proved to be labeled by the same procedure, suggesting that the modification site with this irreversible substrate might depend both on the nucleophilicity of the amino acids and their spatial arrangement in the individual catalytic cavity. Similarly, a Glu-259 in 257TLEE260 was selectively labeled using B. circulans beta-galactosidase, indicating that Glu

  14. Potential of the virion-associated peptidoglycan hydrolase HydH5 and its derivative fusion proteins in milk biopreservation.

    Directory of Open Access Journals (Sweden)

    Lorena Rodríguez-Rubio

    Full Text Available Bacteriophage lytic enzymes have recently attracted considerable interest as novel antimicrobials against Gram-positive bacteria. In this work, antimicrobial activity in milk of HydH5 [a virion-associated peptidoglycan hydrolase (VAPGH encoded by the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88], and three different fusion proteins created between HydH5 and lysostaphin has been assessed. The lytic activity of the five proteins (HydH5, HydH5Lyso, HydH5SH3b, CHAPSH3b and lysostaphin was confirmed using commercial whole extended shelf-life milk (ESL in challenge assays with 10(4 CFU/mL of the strain S. aureus Sa9. HydH5, HydH5Lyso and HydH5SH3b (3.5 µM kept the staphylococcal viable counts below the control cultures for 6 h at 37°C. The effect is apparent just 15 minutes after the addition of the lytic enzyme. Of note, lysostaphin and CHAPSH3b showed the highest staphylolytic protection as they were able to eradicate the initial staphylococcal challenge immediately or 15 min after addition, respectively, at lower concentration (1 µM at 37°C. CHAPSH3b showed the same antistaphyloccal effect at room temperature (1.65 µM. No re-growth was observed for the remainder of the experiment (up to 6 h. CHAPSH3b activity (1.65 µM was also assayed in raw (whole and skim and pasteurized (whole and skim milk. Pasteurization of milk clearly enhanced CHAPSH3b staphylolytic activity in both whole and skim milk at both temperatures. This effect was most dramatic at room temperature as this protein was able to reduce S. aureus viable counts to undetectable levels immediately after addition with no re-growth detected for the duration of the experiment (360 min. Furthermore, CHAPSH3b protein is known to be heat tolerant and retained some lytic activity after pasteurization treatment and after storage at 4°C for 3 days. These results might facilitate the use of the peptidoglycan hydrolase HydH5 and its derivative fusions, particularly CHAPSH3b, as

  15. Potential of the Virion-Associated Peptidoglycan Hydrolase HydH5 and Its Derivative Fusion Proteins in Milk Biopreservation

    Science.gov (United States)

    Rodríguez-Rubio, Lorena; Martínez, Beatriz; Donovan, David M.; García, Pilar; Rodríguez, Ana

    2013-01-01

    Bacteriophage lytic enzymes have recently attracted considerable interest as novel antimicrobials against Gram-positive bacteria. In this work, antimicrobial activity in milk of HydH5 [a virion-associated peptidoglycan hydrolase (VAPGH) encoded by the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88], and three different fusion proteins created between HydH5 and lysostaphin has been assessed. The lytic activity of the five proteins (HydH5, HydH5Lyso, HydH5SH3b, CHAPSH3b and lysostaphin) was confirmed using commercial whole extended shelf-life milk (ESL) in challenge assays with 104 CFU/mL of the strain S. aureus Sa9. HydH5, HydH5Lyso and HydH5SH3b (3.5 µM) kept the staphylococcal viable counts below the control cultures for 6 h at 37°C. The effect is apparent just 15 minutes after the addition of the lytic enzyme. Of note, lysostaphin and CHAPSH3b showed the highest staphylolytic protection as they were able to eradicate the initial staphylococcal challenge immediately or 15 min after addition, respectively, at lower concentration (1 µM) at 37°C. CHAPSH3b showed the same antistaphyloccal effect at room temperature (1.65 µM). No re-growth was observed for the remainder of the experiment (up to 6 h). CHAPSH3b activity (1.65 µM) was also assayed in raw (whole and skim) and pasteurized (whole and skim) milk. Pasteurization of milk clearly enhanced CHAPSH3b staphylolytic activity in both whole and skim milk at both temperatures. This effect was most dramatic at room temperature as this protein was able to reduce S. aureus viable counts to undetectable levels immediately after addition with no re-growth detected for the duration of the experiment (360 min). Furthermore, CHAPSH3b protein is known to be heat tolerant and retained some lytic activity after pasteurization treatment and after storage at 4°C for 3 days. These results might facilitate the use of the peptidoglycan hydrolase HydH5 and its derivative fusions, particularly CHAPSH3b, as biocontrol agents

  16. Omeprazole increases the efficacy of a soluble epoxide hydrolase inhibitor in a PGE{sub 2} induced pain model

    Energy Technology Data Exchange (ETDEWEB)

    Goswami, Sumanta Kumar; Inceoglu, Bora; Yang, Jun; Wan, Debin; Kodani, Sean D. [Department of Entomology and Nematology, UC Davis Comprehensive Cancer Center, University of California, Davis, CA (United States); Trindade da Silva, Carlos Antonio [Department of Entomology and Nematology, UC Davis Comprehensive Cancer Center, University of California, Davis, CA (United States); Department of Genetics and Biochemistry, Federal University of Uberlandia, MG (Brazil); Morisseau, Christophe [Department of Entomology and Nematology, UC Davis Comprehensive Cancer Center, University of California, Davis, CA (United States); Hammock, Bruce D., E-mail: bdhammock@ucdavis.edu [Department of Entomology and Nematology, UC Davis Comprehensive Cancer Center, University of California, Davis, CA (United States)

    2015-12-15

    Epoxyeicosatrienoic acids (EETs) are potent endogenous analgesic metabolites produced from arachidonic acid by cytochrome P450s (P450s). Metabolism of EETs by soluble epoxide hydrolase (sEH) reduces their activity, while their stabilization by sEH inhibition decreases both inflammatory and neuropathic pain. Here, we tested the complementary hypothesis that increasing the level of EETs through induction of P450s by omeprazole (OME), can influence pain related signaling by itself, and potentiate the anti-hyperalgesic effect of sEH inhibitor. Rats were treated with OME (100 mg/kg/day, p.o., 7 days), sEH inhibitor TPPU (3 mg/kg/day, p.o.) and OME (100 mg/kg/day, p.o., 7 days) + TPPU (3 mg/kg/day, p.o., last 3 days of OME dose) dissolved in vehicle PEG400, and their effect on hyperalgesia (increased sensitivity to pain) induced by PGE{sub 2} was monitored. While OME treatment by itself exhibited variable effects on PGE{sub 2} induced hyperalgesia, it strongly potentiated the effect of TPPU in the same assay. The significant decrease in pain with OME + TPPU treatment correlated with the increased levels of EETs in plasma and increased activities of P450 1A1 and P450 1A2 in liver microsomes. The results show that reducing catabolism of EETs with a sEH inhibitor yielded a stronger analgesic effect than increasing generation of EETs by OME, and combination of both yielded the strongest pain reducing effect under the condition of this study. - Highlights: • The soluble epoxide hydrolase (sEH) inhibitor TPPU is anti-hyperalgesic. • Omeprazole potentiates the anti-hyperalgesic actions of TPPU. • This potentiation is associated with increased P450 activity. • The potentiation is associated with an increase in fatty acid epoxide/diol ratio. • Joint use of sEH inhibitors and P450 inducers could result in drug–drug interactions.

  17. The Vital Function of Fe3O4@Au nanocomposites for Hydrolase Biosensor Design and Its Application in Detection of Methyl Parathion

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Yuting; Zhang, Weiying; Lin, Yuehe; Du, Dan

    2013-02-04

    A nanocomposite of gold nanoparticles (AuNPs) decorating a magnetic Fe3O4 core was synthesized using cysteamine (SH–NH2) as linker, and characterized by TEM, XPS, UV and electrochemistry. Then a hydrolase biosensor, based on self-assembly of methyl parathion hydrolase (MPH) on the Fe3O4@Au nanocomposite, was developed for sensitive and selective detection of the organophosphorus pesticide (OP) methyl parathion. The magnetic nanocomposite provides an easy way to construct the enzyme biosensor by simply exerting an external magnetic field, and also provides a simple way to renew the electrode surface by removing the magnet. Unlike inhibition-based enzyme biosensors, the hydrolase is not poisoned by OPs and thus is reusable for continuous measurement. AuNPs not only provide a large surface area, high loading efficiency and fast electron transfer, but also stabilize the enzyme through electrostatic interactions. The MPH biosensor shows rapid response and high selectivity for detection of methyl parathion, with a linear range from 0.5 to 1000 ng/mL and a detection limit of 0.1 ng/mL. It also shows acceptable reproducibility and stability. The simplicity and ease of operation of the proposed method has great potential for on-site detection of P–S containing pesticides and provides a promising strategy to construct a robust biosensor.

  18. Evaluation of the Stability of the Total Antioxidant Capacity, Polyphenol Contents, and Starch Hydrolase Inhibitory Activities of Kombucha Teas Using an In Vitro Model of Digestion

    Directory of Open Access Journals (Sweden)

    Mindani I. Watawana

    2015-01-01

    Full Text Available The objective of this study was to evaluate and compare antioxidant and starch hydrolase inhibitory activity of three different types of Kombucha beverages prepared by three pellicles with different microbial compositions. The fermentation process was carried out for 7 days and the assessments of antioxidant and starch hydrolase inhibitory activities as well as tea phenolic compounds were carried out. These parameters were also evaluated after subjecting the final fermented samples to gastric and duodenal digestion in an in vitro digestion model. The pH had a statistically significant decrease during the period of fermentation. The total phenolics content and antioxidant activities had increased during the fermentation process as well as when subjected to digestion. The starch hydrolase inhibitory activities also increased in a similar manner during the different phases. The α-amylase and α-glucosidase inhibitory activities showed statistically significant increases (P<0.05 as the fermentation progressed, while an increase was observed after being subjected to pancreatic and duodenal digestion as well. All three types of tea showed a higher α-amylase inhibitory activity than α-glucosidase inhibitory activity.

  19. The Antioxidant and Starch Hydrolase Inhibitory Activity of Ten Spices in an In Vitro Model of Digestion: Bioaccessibility of Anthocyanins and Carotenoids

    Directory of Open Access Journals (Sweden)

    Nilakshi Jayawardena

    2015-01-01

    Full Text Available The antioxidant and starch hydrolase inhibitory activities of cardamom, cloves, coriander, cumin seeds, curry leaves, fenugreek, mustard seeds, nutmeg, sweet cumin, and star anise extracts were investigated in an in vitro model of digestion mimicking the gastric and duodenal conditions. The total phenolic contents in all spice extracts had statistically significantly (P<0.05 increased following both gastric and duodenal digestion. This was also in correlation with the antioxidant assays quantifying the water-soluble antioxidant capacity of the extracts. The lipophilic Oxygen Radical Absorbance Capacity assay did not indicate a statistically significant change in the values during any of the digestion phases. Statistically significant (P<0.05 reductions in the anthocyanin contents were observed during the digestion phases in contrast to the carotenoid contents. With the exception of the cumin seed extract, none of the spice extracts showed statistically significant changes in the initial starch hydrolase enzyme inhibitory values prior to gastric and duodenal digestion. In conclusion, this study was able to prove that the 10 spices were a significant source of total phenolics, antioxidant, and starch hydrolase inhibitory activities.

  20. Solution Structure of Archaeoglobus fulgidis Peptidyl-tRNA Hydrolase(Pth2) Provides Evidence for an Extensive Conserved Family of Pth2 Enzymes in Archaea, Bacteria and Eukaryotes.

    Energy Technology Data Exchange (ETDEWEB)

    Powers, Robert; Mirkovic, Nebojsa; Goldsmith-Fischman, Sharon; Acton, Thomas; Chiang, Yiwen; Huang, Yuanpeng; Ma, LiChung; Rajan, Paranji K.; Cort, John R.; Kennedy, Michael A.; Liu, Jinfeng; Rost, Burkhard; Honig, Barry; Murray, Diana; Montelione, Gaetano

    2005-11-01

    The solution structure of protein AF2095 from the thermophilic archaea Archaeglobus fulgidis, a 123-residue (13.6 kDa) protein, has been determined by NMR methods. The structure of AF2095 is comprised of four a-helices and a mixed b-sheet consisting of four parallel and anti-parallel b-strands, where the a-helices sandwich the b-sheet. Sequence and structural comparison of AF2095 with proteins from Homo sapiens, Methanocaldococcus jannaschii and Sulfolobus solfataricus, reveals that AF2095 is a peptidyl-tRNA hydrolase (Pth2). This structural comparison also identifies putative catalytic residues and a tRNA interaction region for AF2095. The structure of AF2095 is also similar to the structure of protein TA0108 from archaea Thermoplasma acidophilum, which is deposited in the Protein Database but not functionally annotated. The NMR structure of AF2095 has been further leveraged to obtain good quality structural models for 55 other proteins. Although earlier studies have proposed that the Pth2 protein family is restricted to archeal and eukaryotic organisms, the similarity of the AF2095 structure to human Pth2, the conservation of key active-site residues, and the good quality of the resulting homology models demonstrate a large family of homologous Pth2 proteins that are conserved in eukaryotic, archaeal and bacterial organisms, providing novel insights in the evolution of the Pth and Pth2 enzyme families.

  1. Mutational and structural analyses of Caldanaerobius polysaccharolyticus Man5B reveal novel active site residues for family 5 glycoside hydrolases.

    Science.gov (United States)

    Oyama, Takuji; Schmitz, George E; Dodd, Dylan; Han, Yejun; Burnett, Alanna; Nagasawa, Naoko; Mackie, Roderick I; Nakamura, Haruki; Morikawa, Kosuke; Cann, Isaac

    2013-01-01

    CpMan5B is a glycoside hydrolase (GH) family 5 enzyme exhibiting both β-1,4-mannosidic and β-1,4-glucosidic cleavage activities. To provide insight into the amino acid residues that contribute to catalysis and substrate specificity, we solved the structure of CpMan5B at 1.6 Å resolution. The structure revealed several active site residues (Y12, N92 and R196) in CpMan5B that are not present in the active sites of other structurally resolved GH5 enzymes. Residue R196 in GH5 enzymes is thought to be strictly conserved as a histidine that participates in an electron relay network with the catalytic glutamates, but we show that an arginine fulfills a functionally equivalent role and is found at this position in every enzyme in subfamily GH5_36, which includes CpMan5B. Residue N92 is required for full enzymatic activity and forms a novel bridge over the active site that is absent in other family 5 structures. Our data also reveal a role of Y12 in establishing the substrate preference for CpMan5B. Using these molecular determinants as a probe allowed us to identify Man5D from Caldicellulosiruptor bescii as a mannanase with minor endo-glucanase activity.

  2. Allelic Variation of Bile Salt Hydrolase Genes in Lactobacillus salivarius Does Not Determine Bile Resistance Levels▿ †

    Science.gov (United States)

    Fang, Fang; Li, Yin; Bumann, Mario; Raftis, Emma J.; Casey, Pat G.; Cooney, Jakki C.; Walsh, Martin A.; O'Toole, Paul W.

    2009-01-01

    Commensal lactobacilli frequently produce bile salt hydrolase (Bsh) enzymes whose roles in intestinal survival are unclear. Twenty-six Lactobacillus salivarius strains from different sources all harbored a bsh1 allele on their respective megaplasmids. This allele was related to the plasmid-borne bsh1 gene of the probiotic strain UCC118. A second locus (bsh2) was found in the chromosomes of two strains that had higher bile resistance levels. Four Bsh1-encoding allele groups were identified, defined by truncations or deletions involving a conserved residue. In vitro analyses showed that this allelic variation was correlated with widely varying bile deconjugation phenotypes. Despite very low activity of the UCC118 Bsh1 enzyme, a mutant lacking this protein had significantly lower bile resistance, both in vitro and during intestinal transit in mice. However, the overall bile resistance phenotype of this and other strains was independent of the bsh1 allele type. Analysis of the L. salivarius transcriptome upon exposure to bile and cholate identified a multiplicity of stress response proteins and putative efflux proteins that appear to broadly compensate for, or mask, the effects of allelic variation of bsh genes. Bsh enzymes with different bile-degrading kinetics, though apparently not the primary determinants of bile resistance in L. salivarius, may have additional biological importance because of varying effects upon bile as a signaling molecule in the host. PMID:19592587

  3. Enhancement of epoxide hydrolase production by 60 Co gamma and UV irradiation mutagenesis of Aspergillus niger ZJB-09103.

    Science.gov (United States)

    Jin, Huo-Xi; OuYang, Xiao-Kun; Hu, Zhong-Ce

    2017-05-01

    An effective epoxide hydrolase (EH) production strain was mutagenized using 60 Co gamma and UV irradiation. Among positive mutant strains, the EH activity of C2-44 reached 33.7 U/g, which was 267% as much as that of the original Aspergillus niger ZJB-09103. Compared with the wild type, there were significant changes in morphology for C2-44, including the color of mycelia on the slants and the shape of conidial head. In addition, glucose and soybean cake were the optimal carbon and nitrogen source in terms of EH activity for the mutant C2-44 instead of soluble starch and peptone for the wild-type strain. The reaction time required to reach 99% enantiomeric excesses of (S)-epichlorohydrin from racemic substrate was shortened significantly by the mutant C2-44. This phenomenon was probably explained by the higher V max for hydrolysis of racemic epichlorohydrin by C2-44 compared with Aspergillus niger ZJB-09103. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

  4. Optical Detection of Paraoxon Using Single-Walled Carbon Nanotube Films with Attached Organophosphorus Hydrolase-Expressed Escherichia coli

    Directory of Open Access Journals (Sweden)

    Intae Kim

    2015-05-01

    Full Text Available In whole-cell based biosensors, spectrophotometry is one of the most commonly used methods for detecting organophosphates due to its simplicity and reliability. The sensor performance is directly affected by the cell immobilization method because it determines the amount of cells, the mass transfer rate, and the stability. In this study, we demonstrated that our previously-reported microbe immobilization method, a microbe-attached single-walled carbon nanotube film, can be applied to whole-cell-based organophosphate sensors. This method has many advantages over other whole-cell organophosphate sensors, including high specific activity, quick cell immobilization, and excellent stability. A device with circular electrodes was fabricated for an enlarged cell-immobilization area. Escherichia coli expressing organophosphorus hydrolase in the periplasmic space and single-walled carbon nanotubes were attached to the device by our method. Paraoxon was hydrolyzed using this device, and detected by measuring the concentration of the enzymatic reaction product, p-nitrophenol. The specific activity of our device was calculated, and was shown to be over 2.5 times that reported previously for other whole-cell organophosphate sensors. Thus, this method for generation of whole-cell-based OP biosensors might be optimal, as it overcomes many of the caveats that prevent the widespread use of other such devices.

  5. Identification of N-ethylmethylamine as a novel scaffold for inhibitors of soluble epoxide hydrolase by crystallographic fragment screening.

    Science.gov (United States)

    Amano, Yasushi; Tanabe, Eiki; Yamaguchi, Tomohiko

    2015-05-15

    Soluble epoxide hydrolase (sEH) is a potential target for the treatment of inflammation and hypertension. X-ray crystallographic fragment screening was used to identify fragment hits and their binding modes. Eight fragment hits were identified via soaking of sEH crystals with fragment cocktails, and the co-crystal structures of these hits were determined via individual soaking. Based on the binding mode, N-ethylmethylamine was identified as a promising scaffold that forms hydrogen bonds with the catalytic residues of sEH, Asp335, Tyr383, and Tyr466. Compounds containing this scaffold were selected from an in-house chemical library and assayed. Although the starting fragment had a weak inhibitory activity (IC50: 800μM), we identified potent inhibitors including 2-({[2-(adamantan-1-yl)ethyl]amino}methyl)phenol exhibiting the highest inhibitory activity (IC50: 0.51μM). This corresponded to a more than 1500-fold increase in inhibitory activity compared to the starting fragment. Co-crystal structures of the hit compounds demonstrate that the binding of N-ethylmethylamine to catalytic residues is similar to that of the starting fragment. We therefore consider crystallographic fragment screening to be appropriate for the identification of weak but promising fragment hits. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Isolation of oxamyl-degrading bacteria and identification of cehA as a novel oxamyl hydrolase gene

    Directory of Open Access Journals (Sweden)

    Konstantina eRousidou

    2016-04-01

    Full Text Available Microbial degradation is the main process controlling the environmental dissipation of the nematicide oxamyl. Despite that, little is known regarding the microorganisms involved in its biotransformation. We report the isolation of four oxamyl-degrading bacterial strains from an agricultural soil exhibiting enhanced biodegradation of oxamyl. Multilocus sequence analysis (MLSA assigned the isolated bacteria to different subgroups of the genus Pseudomonas. The isolated bacteria hydrolyzed oxamyl to oxamyl oxime, which was not further transformed, and utilized methylamine as a C and N source. This was further supported by the detection of methylamine dehydrogenase in three of the four isolates. All oxamyl-degrading strains carried a gene highly homologous to a carbamate-hydrolase gene cehA previously identified in carbaryl- and carbofuran-degrading strains. Transcription analysis verified its direct involvement in the hydrolysis of oxamyl. Selected isolates exhibited relaxed degrading specificity and transformed all carbamates tested including the oximino carbamates aldicarb and methomyl (structurally related to oxamyl and the aryl-methyl carbamates carbofuran and carbaryl which share with oxamyl only the carbamate moiety

  7. Metal ion coordination in the E. coli Nudix hydrolase dihydroneopterin triphosphate pyrophosphatase: New clues into catalytic mechanism.

    Directory of Open Access Journals (Sweden)

    Shannon E Hill

    Full Text Available Dihydroneopterin triphosphate pyrophosphatase (DHNTPase, a member of the Mg2+ dependent Nudix hydrolase superfamily, is the recently-discovered enzyme that functions in the second step of the pterin branch of the folate biosynthetic pathway in E. coli. DHNTPase is of interest because inhibition of enzymes in bacterial folate biosynthetic pathways is a strategy for antibiotic development. We determined crystal structures of DHNTPase with and without activating, Mg2+-mimicking metals Co2+ and Ni2+. Four metal ions, identified by anomalous scattering, and stoichiometrically confirmed in solution by isothermal titration calorimetry, are held in place by Glu56 and Glu60 within the Nudix sequence motif, Glu117, waters, and a sulfate ion, of which the latter is further stabilized by a salt bridge with Lys7. In silico docking of the DHNTP substrate reveals a binding mode in which the pterin ring moiety is nestled in a largely hydrophobic pocket, the β-phosphate activated for nucleophilic attack overlays with the crystallographic sulfate and is in line with an activated water molecule, and remaining phosphate groups are stabilized by all four identified metal ions. The structures and binding data provide new details regarding DHNTPase metal requirements, mechanism, and suggest a strategy for efficient inhibition.

  8. Inhibition of soluble epoxide hydrolase lowers portal hypertension in cirrhotic rats by ameliorating endothelial dysfunction and liver fibrosis.

    Science.gov (United States)

    Deng, Wensheng; Zhu, Yiming; Lin, Jiayun; Zheng, Lei; Zhang, Chihao; Luo, Meng

    2017-07-01

    Epoxyeicostrienoic acids (EETs) are arachidonic acid derived meditators which are catalyzed by soluble epoxide hydrolase (sEH) to less active dihydroeicostrienoics acids (DHETS). The aim of our study is to investigate the effects of sEH inhibition on hepatic and systemic hemodynamics, hepatic endothelial dysfunction, and hepatic fibrosis in CCl4 cirrhotic rats. The sEH inhibitor,trans-4-{4-[3-(4-trifluoromethoxyphenyl)-ureido]cyclohexyloxy}benzoic acid (t-TUCB) was administered to stabilize hepatic EETs by gavage at a dose of 1mg/kg/d. Our results showed that hepatic sEH expression was markedly increased in portal hypertension, and led to a lower ratio of EETs/DHETs which was effectively reversed by t-TUCB administration. t-TUCB significantly decreased portal pressure without significant changes in systemic hemodynamics, which was associated with the attenuation of intrahepatic vascular resistance (IHVR) and liver fibrosis. t-TUCB ameliorated endothelial dysfunction, increased hepatic endothelial nitric oxide synthase (eNOS) phosphorylation and nitric oxide (NO) production. In addition, t-TUCB significantly reduced alpha-Smooth Muscle Actin (α-SMA) expression and liver fibrosis, which was associated with a decrease in NF-κB signaling. Taken together, inhibition of sEH reduces portal pressure, liver fibrosis and attenuates hepatic endothelial dysfunction in cirrhotic rats. Our results indicate that sEH inhbitors may be useful in the treatment of portal hypertension in patients with cirrhosis. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Anaerobic accumulation of short-chain fatty acids from algae enhanced by damaging cell structure and promoting hydrolase activity.

    Science.gov (United States)

    Feng, Leiyu; Chen, Yunzhi; Chen, Xutao; Duan, Xu; Xie, Jing; Chen, Yinguang

    2018-02-01

    Short-chain fatty acid (SCFAs) produced from harvested algae by anaerobic fermentation with uncontrolled pH was limited due to the solid cell structure of algae. This study, therefore, was undertaken to enhance the generation of SCFAs from algae by controlling the fermentation pH. pH influenced not only the total SCFAs production, but the percentage of individual SCFA. The maximal yield of SCFAs occurred at pH 10.0 and fermentation time of 6 d (3161 mg COD/L), which mainly contained acetic and iso-valeric acids and was nearly eight times that at uncontrolled pH (392 mg COD/L). Mechanism exploration revealed at alkaline pH, especially at pH 10.0, not only the cell structure of algae was damaged effectively, but also activities and relative quantification of hydrolases as well as the abundance of microorganisms responsible for organics hydrolysis and SCFAs production were improved. Also, the released microcystins from algae were removed efficiently during alkaline anaerobic fermentation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Activity of xyloglucan endotransglucosylases/hydrolases suggests a role during host invasion by the parasitic plant Cuscuta reflexa.

    Science.gov (United States)

    Olsen, Stian; Krause, Kirsten

    2017-01-01

    The parasitic vines of the genus Cuscuta form haustoria that grow into other plants and connect with their vascular system, thus allowing the parasite to feed on its host. A major obstacle that meets the infection organ as it penetrates the host tissue is the rigid plant cell wall. In the present study, we examined the activity of xyloglucan endotransglucosylases/hydrolases (XTHs) during the host-invasive growth of the haustorium. The level of xyloglucan endotransglucosylation (XET) activity was found to peak at the penetrating stage of Cuscuta reflexa on its host Pelargonium zonale. In vivo colocalization of XET activity and donor substrate demonstrated XET activity at the border between host and parasite. A test for secretion of XET-active enzymes from haustoria of C. reflexa corroborated this and further indicated that the xyloglucan-modifying enzymes originated from the parasite. A known inhibitor of XET, Coomassie Brilliant Blue R250, was shown to reduce the level of XET in penetrating haustoria of C. reflexa. Moreover, the coating of P. zonale petioles with the inhibitor compound lowered the number of successful haustorial invasions of this otherwise compatible host plant. The presented data indicate that the activity of Cuscuta XTHs at the host-parasite interface is essential to penetration of host plant tissue.

  11. Metal ion coordination in the E. coli Nudix hydrolase dihydroneopterin triphosphate pyrophosphatase: New clues into catalytic mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Hill, Shannon E.; Nguyen, Elaine; Ukachukwu, Chiamaka U.; Freeman, Dana M.; Quirk, Stephen; Lieberman, Raquel L.; Boggon, Titus J.

    2017-07-25

    Dihydroneopterin triphosphate pyrophosphatase (DHNTPase), a member of the Mg2+ dependent Nudix hydrolase superfamily, is the recently-discovered enzyme that functions in the second step of the pterin branch of the folate biosynthetic pathway in E. coli. DHNTPase is of interest because inhibition of enzymes in bacterial folate biosynthetic pathways is a strategy for antibiotic development. We determined crystal structures of DHNTPase with and without activating, Mg2+-mimicking metals Co2+ and Ni2+. Four metal ions, identified by anomalous scattering, and stoichiometrically confirmed in solution by isothermal titration calorimetry, are held in place by Glu56 and Glu60 within the Nudix sequence motif, Glu117, waters, and a sulfate ion, of which the latter is further stabilized by a salt bridge with Lys7. In silico docking of the DHNTP substrate reveals a binding mode in which the pterin ring moiety is nestled in a largely hydrophobic pocket, the β-phosphate activated for nucleophilic attack overlays with the crystallographic sulfate and is in line with an activated water molecule, and remaining phosphate groups are stabilized by all four identified metal ions. The structures and binding data provide new details regarding DHNTPase metal requirements, mechanism, and suggest a strategy for efficient inhibition.

  12. Contribution of the Staphylococcus aureus Atl AM and GL murein hydrolase activities in cell division, autolysis, and biofilm formation.

    Directory of Open Access Journals (Sweden)

    Jeffrey L Bose

    Full Text Available The most prominent murein hydrolase of Staphylococcus aureus, AtlA, is a bifunctional enzyme that undergoes proteolytic cleavage to yield two catalytically active proteins, an amidase (AM and a glucosaminidase (GL. Although the bifunctional nature of AtlA has long been recognized, most studies have focused on the combined functions of this protein in cell wall metabolism and biofilm development. In this study, we generated mutant derivatives of the clinical S. aureus isolate, UAMS-1, in which one or both of the AM and GL domains of AtlA have been deleted. Examination of these strains revealed that each mutant exhibited growth rates comparable to the parental strain, but showed clumping phenotypes and lysis profiles that were distinct from the parental strain and each other, suggesting distinct roles in cell wall metabolism. Given the known function of autolysis in the release of genomic DNA for use as a biofilm matrix molecule, we also tested the mutants in biofilm assays and found both AM and GL necessary for biofilm development. Furthermore, the use of enzymatically inactive point mutations revealed that both AM and GL must be catalytically active for S. aureus to form a biofilm. The results of this study provide insight into the relative contributions of AM and GL in S. aureus and demonstrate the contribution of Atl-mediated lysis in biofilm development.

  13. Contribution of the Staphylococcus aureus Atl AM and GL murein hydrolase activities in cell division, autolysis, and biofilm formation.

    Science.gov (United States)

    Bose, Jeffrey L; Lehman, McKenzie K; Fey, Paul D; Bayles, Kenneth W

    2012-01-01

    The most prominent murein hydrolase of Staphylococcus aureus, AtlA, is a bifunctional enzyme that undergoes proteolytic cleavage to yield two catalytically active proteins, an amidase (AM) and a glucosaminidase (GL). Although the bifunctional nature of AtlA has long been recognized, most studies have focused on the combined functions of this protein in cell wall metabolism and biofilm development. In this study, we generated mutant derivatives of the clinical S. aureus isolate, UAMS-1, in which one or both of the AM and GL domains of AtlA have been deleted. Examination of these strains revealed that each mutant exhibited growth rates comparable to the parental strain, but showed clumping phenotypes and lysis profiles that were distinct from the parental strain and each other, suggesting distinct roles in cell wall metabolism. Given the known function of autolysis in the release of genomic DNA for use as a biofilm matrix molecule, we also tested the mutants in biofilm assays and found both AM and GL necessary for biofilm development. Furthermore, the use of enzymatically inactive point mutations revealed that both AM and GL must be catalytically active for S. aureus to form a biofilm. The results of this study provide insight into the relative contributions of AM and GL in S. aureus and demonstrate the contribution of Atl-mediated lysis in biofilm development.

  14. Binding affinity and adhesion force of organophosphate hydrolase enzyme with soil particles related to the isoelectric point of the enzyme.

    Science.gov (United States)

    Islam, Shah Md Asraful; Yeasmin, Shabina; Islam, Md Saiful; Islam, Md Shariful

    2017-07-01

    The binding affinity of organophosphate hydrolase enzyme (OphB) with soil particles in relation to the isoelectric point (pI) was studied. Immobilization of OphB with soil particles was observed by confocal microscopy, Fourier transform infrared spectroscopy (FT-IR), and Atomic force microscopy (AFM). The calculated pI of OphB enzyme was increased from 8.69 to 8.89, 9.04 and 9.16 by the single, double and triple mutant of OphB enzyme, respectively through the replacement of negatively charged aspartate with positively charged histidine. Practically, the binding affinity was increased to 5.30%, 11.50%, and 16.80% for single, double and triple mutants, respectively. In contrast, enzyme activity of OphB did not change by the mutation of the enzyme. On the other hand, adhesion forces were gradually increased for wild type OphB enzyme (90 pN) to 96, 100 and 104 pN for single, double and triple mutants of OphB enzyme, respectively. There was an increasing trend of binding affinity and adhesion force by the increase of isoelectric point (pI) of OphB enzyme. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Improving the secretion of a methyl parathion hydrolase in Pichia pastoris by modifying its N-terminal sequence.

    Directory of Open Access Journals (Sweden)

    Ping Wang

    Full Text Available Pichia pastoris is commonly used to express and secrete target proteins, although not all recombinant proteins can be successfully produced. In this study, we used methyl parathion hydrolase (MPH from Ochrobactrum sp. M231 as a model to study the importance of the N-terminus of the protein for its secretion. While MPH can be efficiently expressed intracellularly in P. pastoris, it is not secreted into the extracellular environment. Three MPH mutants (N66-MPH, D10-MPH, and N9-MPH were constructed through modification of its N-terminus, and the secretion of each by P. pastoris was improved when compared to wild-type MPH. The level of secreted D10-MPH was increased to 0.21 U/mL, while that of N9-MPH was enhanced to 0.16 U/mL. Although N66-MPH was not enzymatically active, it was secreted efficiently, and was identified by SDS-PAGE. These results demonstrate that the secretion of heterologous proteins in P. pastoris may be improved by modifying their N-terminal structures.

  16. Mutational and structural analyses of Caldanaerobius polysaccharolyticus Man5B reveal novel active site residues for family 5 glycoside hydrolases.

    Directory of Open Access Journals (Sweden)

    Takuji Oyama

    Full Text Available CpMan5B is a glycoside hydrolase (GH family 5 enzyme exhibiting both β-1,4-mannosidic and β-1,4-glucosidic cleavage activities. To provide insight into the amino acid residues that contribute to catalysis and substrate specificity, we solved the structure of CpMan5B at 1.6 Å resolution. The structure revealed several active site residues (Y12, N92 and R196 in CpMan5B that are not present in the active sites of other structurally resolved GH5 enzymes. Residue R196 in GH5 enzymes is thought to be strictly conserved as a histidine that participates in an electron relay network with the catalytic glutamates, but we show that an arginine fulfills a functionally equivalent role and is found at this position in every enzyme in subfamily GH5_36, which includes CpMan5B. Residue N92 is required for full enzymatic activity and forms a novel bridge over the active site that is absent in other family 5 structures. Our data also reveal a role of Y12 in establishing the substrate preference for CpMan5B. Using these molecular determinants as a probe allowed us to identify Man5D from Caldicellulosiruptor bescii as a mannanase with minor endo-glucanase activity.

  17. Immobilization of Glycoside Hydrolase Families GH1, GH13, and GH70: State of the Art and Perspectives

    Directory of Open Access Journals (Sweden)

    Natália G. Graebin

    2016-08-01

    Full Text Available Glycoside hydrolases (GH are enzymes capable to hydrolyze the glycosidic bond between two carbohydrates or even between a carbohydrate and a non-carbohydrate moiety. Because of the increasing interest for industrial applications of these enzymes, the immobilization of GH has become an important development in order to improve its activity, stability, as well as the possibility of its reuse in batch reactions and in continuous processes. In this review, we focus on the broad aspects of immobilization of enzymes from the specific GH families. A brief introduction on methods of enzyme immobilization is presented, discussing some advantages and drawbacks of this technology. We then review the state of the art of enzyme immobilization of families GH1, GH13, and GH70, with special attention on the enzymes β-glucosidase, α-amylase, cyclodextrin glycosyltransferase, and dextransucrase. In each case, the immobilization protocols are evaluated considering their positive and negative aspects. Finally, the perspectives on new immobilization methods are briefly presented.

  18. Engineering of family-5 glycoside hydrolase (Cel5A from an uncultured bacterium for efficient hydrolysis of cellulosic substrates.

    Directory of Open Access Journals (Sweden)

    Amar A Telke

    Full Text Available Cel5A, an endoglucanase, was derived from the metagenomic library of vermicompost. The deduced amino acid sequence of Cel5A shows high sequence homology with family-5 glycoside hydrolases, which contain a single catalytic domain but no distinct cellulose-binding domain. Random mutagenesis and cellulose-binding module (CBM fusion approaches were successfully applied to obtain properties required for cellulose hydrolysis. After two rounds of error-prone PCR and screening of 3,000 mutants, amino acid substitutions were identified at various positions in thermotolerant mutants. The most heat-tolerant mutant, Cel5A_2R2, showed a 7-fold increase in thermostability. To enhance the affinity and hydrolytic activity of Cel5A on cellulose substrates, the family-6 CBM from Saccharophagus degradans was fused to the C-terminus of the Cel5A_2R2 mutant using overlap PCR. The Cel5A_2R2-CBM6 fusion protein showed 7-fold higher activity than the native Cel5A on Avicel and filter paper. Cellobiose was a major product obtained from the hydrolysis of cellulosic substrates by the fusion enzyme, which was identified by using thin layer chromatography analysis.

  19. Dysfunction in fatty acid amide hydrolase is associated with depressive-like behavior in Wistar Kyoto rats.

    Science.gov (United States)

    Vinod, K Yaragudri; Xie, Shan; Psychoyos, Delphine; Hungund, Basalingappa L; Cooper, Thomas B; Tejani-Butt, Shanaz M

    2012-01-01

    While the etiology of depression is not clearly understood at the present time, this mental disorder is thought be a complex and multifactorial trait with important genetic and environmental contributing factors. The role of the endocannabinoid (eCB) system in depressive behavior was examined in Wistar Kyoto (WKY) rat strain, a genetic model of depression. Our findings revealed selective abnormalities in the eCB system in the brains of WKY rats compared to Wistar (WIS) rats. Immunoblot analysis indicated significantly higher levels of fatty acid amide hydrolase (FAAH) in frontal cortex and hippocampus of WKY rats with no alteration in the level of N-arachidonyl phosphatidyl ethanolamine specific phospholipase-D (NAPE-PLD). Significantly higher levels of CB1 receptor-mediated G-protein coupling and lower levels of anandamide (AEA) were found in frontal cortex and hippocampus of WKY rats. While the levels of brain derived neurotropic factor (BDNF) were significantly lower in frontal cortex and hippocampus of WKY rats compared to WIS rats, pharmacological inhibition of FAAH elevated BDNF levels in WKY rats. Inhibition of FAAH enzyme also significantly increased sucrose consumption and decreased immobility in the forced swim test in WKY rats. These findings suggest a critical role for the eCB system and BDNF in the genetic predisposition to depressive-like behavior in WKY rats and point to the potential therapeutic utility of eCB enhancing agents in depressive disorder.

  20. Hydrolase and fructosyltransferase activities implicated in the accumulation of different chain size fructans in three Asteraceae species.

    Science.gov (United States)

    Itaya, Nair M; Asega, Amanda F; Carvalho, Maria Angela M; Figueiredo-Ribeiro, Rita de Cássia L

    2007-09-01

    Fructans are widely distributed in Asteraceae from floras with seasonal growth and are thought to be involved in drought and freezing tolerance, in addition to storage function. Reserve organs of Vernonia herbacea and Viguiera discolor, from the cerrado, and of the perennial herb Smallanthus sonchifolius, endemic to Andean region, store over 80% inulin, with different DP (35, 150, and 15, respectively). The fructan pattern in Asteraceae species could be explained by characteristics of their respective 1-FFTs. Hydrolases and fructosyltransferases from S. sonchifolius, V. herbacea and V. discolor were analyzed in plants at the same environmental conditions. The higher 1-FEH activities found in the species with lower DP, S. sonchifolius and V. herbacea reinforce the hypothesis of the involvement of 1-FEH in fructan profile and suggest that the high DP fructan of V. discolor is a consequence of the low affinity of its 1-FEH to the native long chain inulin. Long term incubation with sucrose suggested that the affinity of 1-FFT of V. discolor for 1-kestose is low when compared to that of V. herbacea. Indeed 1-FFT from V. discolor was shown to be an hDP 1-FFT, preferring longer inulins as acceptors. Conversely, 1-FFT from V. herbacea seems to have a higher affinity for short fructo-oligosaccharides, including 1-kestose, as acceptor substrates. Differences in fructan enzymes of the three Asteraceae provide new information towards the understanding of fructan metabolism and control of carbon flow between low and high DP fructans.

  1. Design of Selective Substrates and Activity-Based Probes for Hydrolase Important for Pathogenesis 1 (HIP1) from Mycobacterium tuberculosis.

    Science.gov (United States)

    Lentz, Christian S; Ordonez, Alvaro A; Kasperkiewicz, Paulina; La Greca, Florencia; O'Donoghue, Anthony J; Schulze, Christopher J; Powers, James C; Craik, Charles S; Drag, Marcin; Jain, Sanjay K; Bogyo, Matthew

    2016-11-11

    Although serine proteases are important mediators of Mycobacterium tuberculosis (Mtb) virulence, there are currently no tools to selectively block or visualize members of this family of enzymes. Selective reporter substrates or activity-based probes (ABPs) could provide a means to monitor infection and response to therapy using imaging methods. Here, we use a combination of substrate selectivity profiling and focused screening to identify optimized reporter substrates and ABPs for the Mtb "Hydrolase important for pathogenesis 1" (Hip1) serine protease. Hip1 is a cell-envelope-associated enzyme with minimal homology to host proteases, making it an ideal target for probe development. We identified substituted 7-amino-4-chloro-3-(2-bromoethoxy)isocoumarins as irreversible inhibitor scaffolds. Furthermore, we used specificity data to generate selective reporter substrates and to further optimize a selective chloroisocoumarin inhibitor. These new reagents are potentially useful in delineating the roles of Hip1 during pathogenesis or as diagnostic imaging tools for specifically monitoring Mtb infections.

  2. Genetic and biochemical characterization of the cell wall hydrolase activity of the major secreted protein of Lactobacillus rhamnosus GG.

    Directory of Open Access Journals (Sweden)

    Ingmar J J Claes

    Full Text Available Lactobacillus rhamnosus GG (LGG produces two major secreted proteins, designated here Msp1 (LGG_00324 or p75 and Msp2 (LGG_00031 or p40, which have been reported to promote the survival and growth of intestinal epithelial cells. Intriguingly, although each of these proteins shares homology with cell wall hydrolases, a physiological function that correlates with such an enzymatic activity remained to be substantiated in LGG. To investigate the bacterial function, we constructed knock-out mutants in the corresponding genes aiming to establish a genotype to phenotype relation. Microscopic examination of the msp1 mutant showed the presence of rather long and overly extended cell chains, which suggests that normal daughter cell separation is hampered. Subsequent observation of the LGG wild-type cells by immunofluorescence microscopy revealed that the Msp1 protein accumulates at the septum of exponential-phase cells. The cell wall hydrolyzing activity of the Msp1 protein was confirmed by zymogram analysis. Subsequent analysis by RP-HPLC and mass spectrometry of the digestion products of LGG peptidoglycan (PG by Msp1 indicated that the Msp1 protein has D-glutamyl-L-lysyl endopeptidase activity. Immunofluorescence microscopy and the failure to construct a knock-out mutant suggest an indispensable role for Msp2 in priming septum formation in LGG.

  3. Comprehensive functional characterization of the glycoside hydrolase family 3 enzymes from Cellvibrio japonicus reveals unique metabolic roles in biomass saccharification.

    Science.gov (United States)

    Nelson, Cassandra E; Attia, Mohamed A; Rogowski, Artur; Morland, Carl; Brumer, Harry; Gardner, Jeffrey G

    2017-12-01

    Lignocellulose degradation is central to the carbon cycle and renewable biotechnologies. The xyloglucan (XyG), β(1→3)/β(1→4) mixed-linkage glucan (MLG) and β(1→3) glucan components of lignocellulose represent significant carbohydrate energy sources for saprophytic microorganisms. The bacterium Cellvibrio japonicus has a robust capacity for plant polysaccharide degradation, due to a genome encoding a large contingent of Carbohydrate-Active enZymes (CAZymes), many of whose specific functions remain unknown. Using a comprehensive genetic and biochemical approach, we have delineated the physiological roles of the four C. japonicus glycoside hydrolase family 3 (GH3) members on diverse β-glucans. Despite high protein sequence similarity and partially overlapping activity profiles on disaccharides, these β-glucosidases are not functionally equivalent. Bgl3A has a major role in MLG and sophorose utilization, and supports β(1→3) glucan utilization, while Bgl3B underpins cellulose utilization and supports MLG utilization. Bgl3C drives β(1→3) glucan utilization. Finally, Bgl3D is the crucial β-glucosidase for XyG utilization. This study not only sheds the light on the metabolic machinery of C. japonicus, but also expands the repertoire of characterized CAZymes for future deployment in biotechnological applications. In particular, the precise functional analysis provided here serves as a reference for informed bioinformatics on the genomes of other Cellvibrio and related species. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  4. Crystallization and preliminary X-ray analysis of a family 19 glycosyl hydrolase from Carica papaya latex

    Energy Technology Data Exchange (ETDEWEB)

    Huet, Joëlle, E-mail: jhuet@ulb.ac.be [Laboratoire de Chimie Générale (CP 206/4), Institut de Pharmacie, Université Libre de Bruxelles (ULB), Campus de la Plaine, Boulevard du Triomphe, B-1050 Bruxelles (Belgium); Azarkan, Mohamed [Laboratoire de Chimie Générale (CP 609), Faculté de Médecine, Université Libre de Bruxelles (ULB), Campus Erasme, 808 Route de Lennik, B-1070 Bruxelles (Belgium); Looze, Yvan [Laboratoire de Chimie Générale (CP 206/4), Institut de Pharmacie, Université Libre de Bruxelles (ULB), Campus de la Plaine, Boulevard du Triomphe, B-1050 Bruxelles (Belgium); Villeret, Vincent [CNRS-UMR 8161, Institut de Biologie de Lille, Université de Lille 1-Université de Lille 2-Institut Pasteur de Lille, IFR142, 1 Rue du Professeur Calmette, F-59021 Lille (France); Wintjens, René, E-mail: jhuet@ulb.ac.be [Laboratoire de Chimie Générale (CP 206/4), Institut de Pharmacie, Université Libre de Bruxelles (ULB), Campus de la Plaine, Boulevard du Triomphe, B-1050 Bruxelles (Belgium)

    2008-05-01

    A chitinase isolated from the latex of the tropical species Carica papaya has been crystallized. The addition of N-acetyl-d-glucosamine to the crystallization solution has improved the diffraction quality resolution of the crystal to 1.8 Å resolution. A chitinase isolated from the latex of the tropical species Carica papaya has been purified to homogeneity and crystallized. This enzyme belongs to glycosyl hydrolase family 19 and exhibits exceptional resistance to proteolysis. The initially observed crystals, which diffracted to a resolution of 2.0 Å, were improved through modification of the crystallization protocol. Well ordered crystals were subsequently obtained using N-acetyl-d-glucosamine, the monomer resulting from the hydrolysis of chitin, as an additive to the crystallization solution. Here, the characterization of a chitinase crystal that belongs to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 69.08, b = 44.79, c = 76.73 Å, β = 95.33° and two molecules per asymmetric unit, is reported. Diffraction data were collected to a resolution of 1.8 Å. Structure refinement is currently in progress.

  5. Characterization of multimetric variants of ubiquitin carboxyl-terminal hydrolase L1 in water by small-angle neutron scattering

    International Nuclear Information System (INIS)

    Naito, Sachio; Mochizuki, Hideki; Yasuda, Toru; Mizuno, Yoshikuni; Furusaka, Michihiro; Ikeda, Susumu; Adachi, Tomohiro; Shimizu, Hirohiko M.; Suzuki, Junichi; Fujiwara, Satoru; Okada, Tomoko; Nishikawa, Kaori; Aoki, Shunsuke; Wada, Keiji

    2006-01-01

    Here, we illustrated that the morphological structures of ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) variants and Parkinson's disease (PD) exhibit good pathological correlation by a small-angle neutron scattering (SANS). UCH-L1 is a neuro-specific multiple functional enzyme, deubiquitinating, ubiquityl ligase, and also involved in stabilization of mono-ubiquitin. To examine the relationship between multiple functions of UCH-L1 and the configuration of its variants [wild-type, I93M (linked to familial Parkinson's disease), and S18Y (linked to reduced risk of Parkinson's disease)], in this report, we proposed that these were all self-assembled dimers by an application of a rotating ellipsoidal model; the configurations of these dimers were quite different. The wild-type was a rotating ellipsoidal. The globular form of the monomeric component deformed by the I93M mutation. Conversely, the S18Y polymorphism promoted the globularity. Thus, the multiple functional balance is closely linked to the intermolecular interactions between the UCH-L1 monomer and the final dimeric configuration

  6. Isolation, Identification and Partial Characterization of a Lactobacillus casei Strain with Bile Salt Hydrolase Activity from Pulque.

    Science.gov (United States)

    González-Vázquez, R; Azaola-Espinosa, A; Mayorga-Reyes, L; Reyes-Nava, L A; Shah, N P; Rivera-Espinoza, Y

    2015-12-01

    The aim of this study was to isolate, from pulque, Lactobacillus spp. capable of survival in simulated gastrointestinal stress conditions. Nine Gram-positive rods were isolated; however, only one strain (J57) shared identity with Lactobacillus and was registered as Lactobacillus casei J57 (GenBank accession: JN182264). The other strains were identified as Bacillus spp. The most significant observation during the test of tolerance to simulated gastrointestinal conditions (acidity, gastric juice and bile salts) was that L. casei J57 showed a rapid decrease (p ≤ 0.05) in the viable population at 0 h. Bile salts were the stress condition that most affected its survival, from which deoxycholic acid and the mix of bile salts (oxgall) were the most toxic. L. casei J57 showed bile salt hydrolase activity over primary and secondary bile salts as follows: 44.91, 671.72, 45.27 and 61.57 U/mg to glycocholate, taurocholate, glycodeoxycholate and taurodeoxycholate. In contrast, the control strain (L. casei Shirota) only showed activity over tauroconjugates. These results suggest that L. casei J57 shows potential for probiotic applications.

  7. Application of the Kombucha 'tea fungus' for the enhancement of antioxidant and starch hydrolase inhibitory properties of ten herbal teas.

    Science.gov (United States)

    Watawana, Mindani I; Jayawardena, Nilakshi; Choo, Candy; Waisundara, Viduranga Y

    2016-03-01

    Ten herbal teas (Acacia arabica, Aegle marmelos flower, A. marmelos root bark, Aerva lanata, Asteracantha longifolia, Cassia auriculata, Hemidesmus indicus, Hordeum vulgare, Phyllanthus emblica, Tinospora cordifolia) were fermented with the Kombucha 'tea fungus'. The pH values of the fermented beverages ranged from 4.0 to 6.0 by day 7, while the titratable acidity ranged from 2.5 to 5.0g/mL (PKombucha beverages to have statistically significant increases (P<0.05) by day 7. The α-amylase inhibitory activities ranged from 52.5 to 67.2μg/mL in terms of IC50 values following fermentation, while the α-glucosidase inhibitory activities ranged from 95.2 to 196.1μg/mL. In conclusion, an enhancement of the antioxidant and starch hydrolase inhibitory potential of the herbal teas was observed by adding the tea fungus. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Plant nucleoside 5'-phosphoramidate hydrolase; simple purification from yellow lupin (Lupinus luteus) seeds and properties of homogeneous enzyme.

    Science.gov (United States)

    Guranowski, Andrzej; Wojdyła, Anna M; Rydzik, Anna M; Stepiński, Janusz; Jemielity, Jacek

    2011-01-01

    Adenosine 5'-phosphoramidate (NH₂-pA) is an uncommon natural nucleotide of poorly understood biochemistry and function. We studied a plant enzyme potentially involved in the catabolism of NH₂-pA. A fast and simple method comprising extraction of yellow lupin (Lupinus luteus) seed-meal with a low ionic strength buffer, ammonium sulfate and acetone fractionations, removal of contaminating proteins by heat denaturation, and affinity chromatography on AMP-agarose, yielded homogenous nucleoside 5'-phosphoramidase. Mass spectrometric analysis showed that the lupin hydrolase exhibits closest similarity to Arabidopsis thaliana Hint1 protein. The substrate specificity of the lupin enzyme, in particular its ability to split the P-S bond in adenosine 5'-phosphorothioate, is typical of known Hint1 proteins. Adenosine 5'-phosphofluoride and various derivatives of guanosine 5'-phosphoramidate were also substrates. Neither common divalent metal cations nor 10 mM EDTA or EGTA affected the hydrolysis of NH₂-pA. The enzyme functions as a homodimer (2 x 15,800 Da). At the optimum pH of 7.0, the K(m) for NH₂-pA was 0.5 µM and k(cat) 0.8 s⁻¹ (per monomer active site). The properties of the lupin nucleoside 5'-phosphoramidase are compared with those of its counterparts from other organisms.

  9. [Blue-light induced expression of S-adenosy-L-homocysteine hydrolase-like gene in Mucor amphibiorum RCS1].

    Science.gov (United States)

    Gao, Ya; Wang, Shu; Fu, Mingjia; Zhong, Guolin

    2013-09-04

    To determine blue-light induced expression of S-adenosyl-L-homocysteine hydrolase-like (sahhl) gene in fungus Mucor amphibiorum RCS1. In the random process of PCR, a sequence of 555 bp was obtained from M. amphibiorum RCS1. The 555 bp sequence was labeled with digoxin to prepare the probe for northern hybridization. By northern hybridization, the transcription of sahhl gene was analyzed in M. amphibiorum RCS1 mycelia culture process from darkness to blue light to darkness. Simultaneously real-time PCR method was used to the sahhl gene expression analysis. Compared with the sequence of sahh gene from Homo sapiens, Mus musculus and some fungi species, a high homology of the 555 bp sequence was confirmed. Therefore, the preliminary confirmation has supported that the 555 bp sequence should be sahhl gene from M. amphibiorum RCS1. Under the dark pre-culture in 24 h, a large amounts of transcript of sahhl gene in the mycelia can be detected by northern hybridization and real-time PCR in the condition of 24 h blue light. But a large amounts of transcript of sahhl gene were not found in other detection for the dark pre-culture of 48 h, even though M. amphibiorum RCS1 mycelia were induced by blue light. Blue light can induce the expression of sahhl gene in the vigorous growth of M. amphibiorum RCS1 mycelia.

  10. Dysfunction in fatty acid amide hydrolase is associated with depressive-like behavior in Wistar Kyoto rats.

    Directory of Open Access Journals (Sweden)

    K Yaragudri Vinod

    Full Text Available BACKGROUND: While the etiology of depression is not clearly understood at the present time, this mental disorder is thought be a complex and multifactorial trait with important genetic and environmental contributing factors. METHODOLOGY/PRINCIPAL FINDINGS: The role of the endocannabinoid (eCB system in depressive behavior was examined in Wistar Kyoto (WKY rat strain, a genetic model of depression. Our findings revealed selective abnormalities in the eCB system in the brains of WKY rats compared to Wistar (WIS rats. Immunoblot analysis indicated significantly higher levels of fatty acid amide hydrolase (FAAH in frontal cortex and hippocampus of WKY rats with no alteration in the level of N-arachidonyl phosphatidyl ethanolamine specific phospholipase-D (NAPE-PLD. Significantly higher levels of CB1 receptor-mediated G-protein coupling and lower levels of anandamide (AEA were found in frontal cortex and hippocampus of WKY rats. While the levels of brain derived neurotropic factor (BDNF were significantly lower in frontal cortex and hippocampus of WKY rats compared to WIS rats, pharmacological inhibition of FAAH elevated BDNF levels in WKY rats. Inhibition of FAAH enzyme also significantly increased sucrose consumption and decreased immobility in the forced swim test in WKY rats. CONCLUSIONS/SIGNIFICANCE: These findings suggest a critical role for the eCB system and BDNF in the genetic predisposition to depressive-like behavior in WKY rats and point to the potential therapeutic utility of eCB enhancing agents in depressive disorder.

  11. Crystallization of mutants of Turnip yellow mosaic virus protease/ubiquitin hydrolase designed to prevent protease self-recognition.

    Science.gov (United States)

    Ayach, Maya; Bressanelli, Stéphane

    2015-04-01

    Processing of the polyprotein of Turnip yellow mosaic virus is mediated by the protease PRO. PRO cleaves at two places, one of which is at the C-terminus of the PRO domain of another polyprotein molecule. In addition to this processing activity, PRO possesses an ubiquitin hydrolase (DUB) activity. The crystal structure of PRO has previously been reported in its polyprotein-processing mode with the C-terminus of one PRO inserted into the catalytic site of the next PRO, generating PRO polymers in the crystal packing of the trigonal space group. Here, two mutants designed to disrupt specific PRO-PRO interactions were generated, produced and purified. Crystalline plates were obtained by seeding and cross-seeding from initial `sea urchin'-like microcrystals of one mutant. The plates diffracted to beyond 2 Å resolution at a synchrotron source and complete data sets were collected for the two mutants. Data processing and analysis indicated that both mutant crystals belonged to the same monoclinic space group, with two molecules of PRO in the asymmetric unit.

  12. The Soluble Epoxide Hydrolase Inhibitor AR9281 Decreases Blood Pressure, Ameliorates Renal Injury and Improves Vascular Function in Hypertension

    Directory of Open Access Journals (Sweden)

    Sean Shaw

    2009-12-01

    Full Text Available Soluble epoxide hydrolase inhibitors (sEHIs are demonstrating promise as potential pharmaceutical agents for the treatment of cardiovascular disease, diabetes, inflammation, and kidney disease. The present study determined the ability of a first-inclass sEHI, AR9281, to decrease blood pressure, improve vascular function, and decrease renal inflammation and injury in angiotensin hypertension. Rats were infused with angiotensin and AR9281 was given orally during the 14-day infusion period. Systolic blood pressure averaged 180 ± 5 mmHg in vehicle treated and AR9281 treatment significantly lowered blood pressure to 142 ± 7 mmHg in angiotensin hypertension. Histological analysis demonstrated decreased injury to the juxtamedullary glomeruli. Renal expression of inflammatory genes was increased in angiotensin hypertension and two weeks of AR9281 treatment decreased this index of renal inflammation. Vascular function in angiotensin hypertension was also improved by AR9281 treatment. Decreased afferent arteriolar and mesenteric resistance endothelial dependent dilator responses were ameliorated by AR9281 treatment of angiotensin hypertensive rats. These data demonstrate that the first-in-class sEHI, AR9281, lowers blood pressure, improves vascular function and reduces renal damage in angiotensin hypertension.

  13. Structural Analysis of a Family 81 Glycoside Hydrolase Implicates Its Recognition of β-1,3-Glucan Quaternary Structure.

    Science.gov (United States)

    Pluvinage, Benjamin; Fillo, Alexander; Massel, Patricia; Boraston, Alisdair B

    2017-09-05

    Family 81 glycoside hydrolases (GHs), which are known to cleave β-1,3-glucans, are found in archaea, bacteria, eukaryotes, and viruses. Here we examine the structural and functional features of the GH81 catalytic module, BhGH81, from the Bacillus halodurans protein BH0236 to probe the molecular basis of β-1,3-glucan recognition and cleavage. BhGH81 displayed activity on laminarin, curdlan, and pachyman, but not scleroglucan; the enzyme also cleaved β-1,3-glucooligosaccharides as small as β-1,3-glucotriose. The crystal structures of BhGH81 in complex with various β-1,3-glucooligosaccharides revealed distorted sugars in the -1 catalytic subsite and an arrangement consistent with an inverting catalytic mechanism having a proposed conformational itinerary of 2 S 0 → 2,5 B ‡ → 5 S 1 . Notably, the architecture of the catalytic site, location of an adjacent ancillary β-1,3-glucan binding site, and the surface properties of the enzyme indicate the likely ability to recognize the double and/or triple-helical quaternary structures adopted by β-1,3-glucans. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Proteomic analysis of Oesophagostomum dentatum (Nematoda during larval transition, and the effects of hydrolase inhibitors on development.

    Directory of Open Access Journals (Sweden)

    Martina Ondrovics

    Full Text Available In this study, in vitro drug testing was combined with proteomic and bioinformatic analyses to identify and characterize proteins involved in larval development of Oesophagostomum dentatum, an economically important parasitic nematode. Four hydrolase inhibitors ο-phenanthroline, sodium fluoride, iodoacetamide and 1,2-epoxy-3-(pnitrophenoxy-propane (EPNP significantly inhibited (≥90% larval development. Comparison of the proteomic profiles of the development-inhibited larvae with those of uninhibited control larvae using two-dimensional gel electrophoresis, and subsequent MALDI-TOF mass spectrometric analysis identified a down-regulation of 12 proteins inferred to be involved in various larval developmental processes, including post-embryonic development and growth. Furthermore, three proteins (i.e. intermediate filament protein B, tropomyosin and peptidyl-prolyl cis-trans isomerase inferred to be involved in the moulting process were down-regulated in moulting- and development-inhibited O. dentatum larvae. This first proteomic map of O. dentatum larvae provides insights in the protein profile of larval development in this parasitic nematode, and significantly improves our understanding of the fundamental biology of its development. The results and the approach used might assist in developing new interventions against parasitic nematodes by blocking or disrupting their key biological pathways.

  15. Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography

    Science.gov (United States)

    Wan, Qun; Parks, Jerry M.; Hanson, B. Leif; Fisher, Suzanne Zoe; Ostermann, Andreas; Schrader, Tobias E.; Graham, David E.; Coates, Leighton; Langan, Paul; Kovalevsky, Andrey

    2015-01-01

    Glycoside hydrolase (GH) enzymes apply acid/base chemistry to catalyze the decomposition of complex carbohydrates. These ubiquitous enzymes accept protons from solvent and donate them to substrates at close to neutral pH by modulating the pKa values of key side chains during catalysis. However, it is not known how the catalytic acid residue acquires a proton and transfers it efficiently to the substrate. To better understand GH chemistry, we used macromolecular neutron crystallography to directly determine protonation and ionization states of the active site residues of a family 11 GH at multiple pD (pD = pH + 0.4) values. The general acid glutamate (Glu) cycles between two conformations, upward and downward, but is protonated only in the downward orientation. We performed continuum electrostatics calculations to estimate the pKa values of the catalytic Glu residues in both the apo- and substrate-bound states of the enzyme. The calculated pKa of the Glu increases substantially when the side chain moves down. The energy barrier required to rotate the catalytic Glu residue back to the upward conformation, where it can protonate the glycosidic oxygen of the substrate, is 4.3 kcal/mol according to free energy simulations. These findings shed light on the initial stage of the glycoside hydrolysis reaction in which molecular motion enables the general acid catalyst to obtain a proton from the bulk solvent and deliver it to the glycosidic oxygen. PMID:26392527

  16. Ubiquitin carboxyl terminal hydrolase L1 negatively regulates TNFα-mediated vascular smooth muscle cell proliferation via suppressing ERK activation

    International Nuclear Information System (INIS)

    Ichikawa, Tomonaga; Li, Jinqing; Dong, Xiaoyu; Potts, Jay D.; Tang, Dong-Qi; Li, Dong-Sheng; Cui, Taixing

    2010-01-01

    Deubiquitinating enzymes (DUBs) appear to be critical regulators of a multitude of processes such as proliferation, apoptosis, differentiation, and inflammation. We have recently demonstrated that a DUB of ubiquitin carboxyl terminal hydrolase L1 (UCH-L1) inhibits vascular lesion formation via suppressing inflammatory responses in vasculature. However, the precise underlying mechanism remains to be defined. Herein, we report that a posttranscriptional up-regulation of UCH-L1 provides a negative feedback to tumor necrosis factor alpha (TNFα)-mediated activation of extracellular signal-regulated kinases (ERK) and proliferation in vascular smooth muscle cells (VSMCs). In rat adult VSMCs, adenoviral over-expression of UCH-L1 inhibited TNFα-induced activation of ERK and DNA synthesis. In contrast, over-expression of UCH-L1 did not affect platelet derived growth factor (PDGF)-induced VSMC proliferation and activation of growth stimulating cascades including ERK. TNFα hardly altered UCH-L1 mRNA expression and stability; however, up-regulated UCH-L1 protein expression via increasing UCH-L1 translation. These results uncover a novel mechanism by which UCH-L1 suppresses vascular inflammation.

  17. Efficient asymmetric hydrolysis of styrene oxide catalyzed by Mung bean epoxide hydrolases in ionic liquid-based biphasic systems.

    Science.gov (United States)

    Chen, Wen-Jing; Lou, Wen-Yong; Zong, Min-Hua

    2012-07-01

    The asymmetric hydrolysis of styrene oxide to (R)-1-phenyl-1,2-ethanediol using Mung bean epoxide hydrolases was, for the first time, successfully conducted in an ionic liquid (IL)-containing biphasic system. Compared to aqueous monophasic system, IL-based biphasic systems could not only dissolve the substrate, but also effectively inhibit the non-enzymatic hydrolysis, and therefore markedly improve the reaction efficiency. Of all the tested ILs, the best results were observed in the biphasic system containing C(4)MIM·PF(6), which exhibited good biocompatibility with the enzyme and was an excellent solvent for the substrate. In the C(4)MIM·PF(6)/buffer biphasic system, it was found that the optimal volume ratio of IL to buffer, reaction temperature, buffer pH and substrate concentration were 1/6, 35°C, 6.5 and 100 mM, respectively, under which the initial reaction rate, the yield and the product e.e. were 18.4 mM/h, 49.4% and 97.0%. The biocatalytic process was shown to be feasible on a 500-mL preparative scale. Copyright © 2011 Elsevier Ltd. All rights reserved.

  18. Phylogenetic diversity and environment-specific distributions of glycosyl hydrolase family 10 xylanases in geographically distant soils.

    Directory of Open Access Journals (Sweden)

    Guozeng Wang

    Full Text Available BACKGROUND: Xylan is one of the most abundant biopolymers on Earth. Its degradation is mediated primarily by microbial xylanase in nature. To explore the diversity and distribution patterns of xylanase genes in soils, samples of five soil types with different physicochemical characters were analyzed. METHODOLOGY/PRINCIPAL FINDINGS: Partial xylanase genes of glycoside hydrolase (GH family 10 were recovered following direct DNA extraction from soil, PCR amplification and cloning. Combined with our previous study, a total of 1084 gene fragments were obtained, representing 366 OTUs. More than half of the OTUs were novel (identities of <65% with known xylanases and had no close relatives based on phylogenetic analyses. Xylanase genes from all the soil environments were mainly distributed in Bacteroidetes, Proteobacteria, Acidobacteria, Firmicutes, Actinobacteria, Dictyoglomi and some fungi. Although identical sequences were found in several sites, habitat-specific patterns appeared to be important, and geochemical factors such as pH and oxygen content significantly influenced the compositions of xylan-degrading microbial communities. CONCLUSION/SIGNIFICANCE: These results provide insight into the GH 10 xylanases in various soil environments and reveal that xylan-degrading microbial communities are environment specific with diverse and abundant populations.

  19. Comprehensive functional characterization of the Glycoside Hydrolase Family 3 enzymes from Cellvibrio japonicus reveals unique metabolic roles in biomass saccharification

    International Nuclear Information System (INIS)

    Nelson, Cassandra E.; Attia, Mohamed A.; Rogowski, Artur; Morland, Carl; Brumer, Harry; Gardner, Jeffrey G.

    2017-01-01

    Here, lignocellulose degradation is central to the carbon cycle and renewable biotechnologies. The xyloglucan (XyG), β(1!3)/β(1!4) mixed-linkage glucan (MLG), and β(1!3) glucan components of lignocellulose represent significant carbohydrate energy sources for saprophytic microorganisms. The bacterium Cellvibrio japonicus has a robust capacity for plant polysaccharide degradation, due to a genome encoding a large contingent of Carbohydrate-Active Enzymes (CAZymes), many of whose specific functions remain unknown. Using a comprehensive genetic and biochemical approach we have delineated the physiological roles of the four C. japonicus Glycoside Hydrolase Family 3 (GH3) members on diverse β-glucans. Despite high protein sequence similarity and partially overlapping activity profiles on disaccharides, these β-glucosidases are not functionally equivalent. Bgl3A has a major role in MLG and sophorose utilization, and supports β(1!3) glucan utilization, while Bgl3B underpins cellulose utilization and supports MLG utilization. Bgl3C drives β(1!3) glucan utilization. Finally, Bgl3D is the crucial β-glucosidase for XyG utilization. This study not only sheds the light on the metabolic machinery of C. japonicus, but also expands the repertoire of characterized CAZymes for future deployment in biotechnological applications. In particular, the precise functional analysis provided here serves as a reference for informed bioinformatics on the genomes of other Cellvibrio and related species.

  20. Glial Fibrillary Acidic Protein and Ubiquitin C-Terminal Hydrolase-L1 as Outcome Predictors in Traumatic Brain Injury.

    Science.gov (United States)

    Takala, Riikka S K; Posti, Jussi P; Runtti, Hilkka; Newcombe, Virginia F; Outtrim, Joanne; Katila, Ari J; Frantzén, Janek; Ala-Seppälä, Henna; Kyllönen, Anna; Maanpää, Henna-Riikka; Tallus, Jussi; Hossain, Md Iftakher; Coles, Jonathan P; Hutchinson, Peter; van Gils, Mark; Menon, David K; Tenovuo, Olli

    2016-03-01

    Biomarkers ubiquitin C-terminal hydrolase-L1 (UCH-L1) and glial fibrillary acidic protein (GFAP) may help detect brain injury, assess its severity, and improve outcome prediction. This study aimed to evaluate the prognostic value of these biomarkers during the first days after brain injury. Serum UCH-L1 and GFAP were measured in 324 patients with traumatic brain injury (TBI) enrolled in a prospective study. The outcome was assessed using the Glasgow Outcome Scale (GOS) or the extended version, Glasgow Outcome Scale-Extended (GOSE). Patients with full recovery had lower UCH-L1 concentrations on the second day and patients with favorable outcome had lower UCH-L1 concentrations during the first 2 days compared with patients with incomplete recovery and unfavorable outcome. Patients with full recovery and favorable outcome had significantly lower GFAP concentrations in the first 2 days than patients with incomplete recovery or unfavorable outcome. There was a strong negative correlation between outcome and UCH-L1 in the first 3 days and GFAP levels in the first 2 days. On arrival, both UCH-L1 and GFAP distinguished patients with GOS score 1-3 from patients with GOS score 4-5, but not patients with GOSE score 8 from patients with GOSE score 1-7. For UCH-L1 and GFAP to predict unfavorable outcome (GOS score ≤ 3), the area under the receiver operating characteristic curve was 0.727, and 0.723, respectively. Neither UCHL-1 nor GFAP was independently able to predict the outcome when age, worst Glasgow Coma Scale score, pupil reactivity, Injury Severity Score, and Marshall score were added into the multivariate logistic regression model. GFAP and UCH-L1 are significantly associated with outcome, but they do not add predictive power to commonly used prognostic variables in a population of patients with TBI of varying severities. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Identification and characterization of a bile salt hydrolase from Lactobacillus salivarius for development of novel alternatives to antibiotic growth promoters.

    Science.gov (United States)

    Wang, Zhong; Zeng, Ximin; Mo, Yiming; Smith, Katie; Guo, Yuming; Lin, Jun

    2012-12-01

    Antibiotic growth promoters (AGPs) have been used as feed additives to improve average body weight gain and feed efficiency in food animals for more than 5 decades. However, there is a worldwide trend to limit AGP use to protect food safety and public health, which raises an urgent need to discover effective alternatives to AGPs. The growth-promoting effect of AGPs has been shown to be highly correlated with the decreased activity of intestinal bile salt hydrolase (BSH), an enzyme that is produced by various gut microflora and involved in host lipid metabolism. Thus, BSH inhibitors are likely promising feed additives to AGPs to improve animal growth performance. In this study, the genome of Lactobacillus salivarius NRRL B-30514, a BSH-producing strain isolated from chicken, was sequenced by a 454 GS FLX sequencer. A BSH gene identified by genome analysis was cloned and expressed in an Escherichia coli expression system for enzymatic analyses. The BSH displayed efficient hydrolysis activity for both glycoconjugated and tauroconjugated bile salts, with slightly higher catalytic efficiencies (k(cat)/K(m)) on glycoconjugated bile salts. The optimal pH and temperature for the BSH activity were 5.5 and 41°C, respectively. Examination of a panel of dietary compounds using the purified BSH identified some potent BSH inhibitors, in which copper and zinc have been recently demonstrated to promote feed digestion and body weight gain in different food animals. In sum, this study identified and characterized a BSH with broad substrate specificity from a chicken L. salivarius strain and established a solid platform for us to discover novel BSH inhibitors, the promising feed additives to replace AGPs for enhancing the productivity and sustainability of food animals.

  2. Streptococcus pneumoniae Endohexosaminidase D, Structural and Mechanistic Insight into Substrate-Assisted Catalysis in Family 85 Glycoside Hydrolases

    International Nuclear Information System (INIS)

    Abbott, D.; Macauley, M.; Vocadlo, D.; Boraston, A.

    2009-01-01

    Endo-?-d-glucosaminidases from family 85 of glycoside hydrolases (GH85 endohexosaminidases) act to cleave the glycosidic linkage between the two N-acetylglucosamine units that make up the chitobiose core of N-glycans. Endohexosaminidase D (Endo-D), produced by Streptococcus pneumoniae, is believed to contribute to the virulence of this organism by playing a role in the deglycosylation of IgG antibodies. Endohexosaminidases have received significant attention for this reason and, moreover, because they are powerful tools for chemoenzymatic synthesis of proteins having defined glycoforms. Here we describe mechanistic and structural studies of the catalytic domain (SpGH85) of Endo-D that provide compelling support for GH85 enzymes using a catalytic mechanism involving substrate-assisted catalysis. Furthermore, the structure of SpGH85 in complex with the mechanism-based competitive inhibitor NAG-thiazoline (Kd = 28 ?m) provides a coherent rationale for previous mutagenesis studies of Endo-D and other related GH85 enzymes. We also find GH85, GH56, and GH18 enzymes have a similar configuration of catalytic residues. Notably, GH85 enzymes have an asparagine in place of the aspartate residue found in these other families of glycosidases. We propose that this residue, as the imidic acid tautomer, acts analogously to the key catalytic aspartate of GH56 and GH18 enzymes. This topographically conserved arrangement of the asparagine residue and a conserved glutamic acid, coupled with previous kinetic studies, suggests these enzymes may use an unusual proton shuttle to coordinate effective general acid and base catalysis to aid cleavage of the glycosidic bond. These results collectively provide a blueprint that may be used to facilitate protein engineering of these enzymes to improve their function as biocatalysts for synthesizing glycoproteins having defined glycoforms and also may serve as a guide for generating inhibitors of GH85 enzymes.

  3. Inhibition of soluble epoxide hydrolase attenuates hepatic fibrosis and endoplasmic reticulum stress induced by carbon tetrachloride in mice

    International Nuclear Information System (INIS)

    Harris, Todd R.; Bettaieb, Ahmed; Kodani, Sean; Dong, Hua; Myers, Richard; Chiamvimonvat, Nipavan; Haj, Fawaz G.; Hammock, Bruce D.

    2015-01-01

    Liver fibrosis is a pathological condition in which chronic inflammation and changes to the extracellular matrix lead to alterations in hepatic tissue architecture and functional degradation of the liver. Inhibitors of the enzyme soluble epoxide hydrolase (sEH) reduce fibrosis in the heart, pancreas and kidney in several disease models. In this study, we assess the effect of sEH inhibition on the development of fibrosis in a carbon tetrachloride (CCl 4 )-induced mouse model by monitoring changes in the inflammatory response, matrix remolding and endoplasmic reticulum stress. The sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) was administered in drinking water. Collagen deposition in the liver was increased five-fold in the CCl 4 -treated group, and this was returned to control levels by TPPU treatment. Hepatic expression of Col1a2 and 3a1 mRNA was increased over fifteen-fold in the CCl 4 -treated group relative to the Control group, and this increase was reduced by 50% by TPPU treatment. Endoplasmic reticulum (ER) stress observed in the livers of CCl 4 -treated animals was attenuated by TPPU treatment. In order to support the hypothesis that TPPU is acting to reduce the hepatic fibrosis and ER stress through its action as a sEH inhibitor we used a second sEH inhibitor, trans-4-(4-[3-(4-trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy)-benzoic acid (t-TUCB), and sEH null mice. Taken together, these data indicate that the sEH may play an important role in the development of hepatic fibrosis induced by CCl 4 , presumably by reducing endogenous fatty acid epoxide chemical mediators acting to reduce ER stress. - Highlights: • We administer an inhibitor of sEH in a CCl4 murine model. • sEH inhibition reduces liver collagen deposition and pro-fibrotic gene expression. • sEH inhibition induces MMP-1a activity

  4. Fatty acid amide hydrolase (FAAH) regulates hypercapnia/ischemia-induced increases in n-acylethanolamines in mouse brain.

    Science.gov (United States)

    Lin, Lin; Metherel, Adam H; Jones, Peter J; Bazinet, Richard P

    2017-09-01

    N-acylethanolamines (NAEs) are endogenous lipid ligands for several receptors including cannabinoid receptors and peroxisome proliferator-activated receptor-alpha (PPAR-α), which regulate numerous physiological functions. Fatty acid amide hydrolase (FAAH) is largely responsible for the degradation of NAEs. However, at high concentrations of ethanolamines and unesterified fatty acids, FAAH can also catalyze the reverse reaction, producing NAEs. Several brain insults such as ischemia and hypoxia increase brain unesterified fatty acids. Because FAAH can catalyze the synthesis of NAE, we aimed to test whether FAAH was necessary for CO 2 -induced hypercapnia/ischemia increases in NAE. To test this, we examined levels of NAEs, 1- and 2-arachidonoylglycerols as well as their corresponding fatty acid precursors in wild-type and mice lacking FAAH (FAAH-KO) with three Kill methods: (i) head-focused, high-energy microwave irradiation (microwave), (ii) 5 min CO 2 followed by microwave irradiation (CO 2 + microwave), and (iii) 5 min CO 2 only (CO 2 ). Both CO 2 -induced groups increased, to a similar extent, brain levels of unesterified oleic, arachidonic, and docosahexaenoic acid and 1- and 2-arachidonoylglycerols compared to the microwave group in both wild-type and FAAH-KO mice. Oleoylethanolamide (OEA), arachidonoylethanolamide (AEA), and docosahexaenoylethanolamide (DHEA) levels were about 8-, 7-, and 2.5-fold higher, respectively, in the FAAH-KO mice compared with the wild-type mice. Interestingly, the concentrations of OEA, AEA, and DHEA increased 2.5- to 4-fold in response to both CO 2 -induced groups in wild-type mice, but DHEA increased only in the CO 2 group in FAAH-KO mice. Our study demonstrates that FAAH is necessary for CO 2 - induced increases in OEA and AEA but not DHEA. Targeting brain FAAH could impair the production of NAEs in response to brain injuries. © 2017 International Society for Neurochemistry.

  5. Improvement of Aspergillus flavus saponin hydrolase thermal stability and productivity via immobilization on a novel carrier based on sugarcane bagasse

    Directory of Open Access Journals (Sweden)

    Hala A. Amin

    2018-03-01

    Full Text Available Soyasapogenol B (SB is known to have many biological activities such as hepatoprotective, anti-inflammatory, anti-mutagenic, antiviral and anticancer activities. Enzymatic conversion of soyasaponins to SB was carried out using saponin hydrolase (SH extracted from Aspergillus flavus. The partially purified enzyme was immobilized on different carriers by physical adsorption, covalent binding or entrapment. Among the investigated carriers, Eupergit C and sugarcane bagasse (SCB activated by DIC and NHS were the most suitable two carriers for immobilization (the immobilized forms recovered 46.5 and 37.1% of the loaded enzyme activity, respectively. Under optimized immobilization conditions, immobilized SH on Eupergit C and on activated SBC recovered 87.7 and 83.3% of its original activity, respectively. Compared to free SH, immobilized SH on Eupergit C and on activated SCB showed higher optimum pH, activation energy, half-lives and lower deactivation constant rate. Also, their SB productivities were improved by 2.3- and 2.2-folds compared to free SH (87.7 and 83.3 vs. 37.5%, respectively. Hence, being SCB more sustainable and an inexpensive material, it can be considered a good alternative to Eupergit C as a support for SH immobilization. SH immobilization on industrially applicable and inexpensive carrier is necessary to improve SB yield and reduce its production cost. The chemical structure of SCB and the resulting cellulose derivatives were studied by ATR-IR spectroscopy. The thermal analysis technique was used to study the chemical treatment of SCB and coupling with the enzyme. This technique confirmed the removal of lignin and hemicellulose by chemical treatment of SCB.

  6. Characterization of a novel theme C glycoside hydrolase family 9 cellulase and its CBM-chimeric enzymes.

    Science.gov (United States)

    Duan, Cheng-Jie; Huang, Ming-Yue; Pang, Hao; Zhao, Jing; Wu, Chao-Xing; Feng, Jia-Xun

    2017-07-01

    In bacterial cellulase systems, glycoside hydrolase family 9 (GH9) cellulases are generally regarded as the major cellulose-degrading factors besides GH48 exoglucanase. In this study, umcel9A, which was cloned from uncultured microorganisms from compost, with the encoded protein being theme C GH9 cellulase, was heterologously expressed in Escherichia coli, and the biochemical properties of the purified enzyme were characterized. Hydrolysis of carboxylmethylcellulose (CMC) by Umcel9A led to the decreased viscosity of CMC solution and production of reducing sugars. Interestingly, cellobiose was the major product when cellulosic materials were hydrolyzed by Umcel9A. Six representative carbohydrate-binding modules (CBMs) from different CBM families (CBM1, CBM2, CBM3, CBM4, CBM10, and CBM72) were fused with Umcel9A at the natural terminal position, resulting in significant enhancement of the binding capacity of the chimeric enzymes toward four different insoluble celluloses as compared with that of Umcel9A. Catalytic activity of the chimeric enzymes against insoluble celluloses, including phosphoric acid-swollen cellulose (PASC), alkali-pretreated sugarcane bagasse (ASB), filter paper powder (FPP), and Avicel, was higher than that of Umcel9A, except for Umcel9A-CBM3. In these chimeric enzymes, CBM4-Umcel9A exhibited the highest activity toward the four tested insoluble celluloses and displayed 4.2-, 3.0-, 2.4-, and 6.6-fold enhanced activity toward PASC, ASB, FPP, and Avicel, respectively, when compared with that of Umcel9A. CBM4-Umcel9A also showed highest V max and catalytic efficiency (k cat /K M ) against PASC. Construction of chimeric enzymes may have potential applications in biocatalytic processes and provides insight into the evolution of the molecular architecture of catalytic module and CBM in GH9 cellulases.

  7. Growth, hydrolases and ultrastructure of Fusarium oxysporum as affected by phenolic rich extracts from several xerophytic plants.

    Science.gov (United States)

    Mohamed, Mahmoud S M; Saleh, Ahmed M; Abdel-Farid, Ibrahim B; El-Naggar, Sabry A

    2017-09-01

    Fusarium oxysporum, the causal agent of rot and wilt diseases, is one of the most detrimental phytopathogens for the productivity of many economic crops. The present study was conducted to evaluate the potentiality of some xerophytic plants as eco-friendly approach for management of F. oxysporum. Phenolic rich extracts from five plants namely: Horwoodia dicksoniae, Citrullus colocynthis, Gypsophila capillaris, Pulicaria incisa and Rhanterium epapposum were examined in vitro. The different extracts showed high variability in their phenolic and flavonoid contents as well as total antioxidant capacity. A strong positive correlation existed between the antifungal activity of the tested extracts and their contents of both total phenolics and flavonoids (r values are 0.91 and 0.82, respectively). Extract of P. incisa was the most effective in reducing the mycelial growth (IC 50 =0.92mg/ml) and inhibiting the activities of CMCase, pectinase, amylase and protease by 36, 42, 58 and 55%, respectively. The high performance liquid chromatography analysis of P. incisa extract revealed the presence of eight phenolic acids along with five polyphenolic compounds. The flavonol, quercetin and its glycosides rutin and quercetrin were the most abundant followed by the phenolic acids, t-cinnamic, caffeic, ferulic and vanillic. P. incisa extract not only affects the growth and hydrolases of F. oxysporum but also induces ultrastructure changes in the mycelium, as revealed by transmission electron microscopy. To our knowledge, this is the first study to investigate the mechanisms underlying the antifungal activity of P. incisa. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. A coupled photometric assay for characterization of S-adenosyl-l-homocysteine hydrolases in the physiological hydrolytic direction.

    Science.gov (United States)

    Kailing, Lyn L; Bertinetti, Daniela; Herberg, Friedrich W; Pavlidis, Ioannis V

    2017-10-25

    S-Adenosyl-l-homocysteine hydrolases (SAHases) are important metabolic enzymes and their dysregulation is associated with some severe diseases. In vivo they catalyze the hydrolysis of S-adenosyl-l-homocysteine (SAH), the by-product of methylation reactions in various organisms. SAH is a potent inhibitor of methyltransferases, thus its removal from the equilibrium is an important requirement for methylation reactions. SAH hydrolysis is also the first step in the cellular regeneration process of the methyl donor S-adenosyl-l-methionine (SAM). However, in vitro the equilibrium lies towards the synthetic direction. To enable characterization of SAHases in the physiologically relevant direction, we have developed a coupled photometric assay that shifts the equilibrium towards hydrolysis by removing the product adenosine, using a high affinity adenosine kinase (AK). This converts adenosine to AMP and thereby forms equimolar amounts of ADP, which is phosphorylated by a pyruvate kinase (PK), in turn releasing pyruvate. The readout of the assay is the consumption of NADH during the lactate dehydrogenase (LDH) catalyzed reduction of pyruvate to lactic acid. The applicability of the assay is showcased for the determination of the kinetic constants of an SAHase from Bradyrhizobium elkanii (K M,SAH 41±5μM, v max,SAH 25±1μM/min with 0.13mg/mL enzyme). This assay is a valuable tool for in vitro characterization of SAHases with biotechnological potential, and for monitoring SAHase activity in diagnostics. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Cloning and identification of novel hydrolase genes from a dairy cow rumen metagenomic library and characterization of a cellulase gene

    Directory of Open Access Journals (Sweden)

    Gong Xia

    2012-10-01

    Full Text Available Abstract Background Interest in cellulose degrading enzymes has increased in recent years due to the expansion of the cellulosic biofuel industry. The rumen is a highly adapted environment for the degradation of cellulose and a promising source of enzymes for industrial use. To identify cellulase enzymes that may be of such use we have undertaken a functional metagenomic screen to identify cellulase enzymes from the bacterial community in the rumen of a grass-hay fed dairy cow. Results Twenty five clones specifying cellulose activity were identified. Subcloning and sequence analysis of a subset of these hydrolase-positive clones identified 10 endoglucanase genes. Preliminary characterization of the encoded cellulases was carried out using crude extracts of each of the subclones. Zymogram analysis using carboxymethylcellulose as a substrate showed a single positive band for each subclone, confirming that only one functional cellulase gene was present in each. One cellulase gene, designated Cel14b22, was expressed at a high level in Escherichia coli and purified for further characterization. The purified recombinant enzyme showed optimal activity at pH 6.0 and 50°C. It was stable over a broad pH range, from pH 4.0 to 10.0. The activity was significantly enhanced by Mn2+ and dramatically reduced by Fe3+ or Cu2+. The enzyme hydrolyzed a wide range of beta-1,3-, and beta-1,4-linked polysaccharides, with varying activities. Activities toward microcrystalline cellulose and filter paper were relatively high, while the highest activity was toward Oat Gum. Conclusion The present study shows that a functional metagenomic approach can be used to isolate previously uncharacterized cellulases from the rumen environment.

  10. A Personal Retrospective: Elevating Anandamide (AEA) by Targeting Fatty Acid Amide Hydrolase (FAAH) and the Fatty Acid Binding Proteins (FABPs).

    Science.gov (United States)

    Deutsch, Dale G

    2016-01-01

    This perspective was adapted from a Career Achievement Award talk given at the International Cannabinoid Research Society Symposium in Bukovina, Poland on June 27, 2016. As a biochemist working in the neurosciences, I was always fascinated with neurotransmitter inactivation. In 1993 we identified an enzyme activity that breaks down anandamide. We called the enzyme anandamide amidase, now called FAAH. We and other laboratories developed FAAH inhibitors that were useful reagents that also proved to have beneficial physiological effects and until recently, new generations of inhibitors were in clinical trials. Nearly all neurotransmitters are water soluble and as such, require a transmembrane protein transporter to pass through the lipid membrane for inactivation inside the cell. However, using model systems, we and others have shown that this is unnecessary for anandamide, an uncharged hydrophobic molecule that readily diffuses across the cellular membrane. Interestingly, its uptake is driven by the concentration gradient resulting from its breakdown mainly by FAAH localized in the endoplasmic reticulum. We identified the FABPs as intracellular carriers that "solubilize" anandamide, transporting anandamide to FAAH. Compounds that bind to FABPs block AEA breakdown, raising its level. The cannabinoids (THC and CBD) also were discovered to bind FABPs and this may be one of the mechanisms by which CBD works in childhood epilepsy, raising anandamide levels. Targeting FABPs may be advantageous since they have some tissue specificity and do not require reactive serine hydrolase inhibitors, as does FAAH, with potential for off-target reactions. At the International Cannabis Research Society Symposium in 1992, Raphe Mechoulam revealed that his laboratory isolated an endogenous lipid molecule that binds to the CB1 receptor (cannabinoid receptor type 1) and this became the milestone paper published in December of that year describing anandamide (AEA, Devane et al., 1992). As to

  11. Hint2, the mitochondrial nucleoside 5'-phosphoramidate hydrolase; properties of the homogeneous protein from sheep (Ovis aries) liver.

    Science.gov (United States)

    Bretes, Ewa; Wojdyła-Mamoń, Anna M; Kowalska, Joanna; Jemielity, Jacek; Kaczmarek, Renata; Baraniak, Janina; Guranowski, Andrzej

    2013-01-01

    Adenosine 5'-phosphoramidate (NH2-pA) is a rare natural nucleotide and its biochemistry and biological functions are poorly recognized. All organisms have proteins that may be involved in the catabolism of NH2-pA. They are members of the HIT protein family and catalyze hydrolytic splitting of NH2-pA to 5'-AMP and ammonia. At least five HIT proteins have been identified in mammals; however, the enzymatic and molecular properties of only Fhit and Hint1 have been comprehensively studied. Our study focuses on the Hint2 protein purified by a simple procedure to homogeneity from sheep liver mitochondrial fraction (OaHint2). Hint1 protein was also prepared from sheep liver (OaHint1) and the molecular and kinetic properties of the two proteins compared. Both function as homodimers and behave as nucleoside 5'-phosphoramidate hydrolases. The molecular mass of the OaHint2 monomer is 16 kDa and that of the OaHint1 monomer 14.9 kDa. Among potential substrates studied, NH2-pA appeared to be the best; the Km and kcat values estimated for this compound are 6.6 μM and 68.3 s⁻¹, and 1.5 μM and 11.0 s⁻¹ per natively functioning dimer of OaHint2 and OaHint1, respectively. Studies of the rates of hydrolysis of different NH2-pA derivatives show that Hint2 is more specific towards compounds with a P-N bond than Hint1. The thermostability of these two proteins is also compared.

  12. InvA protein is a Nudix hydrolase required for infection by pathogenic Leptospira in cell lines and animals.

    Science.gov (United States)

    Luo, Yihui; Liu, Yan; Sun, Dexter; Ojcius, David M; Zhao, Jinfang; Lin, Xuai; Wu, Dong; Zhang, Rongguang; Chen, Ming; Li, Lanjuan; Yan, Jie

    2011-10-21

    Leptospirosis caused by pathogenic species of the genus Leptospira is a re-emerging zoonotic disease, which affects a wide variety of host species and is transmitted by contaminated water. The genomes of several pathogenic Leptospira species contain a gene named invA, which contains a Nudix domain. However, the function of this gene has never been characterized. Here, we demonstrated that the invA gene was highly conserved in protein sequence and present in all tested pathogenic Leptospira species. The recombinant InvA protein of pathogenic L. interrogans strain Lai hydrolyzed several specific dinucleoside oligophosphate substrates, reflecting the enzymatic activity of Nudix in Leptospira species. Pathogenic leptospires did not express this protein in media but temporarily expressed it at early stages (within 60 min) of infection of macrophages and nephric epithelial cells. Comparing with the wild type, the invA-deficient mutant displayed much lower infectivity and a significantly reduced survival rate in macrophages and nephric epithelial cells. Moreover, the invA-deficient leptospires presented an attenuated virulence in hamsters, caused mild histopathological damage, and were transmitted in lower numbers in the urine, compared with the wild-type strain. The invA revertant, made by complementing the invA-deficient mutant with the invA gene, reacquired virulence similar to the wild type in vitro and in vivo. The LD(50) in hamsters was 1000-fold higher for the invA-deficient mutant than for the invA revertant and wild type. These results demonstrate that the InvA protein is a Nudix hydrolase, and the invA gene is essential for virulence in pathogenic Leptospira species.

  13. Removal of distal protein-water hydrogen bonds in a plant epoxide hydrolase increases catalytic turnover but decreases thermostability.

    Science.gov (United States)

    Thomaeus, Ann; Naworyta, Agata; Mowbray, Sherry L; Widersten, Mikael

    2008-07-01

    A putative proton wire in potato soluble epoxide hydrolase 1, StEH1, was identified and investigated by means of site-directed mutagenesis, steady-state kinetic measurements, temperature inactivation studies, and X-ray crystallography. The chain of hydrogen bonds includes five water molecules coordinated through backbone carbonyl oxygens of Pro(186), Leu(266), His(269), and the His(153) imidazole. The hydroxyl of Tyr(149) is also an integrated component of the chain, which leads to the hydroxyl of Tyr(154). Available data suggest that Tyr(154) functions as a final proton donor to the anionic alkylenzyme intermediate formed during catalysis. To investigate the role of the putative proton wire, mutants Y149F, H153F, and Y149F/H153F were constructed and purified. The structure of the Y149F mutant was solved by molecular replacement and refined to 2.0 A resolution. Comparison with the structure of wild-type StEH1 revealed only subtle structural differences. The hydroxyl group lost as a result of the mutation was replaced by a water molecule, thus maintaining a functioning hydrogen bond network in the proton wire. All mutants showed decreased catalytic efficiencies with the R,R-enantiomer of trans-stilbene oxide, whereas with the S,S-enantiomer, k (cat)/K (M) was similar or slightly increased compared with the wild-type reactions. k (cat) for the Y149F mutant with either TSO enantiomer was increased; thus the lowered enzyme efficiencies were due to increases in K (M). Thermal inactivation studies revealed that the mutated enzymes were more sensitive to elevated temperatures than the wild-type enzyme. Hence, structural alterations affecting the hydrogen bond chain caused increases in k (cat) but lowered thermostability.

  14. High genetic diversity and different distributions of glycosyl hydrolase family 10 and 11 xylanases in the goat rumen.

    Directory of Open Access Journals (Sweden)

    Guozeng Wang

    Full Text Available BACKGROUND: The rumen harbors a complex microbial ecosystem for efficient hydrolysis of plant polysaccharides which are the main constituent of the diet. Xylanase is crucial for hemicellulose hydrolysis and plays an important role in the plant cell wall degradation. Xylanases of ruminal strains were widely studied, but few studies have focused on their diversity in rumen microenvironment. METHODOLOGY/PRINCIPAL FINDINGS: We explored the genetic diversity of xylanases belonging to two major glycosyl hydrolase families (GH 10 and 11 in goat rumen contents by analyzing the amplicons generated with two degenerate primer sets. Fifty-two distinct GH 10 and 35 GH 11 xylanase gene fragments (similarity <95% were retrieved, and most had low identities with known sequences. Based on phylogenetic analysis, all GH 10 xylanase sequences fell into seven clusters, and 88.5% of them were related to xylanases from Bacteroidetes. Five clusters of GH 11 xylanase sequences were identified. Of these, 85.7% were related to xylanases from Firmicutes, and 14.3% were related to those of rumen fungi. Two full-length xylanase genes (one for each family were directly cloned and expressed in Escherichia coli. Both the recombinant enzymes showed substantial xylanase activity, and were purified and characterized. Combined with the results of sheep rumen, Bacteroidetes and Firmicutes are the two major phyla of xylan-degrading microorganisms in rumen, which is distinct from the representatives of other environments such as soil and termite hindgut, suggesting that xylan-degrading microorganisms are environment specific. CONCLUSION/SIGNIFICANCE: The numerous new xylanase genes suggested the functional diversity of xylanase in the rumen microenvironment which may have great potential applications in industry and agriculture. The phylogenetic diversity and different distributions of xylanase genes will help us understand their roles in plant cell wall degradation in the rumen

  15. The crystal structure of an inverting glycoside hydrolase family 9 exo-β-D-glucosaminidase and the design of glycosynthase.

    Science.gov (United States)

    Honda, Yuji; Arai, Sachiko; Suzuki, Kentaro; Kitaoka, Motomitsu; Fushinobu, Shinya

    2016-02-15

    Exo-β-D-glucosaminidase (EC 3.2.1.165) from Photobacterium profundum (PpGlcNase) is an inverting GH (glycoside hydrolase) belonging to family 9. We have determined the three-dimensional structure of PpGlcNase to describe the first structure-function relationship of an exo-type GH9 glycosidase. PpGlcNase has a narrow and straight active-site pocket, in contrast with the long glycan-binding cleft of a GH9 endoglucanase. This is because PpGlcNase has a long loop, which blocks the position corresponding to subsites -4 to -2 of the endoglucanase. The pocket shape of PpGlcNase explains its substrate preference for a β1,4-linkage at the non-reducing terminus. Asp(139), Asp(143) and Glu(555) in the active site were located near the β-O1 hydroxy group of GlcN (D-glucosamine), with Asp(139) and Asp(143) holding a nucleophilic water molecule for hydrolysis. The D139A, D143A and E555A mutants significantly decreased hydrolytic activity, indicating their essential role. Of these mutants, D139A exclusively exhibited glycosynthase activity using α-GlcN-F (α-D-glucosaminyl fluoride) and GlcN as substrates, to produce (GlcN)2. Using saturation mutagenesis at Asp(139), we obtained D139E as the best glycosynthase. Compared with the wild-type, the hydrolytic activity of D139E was significantly suppressed (strategy for creating an effective glycosynthase from inverting GHs. However, for GH9, where two acidic residues seem to share the catalytic base role, mutation of Asp(139) might inevitably reduce F(-)-release activity. © 2016 Authors; published by Portland Press Limited.

  16. The phospholipase PNPLA7 functions as a lysophosphatidylcholine hydrolase and interacts with lipid droplets through its catalytic domain.

    Science.gov (United States)

    Heier, Christoph; Kien, Benedikt; Huang, Feifei; Eichmann, Thomas O; Xie, Hao; Zechner, Rudolf; Chang, Ping-An

    2017-11-17

    Mammalian patatin-like phospholipase domain-containing proteins (PNPLAs) are lipid-metabolizing enzymes with essential roles in energy metabolism, skin barrier development, and brain function. A detailed annotation of enzymatic activities and structure-function relationships remains an important prerequisite to understand PNPLA functions in (patho-)physiology, for example, in disorders such as neutral lipid storage disease, non-alcoholic fatty liver disease, and neurodegenerative syndromes. In this study, we characterized the structural features controlling the subcellular localization and enzymatic activity of PNPLA7, a poorly annotated phospholipase linked to insulin signaling and energy metabolism. We show that PNPLA7 is an endoplasmic reticulum (ER) transmembrane protein that specifically promotes hydrolysis of lysophosphatidylcholine in mammalian cells. We found that transmembrane and regulatory domains in the PNPLA7 N-terminal region cooperate to regulate ER targeting but are dispensable for substrate hydrolysis. Enzymatic activity is instead mediated by the C-terminal domain, which maintains full catalytic competence even in the absence of N-terminal regions. Upon elevated fatty acid flux, the catalytic domain targets cellular lipid droplets and promotes interactions of PNPLA7 with these organelles in response to increased cAMP levels. We conclude that PNPLA7 acts as an ER-anchored lysophosphatidylcholine hydrolase that is composed of specific functional domains mediating catalytic activity, subcellular positioning, and interactions with cellular organelles. Our study provides critical structural insights into an evolutionarily conserved class of phospholipid-metabolizing enzymes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Epoxy fatty acids and inhibition of the soluble epoxide hydrolase selectively modulate GABA mediated neurotransmission to delay onset of seizures.

    Directory of Open Access Journals (Sweden)

    Bora Inceoglu

    Full Text Available In the brain, seizures lead to release of large amounts of polyunsaturated fatty acids including arachidonic acid (ARA. ARA is a substrate for three major enzymatic routes of metabolism by cyclooxygenase, lipoxygenase and cytochrome P450 enzymes. These enzymes convert ARA to potent lipid mediators including prostanoids, leukotrienes and epoxyeicosatrienoic acids (EETs. The prostanoids and leukotrienes are largely pro-inflammatory molecules that sensitize neurons whereas EETs are anti-inflammatory and reduce the excitability of neurons. Recent evidence suggests a GABA-related mode of action potentially mediated by neurosteroids. Here we tested this hypothesis using models of chemically induced seizures. The level of EETs in the brain was modulated by inhibiting the soluble epoxide hydrolase (sEH, the major enzyme that metabolizes EETs to inactive molecules, by genetic deletion of sEH and by direct administration of EETs into the brain. All three approaches delayed onset of seizures instigated by GABA antagonists but not seizures through other mechanisms. Inhibition of neurosteroid synthesis by finasteride partially blocked the anticonvulsant effects of sEH inhibitors while the efficacy of an inactive dose of neurosteroid allopregnanolone was enhanced by sEH inhibition. Consistent with earlier findings, levels of prostanoids in the brain were elevated. In contrast, levels of bioactive EpFAs were decreased following seizures. Overall these results demonstrate that EETs are natural molecules which suppress the tonic component of seizure related excitability through modulating the GABA activity and that exploration of the EET mediated signaling in the brain could yield alternative approaches to treat convulsive disorders.

  18. Comparative Analysis of Glycoside Hydrolases Activities from Phylogenetically Diverse Marine Bacteria of the Genus Arenibacter

    Directory of Open Access Journals (Sweden)

    Valery Mikhailov

    2013-06-01

    Full Text Available A total of 16 marine strains belonging to the genus Arenibacter, recovered from diverse microbial communities associated with various marine habitats and collected from different locations, were evaluated in degradation of natural polysaccharides and chromogenic glycosides. Most strains were affiliated with five recognized species, and some presented three new species within the genus Arenibacter. No strains contained enzymes depolymerizing polysaccharides, but synthesized a wide spectrum of glycosidases. Highly active β-N-acetylglucosaminidases and α-N-acetylgalactosaminidases were the main glycosidases for all Arenibacter. The genes, encoding two new members of glycoside hydrolyses (GH families, 20 and 109, were isolated and characterized from the genomes of Arenibacter latericius. Molecular genetic analysis using glycosidase-specific primers shows the absence of GH27 and GH36 genes. A sequence comparison with functionally-characterized GH20 and GH109 enzymes shows that both sequences are closest to the enzymes of chitinolytic bacteria Vibrio furnissii and Cellulomonas fimi of marine and terrestrial origin, as well as human pathogen Elisabethkingia meningoseptica and simbionts Akkermansia muciniphila, gut and non-gut Bacteroides, respectively. These results revealed that the genus Arenibacter is a highly taxonomic diverse group of microorganisms, which can participate in degradation of natural polymers in marine environments depending on their niche and habitat adaptations. They are new prospective candidates for biotechnological applications due to their production of unique glycosidases.

  19. The dead, hardened floral bracts of dispersal units of wild wheat function as storage for active hydrolases and in enhancing seedling vigor.

    Directory of Open Access Journals (Sweden)

    Buzi Raviv

    Full Text Available It is commonly assumed that the dead, hardened floral bracts of the dispersal unit of grasses have been evolved to protect seeds from predation and / or assist in fruit/caryopsis dispersal. While these structures have important agronomical and economical implications, their adaptive value has not been fully explored. We investigated the hypothesis that the maternally derived hardened floral bracts have been evolved not just as a means for caryopsis protection and dispersal, but also as storage for substances that might affect seed germination and seedling vigor. Dead glumes as well as lemmas and paleas of wild emmer wheat (Triticum turgidum var dicoccoides were found to store and release upon hydration active hydrolases including nucleases and chitinases. High nuclease activity was released upon hydration from glumes derived from wild strains of wheat including Triticum urartu and wild emmer wheat, while very low nuclease activity was detected in glumes derived from domesticated, free-threshing wheat cultivars (e.g., durum wheat. Germination from the intact dispersal unit of wild emmer wheat was delayed, but post germination growth was better than those of separated caryopses. Most notable was a significant increase in lateral root production on seedlings germinated from the intact dispersal unit. Proteome analysis of wild emmer wheat glumes revealed many proteins stored and released upon hydration including S1-type nucleases, peptidases, antifungal hydrolases such as chitinases and β-1,3-glucanase as well as pectin acetylesterase, a protein involved in cell wall degradation and remodeling. Also, reactive oxygen species (ROS-detoxifying enzymes such as superoxide dismutase and ascorbate peroxidase were overrepresented in dead glumes of wild emmer wheat. Thus our study highlighted previously unknown features of the dispersal unit in wild wheat in which the dead, hardened floral bracts enclosing the caryopsis store active hydrolases and

  20. The Activation of Phytophthora Effector Avr3b by Plant Cyclophilin is Required for the Nudix Hydrolase Activity of Avr3b.

    Science.gov (United States)

    Kong, Guanghui; Zhao, Yao; Jing, Maofeng; Huang, Jie; Yang, Jin; Xia, Yeqiang; Kong, Liang; Ye, Wenwu; Xiong, Qin; Qiao, Yongli; Dong, Suomeng; Ma, Wenbo; Wang, Yuanchao

    2015-08-01

    Plant pathogens secrete an arsenal of effector proteins to impair host immunity. Some effectors possess enzymatic activities that can modify their host targets. Previously, we demonstrated that a Phytophthora sojae RXLR effector Avr3b acts as a Nudix hydrolase when expressed in planta; and this enzymatic activity is required for full virulence of P. sojae strain P6497 in soybean (Glycine max). Interestingly, recombinant Avr3b produced by E. coli does not have the hydrolase activity unless it was incubated with plant protein extracts. Here, we report the activation of Avr3b by a prolyl-peptidyl isomerase (PPIase), cyclophilin, in plant cells. Avr3b directly interacts with soybean cyclophilin GmCYP1, which activates the hydrolase activity of Avr3b in a PPIase activity-dependent manner. Avr3b contains a putative Glycine-Proline (GP) motif; which is known to confer cyclophilin-binding in other protein substrates. Substitution of the Proline (P132) in the putative GP motif impaired the interaction of Avr3b with GmCYP1; as a result, the mutant Avr3bP132A can no longer be activated by GmCYP1, and is also unable to promote Phytophthora infection. Avr3b elicits hypersensitive response (HR) in soybean cultivars producing the resistance protein Rps3b, but Avr3bP132A lost its ability to trigger HR. Furthermore, silencing of GmCYP1 rendered reduced cell death triggered by Avr3b, suggesting that GmCYP1-mediated Avr3b maturation is also required for Rps3b recognition. Finally, cyclophilins of Nicotiana benthamiana can also interact with Avr3b and activate its enzymatic activity. Overall, our results demonstrate that cyclophilin is a "helper" that activates the enzymatic activity of Avr3b after it is delivered into plant cells; as such, cyclophilin is required for the avirulence and virulence functions of Avr3b.

  1. Structure of a Nudix hydrolase (MutT) in the Mg2+-bound state from Bartonella henselae, the bacterium responsible for cat scratch fever

    International Nuclear Information System (INIS)

    Buchko, Garry W.; Edwards, Thomas E.; Abendroth, Jan; Arakaki, Tracy L.; Law, Laura; Napuli, Alberto J.; Hewitt, Stephen N.; Van Voorhis, Wesley C.; Stewart, Lance J.; Staker, Bart L.; Myler, Peter J.

    2011-01-01

    B. henselae is the etiological agent responsible for cat scratch fever (bartonellosis). The crystal structure of the smaller of the two Nudix hydrolases encoded in the genome of B. henselae, Bh-MutT, was determined to 2.1 Å resolution. Cat scratch fever (also known as cat scratch disease and bartonellosis) is an infectious disease caused by the proteobacterium Bartonella henselae following a cat scratch. Although the infection usually resolves spontaneously without treatment in healthy adults, bartonellosis may lead to severe complications in young children and immunocompromised patients, and there is new evidence suggesting that B. henselae may be associated with a broader range of clinical symptoms then previously believed. The genome of B. henselae contains genes for two putative Nudix hydrolases, BH02020 and BH01640 (KEGG). Nudix proteins play an important role in regulating the intracellular concentration of nucleotide cofactors and signaling molecules. The amino-acid sequence of BH02020 is similar to that of the prototypical member of the Nudix superfamily, Escherichia coli MutT, a protein that is best known for its ability to neutralize the promutagenic compound 7,8-dihydro-8-oxoguanosine triphosphate. Here, the crystal structure of BH02020 (Bh-MutT) in the Mg 2+ -bound state was determined at 2.1 Å resolution. As observed in all Nudix hydrolase structures, the α-helix of the highly conserved ‘Nudix box’ in Bh-MutT is one of two helices that sandwich a four-stranded mixed β-sheet with the central two β-strands parallel to each other. The catalytically essential divalent cation observed in the Bh-MutT structure, Mg 2+ , is coordinated to the side chains of Glu57 and Glu61. The structure is not especially robust; a temperature melt obtained using circular dichroism spectroscopy shows that Bh-MutT irreversibly unfolds and precipitates out of solution upon heating, with a T m of 333 K

  2. Modification of soil microbial activity and several hydrolases in a forest soil artificially contaminated with copper

    Science.gov (United States)

    Bellas, Rosa; Leirós, Mā Carmen; Gil-Sotres, Fernando; Trasar-Cepeda, Carmen

    2010-05-01

    Soils have long been exposed to the adverse effects of human activities, which negatively affect soil biological activity. As a result of their functions and ubiquitous presence microorganisms can serve as environmental indicators of soil pollution. Some features of soil microorganisms, such as the microbial biomass size, respiration rate, and enzyme activity are often used as bioindicators of the ecotoxicity of heavy metals. Although copper is essential for microorganisms, excessive concentrations have a negative influence on processes mediated by microorganisms. In this study we measured the response of some microbial indicators to Cu pollution in a forest soil, with the aim of evaluating their potential for predicting Cu contamination. Samples of an Ah horizon from a forest soil under oakwood vegetation (Quercus robur L.) were contaminated in the laboratory with copper added at different doses (0, 120, 360, 1080 and 3240 mg kg-1) as CuCl2×2H2O. The soil samples were kept for 7 days at 25 °C and at a moisture content corresponding to the water holding capacity, and thereafter were analysed for carbon and nitrogen mineralization capacity, microbial biomass C, seed germination and root elongation tests, and for urease, phosphomonoesterase, catalase and ß-glucosidase activities. In addition, carbon mineralization kinetics were studied, by plotting the log of residual C against incubation time, and the metabolic coefficient, qCO2, was estimated. Both organic carbon and nitrogen mineralization were lower in polluted samples, with the greatest decrease observed in the sample contaminated with 1080 mg kg-1. In all samples carbon mineralization followed first order kinetics; the C mineralization constant was lower in contaminated than in uncontaminated samples and, in general, decreased with increasing doses of copper. Moreover, it appears that copper contamination not only reduced the N mineralization capacity, but also modified the N mineralization process, since in

  3. [{sup 11}C]CURB: Evaluation of a novel radiotracer for imaging fatty acid amide hydrolase by positron emission tomography

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, Alan A., E-mail: alan.wilson@camhpet.c [PET Centre, Centre for Addiction and Mental Health, Toronto, Ontario, M5T 1R8 (Canada); Human Neurochemical Pathology Laboratory, Centre for Addiction and Mental Health, Toronto, Ontario, M5T 1R8 (Canada); Garcia, Armando; Parkes, Jun [PET Centre, Centre for Addiction and Mental Health, Toronto, Ontario, M5T 1R8 (Canada); Houle, Sylvain [PET Centre, Centre for Addiction and Mental Health, Toronto, Ontario, M5T 1R8 (Canada); Human Neurochemical Pathology Laboratory, Centre for Addiction and Mental Health, Toronto, Ontario, M5T 1R8 (Canada); Tong, Junchao [Department of Psychiatry, University of Toronto, Toronto, Ontario, M5T 1R8 (Canada); Vasdev, Neil [PET Centre, Centre for Addiction and Mental Health, Toronto, Ontario, M5T 1R8 (Canada); Human Neurochemical Pathology Laboratory, Centre for Addiction and Mental Health, Toronto, Ontario, M5T 1R8 (Canada)

    2011-02-15

    Introduction: Fatty acid amide hydrolase (FAAH) is the enzyme responsible for metabolising the endogenous cannabinoid, anandamide, and thus represents an important target for molecular imaging. To date, no radiotracer has been shown to be useful for imaging of FAAH using either positron emission tomography (PET) or single photon emission computed tomography (SPECT). We here determine the suitability of a novel carbon-11-labeled inhibitor of FAAH via ex vivo biodistribution studies in rat brain in conjunction with pharmacological challenges. Methods: A potent irreversible inhibitor of FAAH, URB694, radiolabeled with carbon-11 in the carbonyl position ([{sup 11}C]CURB), was administered to male rats via tail-vein injection. Rats were sacrificed at various time points postinjection, and tissue samples were dissected, counted and weighed. Specific binding to FAAH was investigated by pretreatment of animals with URB694 or URB597. For metabolism and mechanism of binding studies, whole brains were excised post-radiotracer injection, homogenised and extracted exhaustively with 80% aq. acetonitrile to determine the time course and fraction of radioactivity that was irreversibly bound to brain parenchyma. Results: Upon intravenous injection into rats, [{sup 11}C]CURB showed high brain uptake [standard uptake value (SUV) of 1.6-2.4 at 5 min] with little washout over time, which is characteristic of irreversible binding. Highest uptake of radioactivity was seen in the cortex, intermediate in the cerebellum and lowest in the hypothalamus, reflecting the reported distribution of FAAH. Brain uptake of radioactivity was decreased in a dose-dependent manner by pretreatment with increasing amounts of URB694, demonstrating that binding was saturable. Pretreatment with the well-characterised FAAH inhibitor, URB597, reduced binding in all brain regions by 70-80%. Homogenised brain extraction experiments demonstrated unequivocally that [{sup 11}C]CURB was irreversibly bound to FAAH

  4. Functional characterization and target discovery of glycoside hydrolases from the digestome of the lower termite Coptotermes gestroi

    Directory of Open Access Journals (Sweden)

    Franco Cairo João Paulo L

    2011-11-01

    Full Text Available Abstract Background Lignocellulosic materials have been moved towards the forefront of the biofuel industry as a sustainable resource. However, saccharification and the production of bioproducts derived from plant cell wall biomass are complex and lengthy processes. The understanding of termite gut biology and feeding strategies may improve the current state of biomass conversion technology and bioproduct production. Results The study herein shows comprehensive functional characterization of crude body extracts from Coptotermes gestroi along with global proteomic analysis of the termite's digestome, targeting the identification of glycoside hydrolases and accessory proteins responsible for plant biomass conversion. The crude protein extract from C. gestroi was enzymatically efficient over a broad pH range on a series of natural polysaccharides, formed by glucose-, xylose-, mannan- and/or arabinose-containing polymers, linked by various types of glycosidic bonds, as well as ramification types. Our proteomic approach successfully identified a large number of relevant polypeptides in the C. gestroi digestome. A total of 55 different proteins were identified and classified into 29 CAZy families. Based on the total number of peptides identified, the majority of components found in the C. gestroi digestome were cellulose-degrading enzymes. Xylanolytic enzymes, mannan- hydrolytic enzymes, pectinases and starch-degrading and debranching enzymes were also identified. Our strategy enabled validation of liquid chromatography with tandem mass spectrometry recognized proteins, by enzymatic functional assays and by following the degradation products of specific 8-amino-1,3,6-pyrenetrisulfonic acid labeled oligosaccharides through capillary zone electrophoresis. Conclusions Here we describe the first global study on the enzymatic repertoire involved in plant polysaccharide degradation by the lower termite C. gestroi. The biochemical characterization of whole

  5. [11C]CURB: Evaluation of a novel radiotracer for imaging fatty acid amide hydrolase by positron emission tomography

    International Nuclear Information System (INIS)

    Wilson, Alan A.; Garcia, Armando; Parkes, Jun; Houle, Sylvain; Tong, Junchao; Vasdev, Neil

    2011-01-01

    Introduction: Fatty acid amide hydrolase (FAAH) is the enzyme responsible for metabolising the endogenous cannabinoid, anandamide, and thus represents an important target for molecular imaging. To date, no radiotracer has been shown to be useful for imaging of FAAH using either positron emission tomography (PET) or single photon emission computed tomography (SPECT). We here determine the suitability of a novel carbon-11-labeled inhibitor of FAAH via ex vivo biodistribution studies in rat brain in conjunction with pharmacological challenges. Methods: A potent irreversible inhibitor of FAAH, URB694, radiolabeled with carbon-11 in the carbonyl position ([ 11 C]CURB), was administered to male rats via tail-vein injection. Rats were sacrificed at various time points postinjection, and tissue samples were dissected, counted and weighed. Specific binding to FAAH was investigated by pretreatment of animals with URB694 or URB597. For metabolism and mechanism of binding studies, whole brains were excised post-radiotracer injection, homogenised and extracted exhaustively with 80% aq. acetonitrile to determine the time course and fraction of radioactivity that was irreversibly bound to brain parenchyma. Results: Upon intravenous injection into rats, [ 11 C]CURB showed high brain uptake [standard uptake value (SUV) of 1.6-2.4 at 5 min] with little washout over time, which is characteristic of irreversible binding. Highest uptake of radioactivity was seen in the cortex, intermediate in the cerebellum and lowest in the hypothalamus, reflecting the reported distribution of FAAH. Brain uptake of radioactivity was decreased in a dose-dependent manner by pretreatment with increasing amounts of URB694, demonstrating that binding was saturable. Pretreatment with the well-characterised FAAH inhibitor, URB597, reduced binding in all brain regions by 70-80%. Homogenised brain extraction experiments demonstrated unequivocally that [ 11 C]CURB was irreversibly bound to FAAH. Conclusions

  6. Synthesis and preclinical evaluation of [11C-carbonyl]PF-04457845 for neuroimaging of fatty acid amide hydrolase

    International Nuclear Information System (INIS)

    Hicks, Justin W.; Parkes, Jun; Sadovski, Oleg; Tong, Junchao; Houle, Sylvain; Vasdev, Neil; Wilson, Alan A.

    2013-01-01

    Introduction: Fatty acid amide hydrolase (FAAH) has a significant role in regulating endocannabinoid signaling in the central nervous system. As such, FAAH inhibitors are being actively sought for pain, addiction, and other indications. This has led to the recent pursuit of positron emission tomography (PET) radiotracers targeting FAAH. We report herein the preparation and preclinical evaluation of [ 11 C-carbonyl]PF-04457845, an isotopologue of the potent irreversible FAAH inhibitor. Methods: PF-04457845 was radiolabeled at the carbonyl position via automated [ 11 C]CO 2 -fixation. Ex vivo brain biodistribution of [ 11 C-carbonyl]PF-04457845 was carried out in conscious rats. Specificity was determined by pre-administration of PF-04457845 or URB597 prior to [ 11 C-carbonyl]PF-04457845. In a separate experiment, rats injected with the title radiotracer had whole brains excised, homogenized and extracted to examine irreversible binding to brain parenchyma. Results: The title compound was prepared in 5 ± 1% (n = 4) isolated radiochemical yield based on starting [ 11 C]CO 2 (decay uncorrected) within 25 min from end-of-bombardment in > 98% radiochemical purity and a specific activity of 73.5 ± 8.2 GBq/μmol at end-of-synthesis. Uptake of [ 11 C-carbonyl]PF-04457845 into the rat brain was high (range of 1.2–4.4 SUV), heterogeneous, and in accordance with reported FAAH distribution. Saturable binding was demonstrated by a dose-dependent reduction in brain radioactivity uptake following pre-treatment with PF-04457845. Pre-treatment with the prototypical FAAH inhibitor, URB597, reduced the brain radiotracer uptake in all regions by 71–81%, demonstrating specificity for FAAH. The binding of [ 11 C-carbonyl]PF-04457845 to FAAH at 40 min post injection was irreversible as 98% of the radioactivity in the brain could not be extracted. Conclusions: [ 11 C-carbonyl]PF-04457845 was rapidly synthesized via an automated radiosynthesis. Ex vivo biodistribution studies in

  7. Inhibition of soluble epoxide hydrolase attenuates hepatic fibrosis and endoplasmic reticulum stress induced by carbon tetrachloride in mice

    Energy Technology Data Exchange (ETDEWEB)

    Harris, Todd R. [Department of Entomology and Comprehensive Cancer Center, University of California, Davis, CA 95616 (United States); Bettaieb, Ahmed [Department of Nutrition, University of California, Davis, CA 95616 (United States); Kodani, Sean; Dong, Hua [Department of Entomology and Comprehensive Cancer Center, University of California, Davis, CA 95616 (United States); Myers, Richard; Chiamvimonvat, Nipavan [Department of Internal Medicine: Cardiovascular, University of California, Davis, CA 95616 (United States); Haj, Fawaz G. [Department of Nutrition, University of California, Davis, CA 95616 (United States); Department of Internal Medicine: Endocrinology, Diabetes and Metabolism, University of California, Davis, CA 95616 (United States); Hammock, Bruce D., E-mail: bdhammock@ucdavis.edu [Department of Entomology and Comprehensive Cancer Center, University of California, Davis, CA 95616 (United States)

    2015-07-15

    Liver fibrosis is a pathological condition in which chronic inflammation and changes to the extracellular matrix lead to alterations in hepatic tissue architecture and functional degradation of the liver. Inhibitors of the enzyme soluble epoxide hydrolase (sEH) reduce fibrosis in the heart, pancreas and kidney in several disease models. In this study, we assess the effect of sEH inhibition on the development of fibrosis in a carbon tetrachloride (CCl{sub 4})-induced mouse model by monitoring changes in the inflammatory response, matrix remolding and endoplasmic reticulum stress. The sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) was administered in drinking water. Collagen deposition in the liver was increased five-fold in the CCl{sub 4}-treated group, and this was returned to control levels by TPPU treatment. Hepatic expression of Col1a2 and 3a1 mRNA was increased over fifteen-fold in the CCl{sub 4}-treated group relative to the Control group, and this increase was reduced by 50% by TPPU treatment. Endoplasmic reticulum (ER) stress observed in the livers of CCl{sub 4}-treated animals was attenuated by TPPU treatment. In order to support the hypothesis that TPPU is acting to reduce the hepatic fibrosis and ER stress through its action as a sEH inhibitor we used a second sEH inhibitor, trans-4-(4-[3-(4-trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy)-benzoic acid (t-TUCB), and sEH null mice. Taken together, these data indicate that the sEH may play an important role in the development of hepatic fibrosis induced by CCl{sub 4}, presumably by reducing endogenous fatty acid epoxide chemical mediators acting to reduce ER stress. - Highlights: • We administer an inhibitor of sEH in a CCl4 murine model. • sEH inhibition reduces liver collagen deposition and pro-fibrotic gene expression. • sEH inhibition induces MMP-1a activity.

  8. Ovarian expressed microsomal epoxide hydrolase: Role in detoxification of 4-vinylcyclohexene diepoxide and regulation by phosphatidylinositol-3 kinase signaling

    Energy Technology Data Exchange (ETDEWEB)

    Bhattacharya, Poulomi, E-mail: poulomib@iastate.edu [Department of Animal Science, Iowa State University, Ames, IA 50011 (United States); Sen, Nivedita, E-mail: nsen@email.arizona.edu [Department of Physiology, University of Arizona, Tucson, AZ 85724 (United States); Hoyer, Patricia B., E-mail: Hoyer@u.arizona.edu [Department of Physiology, University of Arizona, Tucson, AZ 85724 (United States); Keating, Aileen F., E-mail: akeating@iastate.edu [Department of Animal Science, Iowa State University, Ames, IA 50011 (United States)

    2012-01-01

    4-vinylcyclohexene diepoxide (VCD) is a metabolite of 4-vinylcyclohexene (VCH) which has the potential to be formed in the ovary through CYP2E1 activity. VCD specifically destroys primordial and small primary follicles in the rodent ovary. Mouse ovaries exposed to VCD demonstrate increased mRNA and protein expression of microsomal epoxide hydrolase (mEH), and an inactive tetrol metabolite (4-(1,2-dihydroxy)ethyl-1,2-dihydroxycyclohexane) can be formed in mouse ovarian follicles, potentially through detoxification action of mEH. In contrast, mEH can bioactivate another ovotoxic chemical, 7,12-dimethylbenz[a]anthracene (DMBA) to a more toxic compound, DMBA-3,4-diol-1,2-epoxide. Thus, the present study evaluated a functional role for mEH during detoxification of VCD. Additionally, because inhibition of the phosphatidyinositol-3 kinase (PI3K) signaling pathway in a previous study protected primordial follicles from VCD-induced destruction, but accelerated DMBA-induced ovotoxicity, a role for PI3K in ovarian mEH regulation was evaluated. Using a post-natal day (PND) 4 Fischer 344 rat whole ovary culture system inhibition of mEH using cyclohexene oxide during VCD exposure resulted in a greater (P < 0.05) loss of primordial and small primary follicles relative to VCD-treated ovaries. Also, relative to controls, meh mRNA was increased (P < 0.05) on day 4 of VCD (30 μM) exposure, followed by increased (P < 0.05) mEH protein after 6 days. Furthermore, inhibition of PI3K signaling increased mEH mRNA and protein expression. Thus, these results support a functional role for mEH in the rat ovary, and demonstrate the involvement of PI3K signaling in regulation of ovarian xenobiotic metabolism by mEH. -- Highlights: ► Ovarian mEH functions to metabolize VCD to a less toxic compound. ► mEH expression is increased in a temporal pattern in response to VCD exposure. ► PI3K signaling is involved in regulation of ovarian mEH expression.

  9. Intrinsically disordered regions may lower the hydration free energy in proteins: a case study of nudix hydrolase in the bacterium Deinococcus radiodurans.

    Directory of Open Access Journals (Sweden)

    Omar Awile

    Full Text Available The proteome of the radiation- and desiccation-resistant bacterium D. radiodurans features a group of proteins that contain significant intrinsically disordered regions that are not present in non-extremophile homologues. Interestingly, this group includes a number of housekeeping and repair proteins such as DNA polymerase III, nudix hydrolase and rotamase. Here, we focus on a member of the nudix hydrolase family from D. radiodurans possessing low-complexity N- and C-terminal tails, which exhibit sequence signatures of intrinsic disorder and have unknown function. The enzyme catalyzes the hydrolysis of oxidatively damaged and mutagenic nucleotides, and it is thought to play an important role in D. radiodurans during the recovery phase after exposure to ionizing radiation or desiccation. We use molecular dynamics simulations to study the dynamics of the protein, and study its hydration free energy using the GB/SA formalism. We show that the presence of disordered tails significantly decreases the hydration free energy of the whole protein. We hypothesize that the tails increase the chances of the protein to be located in the remaining water patches in the desiccated cell, where it is protected from the desiccation effects and can function normally. We extrapolate this to other intrinsically disordered regions in proteins, and propose a novel function for them: intrinsically disordered regions increase the "surface-properties" of the folded domains they are attached to, making them on the whole more hydrophilic and potentially influencing, in this way, their localization and cellular activity.

  10. Distribution and function of carbamate hydrolase genes cehA and mcd in soils: the distinct role of soil pH.

    Science.gov (United States)

    Rousidou, Constantina; Karaiskos, Dionysis; Myti, Despoina; Karanasios, Evangelos; Karas, Panagiotis A; Tourna, Maria; Tzortzakakis, Emmanuel A; Karpouzas, Dimitrios G

    2017-01-01

    Synthetic carbamates constitute a significant pesticide group with oxamyl being a leading compound in the nematicide market. Oxamyl degradation in soil is mainly microbially mediated. However, the distribution and function of carbamate hydrolase genes (cehA, mcd, cahA) associated with the soil biodegradation of carbamates is not yet clear. We studied oxamyl degradation in 16 soils from a potato monoculture area in Greece where oxamyl is regularly used. Oxamyl showed low persistence (DT50 2.4-26.7 days). q-PCR detected the cehA and mcd genes in 10 and three soils, respectively. The abundance of the cehA gene was positively correlated with pH, while both cehA abundance and pH were negatively correlated with oxamyl DT50. Amongst the carbamates used in the study region, oxamyl stimulated the abundance and expression only of the cehA gene, while carbofuran stimulated the abundance and expression of both genes. The cehA gene was also detected in pristine soils upon repeated treatments with oxamyl and carbofuran and only in soils with pH ≥7.2, where the most rapid degradation of oxamyl was observed. These results have major implications regarding the maintenance of carbamate hydrolase genes in soils, have practical implications regarding the agricultural use of carbamates, and provide insights into the evolution of cehA. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  11. The structure of a complex of the lactonohydrolase zearalenone hydrolase with the hydrolysis product of zearalenone at 1.60 Å resolution.

    Science.gov (United States)

    Qi, Qi; Yang, Wen Jing; Zhou, Hu Jian; Ming, Deng Ming; Sun, Kai Lei; Xu, Tian Yu; Hu, Xiao Jian; Lv, Hong

    2017-07-01

    Zearalenone hydrolase (ZHD) is an α/β-hydrolase that detoxifies and degrades the lactone zearalenone (ZEN), a naturally occurring oestrogenic mycotoxin that contaminates crops. Several apoenzyme and enzyme-substrate complex structures have been reported in the resolution range 2.4-2.6 Å. However, the properties and mechanism of this enzyme are not yet fully understood. Here, a 1.60 Å resolution structure of a ZHD-product complex is reported which was determined from a C-terminally His 6 -tagged ZHD crystal soaked with 2 mM ZEN for 30 min. It shows that after the lactone-bond cleavage, the phenol-ring region moves closer to residues Leu132, Tyr187 and Pro188, while the lactone-ring region barely moves. Comparisons of the ZHD-substrate and ZHD-product structures show that the hydrophilic interactions change, especially Trp183 N ℇ1 , which shifts from contacting O2 to O12', suggesting that Trp183 is responsible for the unidirectional translational movement of the phenol ring. This structure provides information on the final stage of the catalytic mechanism of zearalenone hydrolysis.

  12. Endo-xylogalacturonan hydrolase

    NARCIS (Netherlands)

    Herweijer, M.A.; Vincken, J.P.; Meeuwsen, P.J.A.; Vlugt-Bergmans, van der C.J.B.; Beldman, G.; Ooyen, van A.J.J.; Voragen, A.G.J.

    2003-01-01

    A cDNA library of Aspergillus tubingensis was constructed in the yeast Kluyveromyces lactis, using a carbon source rich in xylogalacturonan. The library was screened using a hairy regions preparation from apple, and xylogalacturonan prepared from gum tragacanth as substrates. A novel

  13. The gram-negative bacterium Azotobacter chroococcum NCIMB 8003 employs a new glycoside hydrolase family 70 4,6-α-glucanotransferase enzyme (GtfD) to synthesize a reuteran like polymer from maltodextrins and starch

    NARCIS (Netherlands)

    Gangoiti, Joana; van Leeuwen, Sander S; Vafiadi, Christina; Dijkhuizen, Lubbert

    BACKGROUND: Originally the glycoside hydrolase (GH) family 70 only comprised glucansucrases of lactic acid bacteria which synthesize α-glucan polymers from sucrose. Recently we have identified 2 novel subfamilies of GH70 enzymes represented by the Lactobacillus reuteri 121 GtfB and the

  14. α-Synuclein-induced lysosomal dysfunction occurs through disruptions in protein trafficking in human midbrain synucleinopathy models.

    Science.gov (United States)

    Mazzulli, Joseph R; Zunke, Friederike; Isacson, Ole; Studer, Lorenz; Krainc, Dimitri

    2016-02-16

    Parkinson's disease (PD) is an age-related neurodegenerative disorder characterized by the accumulation of protein aggregates comprised of α-synuclein (α-syn). A major barrier in treatment discovery for PD is the lack of identifiable therapeutic pathways capable of reducing aggregates in human neuronal model systems. Mutations in key components of protein trafficking and cellular degradation machinery represent important risk factors for PD; however, their precise role in disease progression and interaction with α-syn remains unclear. Here, we find that α-syn accumulation reduced lysosomal degradation capacity in human midbrain dopamine models of synucleinopathies through disrupting hydrolase trafficking. Accumulation of α-syn at the cell body resulted in aberrant association with cis-Golgi-tethering factor GM130 and disrupted the endoplasmic reticulum-Golgi localization of rab1a, a key mediator of vesicular transport. Overexpression of rab1a restored Golgi structure, improved hydrolase trafficking and activity, and reduced pathological α-syn in patient neurons. Our work suggests that enhancement of lysosomal hydrolase trafficking may prove beneficial in synucleinopathies and indicates that human midbrain disease models may be useful for identifying critical therapeutic pathways in PD and related disorders.

  15. Two intestinal specific nuclear factors binding to the lactase-phlorizin hydrolase and sucrase-isomaltase promoters are functionally related oligomeric molecules

    DEFF Research Database (Denmark)

    Troelsen, J T; Mitchelmore, C; Sjöström, H

    1994-01-01

    Lactase-phlorizin hydrolase (LPH) and sucrase-isomaltase (SI) are enterocyte-specific gene products. The identification of regulatory cis-elements in the promoter of these two genes has enabled us to carry out comparative studies of the corresponding intestinal-specific nuclear factors (NF-LPH1...... and SIF1-BP). Electrophoretic mobility shift assays demonstrated that the two nuclear factors compete for binding on the same cis-elements. The molecular size of the DNA binding polypeptide is estimated to be approximately 50 kDa for both factors. In the native form the factors are found as 250 k......Da oligomeric complexes. Based on these results NF-LPH1 and SIF1-BP are suggested to be either identical or closely related molecules....

  16. From Soil to Structure, a Novel Dimeric β-Glucosidase Belonging to Glycoside Hydrolase Family 3 Isolated from Compost Using Metagenomic Analysis

    Science.gov (United States)

    McAndrew, Ryan P.; Park, Joshua I.; Heins, Richard A.; Reindl, Wolfgang; Friedland, Gregory D.; D'haeseleer, Patrik; Northen, Trent; Sale, Kenneth L.; Simmons, Blake A.; Adams, Paul D.

    2013-01-01

    A recent metagenomic analysis sequenced a switchgrass-adapted compost community to identify enzymes from microorganisms that were specifically adapted to switchgrass under thermophilic conditions. These enzymes are being examined as part of the pretreatment process for the production of “second-generation” biofuels. Among the enzymes discovered was JMB19063, a novel three-domain β-glucosidase that belongs to the GH3 (glycoside hydrolase 3) family. Here, we report the structure of JMB19063 in complex with glucose and the catalytic variant D261N crystallized in the presence of cellopentaose. JMB19063 is first structure of a dimeric member of the GH3 family, and we demonstrate that dimerization is required for catalytic activity. Arg-587 and Phe-598 from the C-terminal domain of the opposing monomer are shown to interact with bound ligands in the D261N structure. Enzyme assays confirmed that these residues are absolutely essential for full catalytic activity. PMID:23580647

  17. Experimental mixture design as a tool to enhance glycosyl hydrolases production by a new Trichoderma harzianum P49P11 strain cultivated under controlled bioreactor submerged fermentation.

    Science.gov (United States)

    Delabona, Priscila da Silva; Farinas, Cristiane Sanchez; Lima, Deise Juliana da Silva; Pradella, José Geraldo da Cruz

    2013-03-01

    This work investigates the glycosyl hydrolase (GH) profile of a new Trichoderma harzianum strain cultivated under controlled bioreactor submerged fermentation. The influence of different medium components (delignified steam-exploded sugarcane bagasse, sucrose, and soybean flour) on GH biosynthesis was assessed using experimental mixture design (EMD). Additionally, the effect of increased component concentrations in culture media selected from the EMD was studied. It was found that that a mixed culture medium could significantly maximize GH biosynthesis rate, especially for xylanase enzymes which achieved a 2-fold increment. Overall, it was demonstrated that T. harzianumP49P11 enzymes have a great potential to be used in the deconstruction of biomass. Copyright © 2012 Elsevier Ltd. All rights reserved.

  18. Multiple rewards from a treasure trove of novel glycoside hydrolase and polysaccharide lyase structures: new folds, mechanistic details, and evolutionary relationships.

    Science.gov (United States)

    Fushinobu, Shinya; Alves, Victor D; Coutinho, Pedro M

    2013-10-01

    Recent progress in three-dimensional structure analyses of glycoside hydrolases (GHs) and polysaccharide lyases (PLs), the historically relevant enzyme classes involved in the cleavage of glycosidic bonds of carbohydrates and glycoconjugates, is reviewed. To date, about 80% and 95% of the GH and PL families, respectively, have a representative crystal structure. New structures have been determined for enzymes acting on plant cell wall polysaccharides, sphingolipids, blood group antigens, milk oligosaccharides, N-glycans, oral biofilms and dietary seaweeds. Some GH enzymes have very unique catalytic residues such as the Asp-His dyad. New methods such as high-speed atomic force microscopy and computational simulation have opened up a path to investigate both the dynamics and the detailed molecular interactions displayed by these enzymes. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Identification and characterization of epoxide hydrolase activity of polycyclic aromatic hydrocarbon-degrading bacteria for biocatalytic resolution of racemic styrene oxide and styrene oxide derivatives.

    Science.gov (United States)

    Woo, Jung-Hee; Kwon, Tae-Hyung; Kim, Jun-Tae; Kim, Choong-Gon; Lee, Eun Yeol

    2013-04-01

    A novel epoxide hydrolase (EHase) from polycyclic aromatic hydrocarbon (PAH)-degrading bacteria was identified and characterized. EHase activity was identified in four strains of PAH-degrading bacteria isolated from commercial gasoline and oil-contaminated sediment based on their growth on styrene oxide and its derivatives, such as 2,3- and 4-chlorostyrene oxides, as a sole carbon source. Gordonia sp. H37 exhibited high enantioselective hydrolysis activity for 4-chlorostyrene oxide with an enantiomeric ratio of 27. Gordonia sp. H37 preferentially hydrolyzed the (R)-enantiomer of styrene oxide derivatives resulting in the preparation of a (S)-enantiomer with enantiomeric excess greater than 99.9 %. The enantioselective EHase activity was identified and characterized in various PAH-degrading bacteria, and whole cell Gordonia sp. H37 was employed as a biocatalyst for preparing enantiopure (S)-styrene oxide derivatives.

  20. 1,3-disubstituted ureas functionalized with ether groups are potent inhibitors of the soluble epoxide hydrolase with improved pharmacokinetic properties.

    Science.gov (United States)

    Kim, In-Hae; Tsai, Hsing-Ju; Nishi, Kosuke; Kasagami, Takeo; Morisseau, Christophe; Hammock, Bruce D

    2007-10-18

    Soluble epoxide hydrolase (sEH) is a therapeutic target for treating hypertension and inflammation. 1,3-Disubstituted ureas functionalized with an ether group are potent sEH inhibitors. However, their relatively low metabolic stability leads to poor pharmacokinetic properties. To improve their bioavailability, we investigated the effect of incorporating various polar groups on the ether function on the inhibition potencies, physical properties, in vitro metabolic stability, and pharmacokinetic properties. The structure-activity relationship studies showed that a hydrophobic linker between the urea group and the ether function is necessary to keep their potency. In addition, urea-ether inhibitors having a polar group such as diethylene glycol or morpholine significantly improved their physical properties and metabolic stability without any loss of inhibitory potency. Furthermore, improved pharmacokinetic properties in murine and canine models were obtained with the resulting inhibitors. These findings will facilitate the usage of sEH inhibitors in animal models of hypertension and inflammation.

  1. Effect of fungal mycelia on the HPLC-UV and UV-vis spectrophotometric assessment of mycelium-bound epoxide hydrolase using glycidyl phenyl ether.

    Science.gov (United States)

    Dolcet, Marta M; Torres, Mercè; Canela, Ramon

    2016-06-25

    The use of mycelia as biocatalysts has technical and economic advantages. However, there are several difficulties in obtaining accurate results in mycelium-catalysed reactions. Firstly, sample extraction, indispensable because of the presence of mycelia, can bring into the extract components with a similar structure to that of the analyte of interest; secondly, mycelia can influence the recovery of the analyte. We prepared calibration standards of 3-phenoxy-1,2-propanediol (PPD) in the pure solvent and in the presence of mycelia (spiked before or after extraction) from five fungi (Aspergillus niger, Aspergillus tubingensis, Penicillium aurantiogriseum, Penicillium sp. and Aspergillus terreus). The quantification of PPD was carried out by HPLC-UV and UV-vis spectrophotometry. The manuscript shows that the last method is as accurate as the HPLC method. However, the colorimetric method led to a higher data throughput, which allowed the study of more samples in a shorter time. Matrix effects were evaluated visually from the plotted calibration data and statistically by simultaneously comparing the intercept and slope of calibration curves performed with solvent, post-extraction spiked standards and pre-extraction spiked standards. Significant differences were found between the post- and pre-extraction spiked matrix-matched functions. Pre-extraction spiked matrix-matched functions based on A. tubingensis mycelia, selected as the reference, were validated and used to compensate for low recoveries. These validated functions were successfully applied to the quantification of PPD achieved during the hydrolysis of glycidyl phenyl ether by mycelium-bound epoxide hydrolases and equivalent hydrolysis yields were determined by HPLC-UV and UV-vis spectrophotometry. This study may serve as starting point to implement matrix effects evaluation when mycelium-bound epoxide hydrolases are studied. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Post-exposure administration of diazepam combined with soluble epoxide hydrolase inhibition stops seizures and modulates neuroinflammation in a murine model of acute TETS intoxication

    International Nuclear Information System (INIS)

    Vito, Stephen T.; Austin, Adam T.; Banks, Christopher N.; Inceoglu, Bora; Bruun, Donald A.; Zolkowska, Dorota; Tancredi, Daniel J.; Rogawski, Michael A.; Hammock, Bruce D.; Lein, Pamela J.

    2014-01-01

    Tetramethylenedisulfotetramine (TETS) is a potent convulsant poison for which there is currently no approved antidote. The convulsant action of TETS is thought to be mediated by inhibition of type A gamma-aminobutyric acid receptor (GABA A R) function. We, therefore, investigated the effects of post-exposure administration of diazepam, a GABA A R positive allosteric modulator, on seizure activity, death and neuroinflammation in adult male Swiss mice injected with a lethal dose of TETS (0.15 mg/kg, ip). Administration of a high dose of diazepam (5 mg/kg, ip) immediately following the second clonic seizure (approximately 20 min post-TETS injection) effectively prevented progression to tonic seizures and death. However, this treatment did not prevent persistent reactive astrogliosis and microglial activation, as determined by GFAP and Iba-1 immunoreactivity and microglial cell morphology. Inhibition of soluble epoxide hydrolase (sEH) has been shown to exert potent anti-inflammatory effects and to increase survival in mice intoxicated with other GABA A R antagonists. The sEH inhibitor TUPS (1 mg/kg, ip) administered immediately after the second clonic seizure did not protect TETS-intoxicated animals from tonic seizures or death. Combined administration of diazepam (5 mg/kg, ip) and TUPS (1 mg/kg, ip, starting 1 h after diazepam and repeated every 24 h) prevented TETS-induced lethality and influenced signs of neuroinflammation in some brain regions. Significantly decreased microglial activation and enhanced reactive astrogliosis were observed in the hippocampus, with no changes in the cortex. Combining an agent that targets specific anti-inflammatory mechanisms with a traditional antiseizure drug may enhance treatment outcome in TETS intoxication. - Highlights: • Acute TETS intoxication causes delayed and persistent neuroinflammation. • Diazepam given post-TETS prevents lethal tonic seizures but not neuroinflammation. • A soluble epoxide hydrolase inhibitor alters

  3. Relative gene expression of bile salt hydrolase and surface proteins in two putative indigenous Lactobacillus plantarum strains under in vitro gut conditions.

    Science.gov (United States)

    Duary, Raj Kumar; Batish, Virender Kumar; Grover, Sunita

    2012-03-01

    Probiotic bacteria must overcome the toxicity of bile salts secreted in the gut and adhere to the epithelial cells to enable their better colonization with extended transit time. Expression of bile salt hydrolase and other proteins on the surface of probiotic bacteria can help in better survivability and optimal functionality in the gut. Two putative Lactobacillus plantarum isolates i.e., Lp9 and Lp91 along with standard strain CSCC5276 were used. A battery of six housekeeping genes viz. gapB, dnaG, gyrA, ldhD, rpoD and 16S rRNA were evaluated by using geNorm 3.4 excel based application for normalizing the expression of bile salt hydrolase (bsh), mucus-binding protein (mub), mucus adhesion promoting protein (mapA), and elongation factor thermo unstable (EF-Tu) in Lp9 and Lp91. The maximal level of relative bsh gene expression was recorded in Lp91 with 2.89 ± 0.14, 4.57 ± 0.37 and 6.38 ± 0.19 fold increase at 2% bile salt concentration after 1, 2 and 3 h, respectively. Similarly, mub and mapA genes were maximally expressed in Lp9 at the level of 20.07 ± 1.28 and 30.92 ± 1.51 fold, when MRS was supplemented with 0.05% mucin and 1% each of bile and pancreatin (pH 6.5). However, in case of EF-Tu, the maximal expression of 42.84 ± 5.64 fold was recorded in Lp91 in the presence of mucin alone (0.05%). Hence, the expression of bsh, mub, mapA and EF-Tu could be considered as prospective biomarkers for screening of novel probiotic lactobacillus strains for optimal functionality in the gut.

  4. Hydroxynitrile Lyases with α/β-Hydrolase Fold: Two Enzymes with Almost Identical 3D Structures but Opposite Enantioselectivities and Different Reaction Mechanisms

    Science.gov (United States)

    Andexer, Jennifer N; Staunig, Nicole; Eggert, Thorsten; Kratky, Christoph; Pohl, Martina; Gruber, Karl

    2012-01-01

    Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrins to yield hydrocyanic acid (HCN) and the respective carbonyl compound and are key enzymes in the process of cyanogenesis in plants. In organic syntheses, HNLs are used as biocatalysts for the formation of enantiopure cyanohydrins. We determined the structure of the recently identified, R-selective HNL from Arabidopsis thaliana (AtHNL) at a crystallographic resolution of 2.5 Å. The structure exhibits an α/β-hydrolase fold, very similar to the homologous, but S-selective, HNL from Hevea brasiliensis (HbHNL). The similarities also extend to the active sites of these enzymes, with a Ser-His-Asp catalytic triad present in all three cases. In order to elucidate the mode of substrate binding and to understand the unexpected opposite enantioselectivity of AtHNL, complexes of the enzyme with both (R)- and (S)-mandelonitrile were modeled using molecular docking simulations. Compared to the complex of HbHNL with (S)-mandelonitrile, the calculations produced an approximate mirror image binding mode of the substrate with the phenyl rings located at very similar positions, but with the cyano groups pointing in opposite directions. A catalytic mechanism for AtHNL is proposed, in which His236 from the catalytic triad acts as a general base and the emerging negative charge on the cyano group is stabilized by main-chain amide groups and an α-helix dipole very similar to α/β-hydrolases. This mechanistic proposal is additionally supported by mutagenesis studies. PMID:22851196

  5. F1 Domain of the Leishmania (Leishmania donovani Nucleoside Hydrolase Promotes a Th1 Response in Leishmania (Leishmania infantum Cured Patients and in Asymptomatic Individuals Living in an Endemic Area of Leishmaniasis

    Directory of Open Access Journals (Sweden)

    Eugenia Carrillo

    2017-07-01

    Full Text Available The Leishmania (Leishmania donovani nucleoside hydrolase NH36 is the main antigen of the Leishmune® vaccine and one of the promising candidates for vaccination against visceral leishmaniasis. The antigenicity of the N-terminal (F1, the central (F2, or the C-terminal recombinant domain (F3 of NH36 was evaluated using peripheral blood mononuclear cells (PBMC from individuals infected with L. (L. infantum from an endemic area of visceral leishmaniasis of Spain. Both NH36 and F1 domains significantly increased the PBMC proliferation stimulation index of cured patients and infected asymptomatic individuals compared to healthy controls. Moreover, F1 induced a 19% higher proliferative response than NH36 in asymptomatic exposed subjects. In addition, in patients cured from visceral leishmaniasis, proliferation in response to NH36 and F1 was accompanied by a significant increase of IFN-γ and TNF-α secretion, which was 42–43% higher, in response to F1 than to NH36. The interleukin 17 (IL-17 secretion was stronger in asymptomatic subjects, in response to F1, as well as in cured cutaneous leishmaniasis after NH36 stimulation. While no IL-10 secretion was determined by F1, a granzyme B increase was detected in supernatants from cured patients after stimulation with either NH36 or F1. These data demonstrate that F1 is the domain of NH36 that induces a recall cellular response in individuals with acquired resistance to the infection by L. (L. infantum. In addition, F1 and NH36 discriminated the IgG3 humoral response in patients with active visceral leishmaniasis due to L. (L. donovani (Ethiopia and L. (L. infantum (Spain from that of endemic and non-endemic area controls. NH36 showed higher reactivity with sera from L. (L. donovani-infected individuals, indicating species specificity. We conclude that the F1 domain, previously characterized as an inducer of the Th1 and Th17 responses in cured/exposed patients infected with L. (L. infantum chagasi, may

  6. Imaging features of type 1 hereditary tyrosinemia: a review of 30 patients

    Energy Technology Data Exchange (ETDEWEB)

    Dubois, J. [Department of Radiology, Hopital Sainte-Justine, Montreal, Que. (Canada); Garel, L. [Department of Radiology, Hopital Sainte-Justine, Montreal, Que. (Canada); Patriquin, H. [Department of Radiology, Hopital Sainte-Justine, Montreal, Que. (Canada); Paradis, K. [Department of Pediatrics, Gastroenterology, Universite de Montreal, Que. (Canada); Forget, S. [Department of Pediatrics, Gastroenterology, Universite de Montreal, Que. (Canada); Filiatrault, D. [Department of Radiology, Hopital Sainte-Justine, Montreal, Que. (Canada); Grignon, A. [Department of Radiology, Hopital Sainte-Justine, Montreal, Que. (Canada); Russo, P. [Department of Pathology, Hopital Sainte-Justine, Montreal, Que. (Canada); St-Vil, D. [Department of Surgery, Hopital Sainte-Justine, Montreal, Que. (Canada)

    1996-12-01

    Hereditary tyrosinemia type 1, a common genetic disorder in the province of Quebec, is characterized by a deficiency of fumarylacetoacetate hydrolase. In this autosomal recessive disorder of tyrosine metabolism, the accumulation of succinylacetone leads to neurologic crises, acute and chronic liver failure, complex renal tubulopathy, rickets and a hemorrhagic syndrome. Liver trans- plantation has dramatically modified the spontaneous course of this lethal disease. The present paper describes the imaging fea- tures of tyrosinemia in 30 patients followed from 1980 to 1995 at Hopital Sainte-Justine, Montreal, Canada. (orig.). With 10 figs., 2 tabs.

  7. Identification of Novel Mutations in FAH Gene and Prenatal Diagnosis of Tyrosinemia in Indian Family

    Directory of Open Access Journals (Sweden)

    Jayesh J. Sheth

    2012-01-01

    Full Text Available Carrier of tyrosinemia type I was diagnosed by sequencing FAH (fumarylacetoacetate hydrolase gene. It leads to the identification of heterozygous status for both c.648C>G (p.Ile216Met and c.1159G>A (p.Gly387Arg mutations in exons 8 and 13, respectively, in the parents. The experimental program PolyPhen, SIFT, and MT predicts former missense point mutation as “benign” that creates a potential donor splice site and later one as “probably damaging” which disrupts secondary structure of protein.

  8. Inhibition of soluble epoxide hydrolase by cis-4-[4-(3-adamantan-1-ylureido)cyclohexyl-oxy]benzoic acid exhibits antihypertensive and cardioprotective actions in transgenic rats with angiotensin II-dependent hypertension

    Czech Academy of Sciences Publication Activity Database

    Neckář, Jan; Kopkan, L.; Husková, Z.; Kolář, František; Papoušek, František; Kramer, H. J.; Hwang, S.H.; Hammock, B.D.; Imig, J. D.; Malý, J.; Netuka, I.; Ošťádal, Bohuslav; Červenka, L.

    2012-01-01

    Roč. 122, č. 11 (2012), s. 513-525 ISSN 0143-5221 R&D Projects: GA AV ČR(CZ) IAAX01110901; GA AV ČR(CZ) KAN200520703; GA MŠk(CZ) 1M0510 Institutional research plan: CEZ:AV0Z50110509 Keywords : hypertension * angiotensin II * kidney * epoxyeicosatrienoic acids * soluble epoxide hydrolase inhibitor * myocardial ischemia/reperfusion injury Subject RIV: FA - Cardiovascular Diseases incl. Cardiotharic Surgery Impact factor: 4.859, year: 2012

  9. Post-exposure administration of diazepam combined with soluble epoxide hydrolase inhibition stops seizures and modulates neuroinflammation in a murine model of acute TETS intoxication

    Energy Technology Data Exchange (ETDEWEB)

    Vito, Stephen T., E-mail: stvito@ucdavis.edu [Department of Entomology, College of Agricultural and Environmental Sciences, University of California-Davis, Davis, CA 95616 (United States); Austin, Adam T., E-mail: aaustin@ucdavis.edu [Department of Pediatrics, School of Medicine, University of California-Davis Medical Center, Sacramento, CA 95817 (United States); Banks, Christopher N., E-mail: Christopher.Banks@oehha.ca.gov [Department of Molecular Biosciences, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616 (United States); Inceoglu, Bora, E-mail: abinceoglu@ucdavis.edu [Department of Entomology, College of Agricultural and Environmental Sciences, University of California-Davis, Davis, CA 95616 (United States); Bruun, Donald A., E-mail: dabruun@ucdavis.edu [Department of Molecular Biosciences, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616 (United States); Zolkowska, Dorota, E-mail: dzolkowska@gmail.com [Department of Neurology, School of Medicine, University of California-Davis, Sacramento, CA 95817 (United States); Tancredi, Daniel J., E-mail: djtancredi@ucdavis.edu [Department of Pediatrics, School of Medicine, University of California-Davis Medical Center, Sacramento, CA 95817 (United States); Rogawski, Michael A., E-mail: rogawski@ucdavis.edu [Department of Neurology, School of Medicine, University of California-Davis, Sacramento, CA 95817 (United States); Hammock, Bruce D., E-mail: bdhammock@ucdavis.edu [Department of Entomology, College of Agricultural and Environmental Sciences, University of California-Davis, Davis, CA 95616 (United States); Lein, Pamela J., E-mail: pjlein@ucdavis.edu [Department of Molecular Biosciences, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616 (United States)

    2014-12-01

    Tetramethylenedisulfotetramine (TETS) is a potent convulsant poison for which there is currently no approved antidote. The convulsant action of TETS is thought to be mediated by inhibition of type A gamma-aminobutyric acid receptor (GABA{sub A}R) function. We, therefore, investigated the effects of post-exposure administration of diazepam, a GABA{sub A}R positive allosteric modulator, on seizure activity, death and neuroinflammation in adult male Swiss mice injected with a lethal dose of TETS (0.15 mg/kg, ip). Administration of a high dose of diazepam (5 mg/kg, ip) immediately following the second clonic seizure (approximately 20 min post-TETS injection) effectively prevented progression to tonic seizures and death. However, this treatment did not prevent persistent reactive astrogliosis and microglial activation, as determined by GFAP and Iba-1 immunoreactivity and microglial cell morphology. Inhibition of soluble epoxide hydrolase (sEH) has been shown to exert potent anti-inflammatory effects and to increase survival in mice intoxicated with other GABA{sub A}R antagonists. The sEH inhibitor TUPS (1 mg/kg, ip) administered immediately after the second clonic seizure did not protect TETS-intoxicated animals from tonic seizures or death. Combined administration of diazepam (5 mg/kg, ip) and TUPS (1 mg/kg, ip, starting 1 h after diazepam and repeated every 24 h) prevented TETS-induced lethality and influenced signs of neuroinflammation in some brain regions. Significantly decreased microglial activation and enhanced reactive astrogliosis were observed in the hippocampus, with no changes in the cortex. Combining an agent that targets specific anti-inflammatory mechanisms with a traditional antiseizure drug may enhance treatment outcome in TETS intoxication. - Highlights: • Acute TETS intoxication causes delayed and persistent neuroinflammation. • Diazepam given post-TETS prevents lethal tonic seizures but not neuroinflammation. • A soluble epoxide hydrolase

  10. An arabinobio-hydrolase (Arb93B) from Fusarium graminearum is associated with wheat head blight disease

    Science.gov (United States)

    Fusarium head blight (FHB), caused by the fungus Fusarium graminearum, is one of the most important diseases of wheat and barley worldwide. FHB not only reduces crop yield, but the fungus also contaminates grains with mycotoxins, which are harmful to humans and animals. A previous study demonstrated...

  11. Genomic analysis of Bacillus subtilis lytic bacteriophage ϕNIT1 capable of obstructing natto fermentation carrying genes for the capsule-lytic soluble enzymes poly-γ-glutamate hydrolase and levanase.

    Science.gov (United States)

    Ozaki, Tatsuro; Abe, Naoki; Kimura, Keitarou; Suzuki, Atsuto; Kaneko, Jun

    2017-01-01

    Bacillus subtilis strains including the fermented soybean (natto) starter produce capsular polymers consisting of poly-γ-glutamate and levan. Capsular polymers may protect the cells from phage infection. However, bacteriophage ϕNIT1 carries a γ-PGA hydrolase gene (pghP) that help it to counteract the host cell's protection strategy. ϕNIT had a linear double stranded DNA genome of 155,631-bp with a terminal redundancy of 5,103-bp, containing a gene encoding an active levan hydrolase. These capsule-lytic enzyme genes were located in the possible foreign gene cluster regions between central core and terminal redundant regions, and were expressed at the late phase of the phage lytic cycle. All tested natto origin Spounavirinae phages carried both genes for capsule degrading enzymes similar to ϕNIT1. A comparative genomic analysis revealed the diversity among ϕNIT1 and Bacillus phages carrying pghP-like and levan-hydrolase genes, and provides novel understanding on the acquisition mechanism of these enzymatic genes.

  12. The ATP-Dependent Protease ClpP Inhibits Biofilm Formation by Regulating Agr and Cell Wall Hydrolase Sle1 in Staphylococcus aureus

    Science.gov (United States)

    Liu, Qian; Wang, Xing; Qin, Juanxiu; Cheng, Sen; Yeo, Won-Sik; He, Lei; Ma, Xiaowei; Liu, Xiaoyun; Li, Min; Bae, Taeok

    2017-01-01

    Biofilm causes hospital-associated infections on indwelling medical devices. In Staphylococcus aureus, Biofilm formation is controlled by intricately coordinated network of regulating systems, of which the ATP-dependent protease ClpP shows an inhibitory effect. Here, we demonstrate that the inhibitory effect of ClpP on biofilm formation is through Agr and the cell wall hydrolase Sle1. Biofilm formed by clpP mutant consists of proteins and extracellular DNA (eDNA). The increase of the protein was, at least in part, due to the reduced protease activity of the mutant, which was caused by the decreased activity of agr. On the other hand, the increase of eDNA was due to increased cell lysis caused by the higher level of Sle1. Indeed, as compared with wild type, the clpP mutant excreted an increased level of eDNA, and showed higher sensitivity to Triton-induced autolysis. The deletion of sle1 in the clpP mutant decreased the biofilm formation, the level of eDNA, and the Triton-induced autolysis to wild-type levels. Despite the increased biofilm formation capability, however, the clpP mutant showed significantly reduced virulence in a murine model of subcutaneous foreign body infection, indicating that the increased biofilm formation capability cannot compensate for the intrinsic functions of ClpP during infection. PMID:28555174

  13. The ATP-Dependent Protease ClpP Inhibits Biofilm Formation by Regulating Agr and Cell Wall Hydrolase Sle1 in Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Qian Liu

    2017-05-01

    Full Text Available Biofilm causes hospital-associated infections on indwelling medical devices. In Staphylococcus aureus, Biofilm formation is controlled by intricately coordinated network of regulating systems, of which the ATP-dependent protease ClpP shows an inhibitory effect. Here, we demonstrate that the inhibitory effect of ClpP on biofilm formation is through Agr and the cell wall hydrolase Sle1. Biofilm formed by clpP mutant consists of proteins and extracellular DNA (eDNA. The increase of the protein was, at least in part, due to the reduced protease activity of the mutant, which was caused by the decreased activity of agr. On the other hand, the increase of eDNA was due to increased cell lysis caused by the higher level of Sle1. Indeed, as compared with wild type, the clpP mutant excreted an increased level of eDNA, and showed higher sensitivity to Triton-induced autolysis. The deletion of sle1 in the clpP mutant decreased the biofilm formation, the level of eDNA, and the Triton-induced autolysis to wild-type levels. Despite the increased biofilm formation capability, however, the clpP mutant showed significantly reduced virulence in a murine model of subcutaneous foreign body infection, indicating that the increased biofilm formation capability cannot compensate for the intrinsic functions of ClpP during infection.

  14. A Trapped Covalent Intermediate of a Glycoside Hydrolase on the Pathway to Transglycosylation. Insights from Experiments and Quantum Mechanics/Molecular Mechanics Simulations.

    Science.gov (United States)

    Raich, Lluís; Borodkin, Vladimir; Fang, Wenxia; Castro-López, Jorge; van Aalten, Daan M F; Hurtado-Guerrero, Ramón; Rovira, Carme

    2016-03-16

    The conversion of glycoside hydrolases (GHs) into transglycosylases (TGs), i.e., from enzymes that hydrolyze carbohydrates to enzymes that synthesize them, represents a promising solution for the large-scale synthesis of complex carbohydrates for biotechnological purposes. However, the lack of knowledge about the molecular details of transglycosylation hampers the rational design of TGs. Here we present the first crystallographic structure of a natural glycosyl-enzyme intermediate (GEI) of Saccharomyces cerevisiae Gas2 in complex with an acceptor substrate and demonstrate, by means of quantum mechanics/molecular mechanics metadynamics simulations, that it is tuned for transglycosylation (ΔG(⧧) = 12 kcal/mol). The 2-OH···nucleophile interaction is found to be essential for catalysis: its removal raises the free energy barrier significantly (11 and 16 kcal/mol for glycosylation and transglycosylation, respectively) and alters the conformational itinerary of the substrate (from (4)C1 → [(4)E](⧧) → (1,4)B/(4)E to (4)C1 → [(4)H3](⧧) → (4)C1). Our results suggest that changes in the interactions involving the 2-position could have an impact on the transglycosylation activity of several GHs.

  15. Isolation of the opdE gene that encodes for a new hydrolase of Enterobacter sp. capable of degrading organophosphorus pesticides.

    Science.gov (United States)

    Chino-Flores, Concepción; Dantán-González, Edgar; Vázquez-Ramos, Alejandra; Tinoco-Valencia, Raunel; Díaz-Méndez, Rafael; Sánchez-Salinas, Enrique; Castrejón-Godínez, Maria Luisa; Ramos-Quintana, Fernando; Ortiz-Hernández, Maria Laura

    2012-06-01

    Microbial enzymes that can hydrolyze organophosphorus compounds have been isolated, identified and characterized from different microbial species in order to use them in biodegradation of organophosphorus compounds. We isolated a bacterial strain Cons002 from an agricultural soil bacterial consortium, which can hydrolyze methyl-parathion (MP) and other organophosphate pesticides. HPLC analysis showed that strain Cons002 is capable of degrading pesticides MP, parathion and phorate. Pulsed-field gel electrophoresis and 16S rRNA amplification were performed for strain characterization and identification, respectively, showing that the strain Cons002 is related to the genus Enterobacter sp. which has a single chromosome of 4.6 Mb and has no plasmids. Genomic library was constructed from DNA of Enterobacter sp. Cons002. A gene called opdE (Organophosphate Degradation from Enterobacter) consists of 753 bp and encodes a protein of 25 kDa, which was isolated using activity methods. This gene opdE had no similarity to any genes reported to degrade organophosphates. When kanamycin-resistance cassette was placed in the gene opdE, hydrolase activity was suppressed and Enterobacter sp. Cons002 had no growth with MP as a nutrients source.

  16. A Personal Retrospective: Elevating Anandamide (AEA by Targeting Fatty Acid Amide Hydrolase (FAAH and the Fatty Acid Binding Proteins (FABPs

    Directory of Open Access Journals (Sweden)

    Dale Deutsch

    2016-10-01

    Full Text Available This perspective was adapted from a Career Achievement Award talk given at the International Cannabinoid Research Society Symposium in Bukovina, Poland on June 27, 2016. As a biochemist working in the neurosciences, I was always fascinated with neurotransmitter inactivation. In 1993 we identified an enzyme activity that breaks down anandamide. We called the enzyme anandamide amidase, now called FAAH. We and other laboratories developed FAAH inhibitors that were useful reagents that also proved to have beneficial physiological effects and, until recently, new generations of inhibitors were in clinical trials. Nearly all neurotransmitters are water soluble and, as such, require a transmembrane protein transporter to pass through the lipid membrane for inactivation inside the cell. However, using model systems, we and others have shown that this is unnecessary for anandamide, an uncharged hydrophobic molecule that readily diffuses across the cellular membrane. Interestingly, its uptake is driven by the concentration gradient resulting from its breakdown mainly by FAAH localized in the endoplasmic reticulum. We identified the FABPs as intracellular carriers that solubilize anandamide, transporting anandamide to FAAH. Compounds that bind to FABPs block AEA breakdown, raising its level. The cannabinoids (THC and CBD also were discovered to bind FABPs and this may be one of the mechanisms by which CBD works in childhood epilepsy, raising anandamide levels. Targeting FABPs may be advantageous since they have some tissue specificity and do not require reactive serine hydrolase inhibitors, as does FAAH, with potential for off-target reactions.

  17. N-3 Polyunsaturated Fatty Acids Decrease the Protein Expression of Soluble Epoxide Hydrolase via Oxidative Stress-Induced P38 Kinase in Rat Endothelial Cells.

    Science.gov (United States)

    Okada, Takashi; Morino, Katsutaro; Nakagawa, Fumiyuki; Tawa, Masashi; Kondo, Keiko; Sekine, Osamu; Imamura, Takeshi; Okamura, Tomio; Ugi, Satoshi; Maegawa, Hiroshi

    2017-06-24

    N -3 polyunsaturated fatty acids (PUFAs) improve endothelial function. The arachidonic acid-derived metabolites (epoxyeicosatrienoic acids (EETs)) are part of the endothelial hyperpolarization factor and are vasodilators independent of nitric oxide. However, little is known regarding the regulation of EET concentration by docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in blood vessels. Sprague-Dawley rats were fed either a control or fish oil diet for 3 weeks. Compared with the control, the fish oil diet improved acetylcholine-induced vasodilation and reduced the protein expression of soluble epoxide hydrolase (sEH), a key EET metabolic enzyme, in aortic strips. Both DHA and EPA suppressed sEH protein expression in rat aorta endothelial cells (RAECs). Furthermore, the concentration of 4-hydroxy hexenal (4-HHE), a lipid peroxidation product of n -3 PUFAs, increased in n -3 PUFA-treated RAECs. In addition, 4-HHE treatment suppressed sEH expression in RAECs, suggesting that 4-HHE (derived from n -3 PUFAs) is involved in this phenomenon. The suppression of sEH was attenuated by the p38 kinase inhibitor (SB203580) and by treatment with the antioxidant N-acetyl-L-cysteine. In conclusion, sEH expression decreased after n -3 PUFAs treatment, potentially through oxidative stress and p38 kinase. Mild oxidative stress induced by n -3 PUFAs may contribute to their cardio-protective effect.

  18. Response of a diuron-degrading community to diuron exposure assessed by real-time quantitative PCR monitoring of phenylurea hydrolase A and B encoding genes.

    Science.gov (United States)

    Pesce, Stéphane; Beguet, Jérémie; Rouard, Nadine; Devers-Lamrani, Marion; Martin-Laurent, Fabrice

    2013-02-01

    A real-time quantitative PCR method was developed to detect and quantify phenlylurea hydrolase genes' (puhA and puhB) sequences from environmental DNA samples to assess diuron-degrading genetic potential in some soil and sediment microbial communities. In the soil communities, mineralization rates (determined with [ring-¹⁴C]-labeled diuron) were linked to diuron-degrading genetic potentials estimated from puhB number copies, which increased following repeated diuron treatments. In the sediment communities, mineralization potential did not depend solely on the quantity of puhB copies, underlining the need to assess gene expression. In the sediment samples, both puhB copy numbers and mineralization capacities were highly conditioned by whether or not diuron-treated soil was added. This points to transfers of degradative potential from soils to sediments. No puhA gene was detected in soil and sediment DNA extracts. Moreover, some sediments exhibited high diuron mineralization potential even though puhB genes were not detected, suggesting the existence of alternative diuron degradation pathways.

  19. Cloning, characterization, and expression of xyloglucan endotransglucosylase/hydrolase and expansin genes associated with petal growth and development during carnation flower opening

    Science.gov (United States)

    Harada, Taro; Torii, Yuka; Morita, Shigeto; Onodera, Reiko; Hara, Yoshinao; Yokoyama, Ryusuke; Nishitani, Kazuhiko; Satoh, Shigeru

    2011-01-01

    Growth of petal cells is a basis for expansion and morphogenesis (outward bending) of petals during opening of carnation flowers (Dianthus caryophyllus L.). Petal growth progressed through elongation in the early stage, expansion with outward bending in the middle stage, and expansion of the whole area in the late stage of flower opening. In the present study, four cDNAs encoding xyloglucan endotransglucosylase/hydrolase (XTH) (DcXTH1–DcXTH4) and three cDNAs encoding expansin (DcEXPA1–DcEXPA3) were cloned from petals of opening carnation flowers and characterized. Real-time reverse transcription-PCR analyses showed that transcript levels of XTH and expansin genes accumulated differently in floral and vegetative tissues of carnation plants with opening flowers, indicating regulated expression of these genes. DcXTH2 and DcXTH3 transcripts were detected in large quantities in petals as compared with other tissues. DcEXPA1 and DcEXPA2 transcripts were markedly accumulated in petals of opening flowers. The action of XTH in growing petal tissues was confirmed by in situ staining of xyloglucan endotransglucosylase (XET) activity using a rhodamine-labelled xyloglucan nonasaccharide as a substrate. Based on the present findings, it is suggested that two XTH genes (DcXTH2 and DcXTH3) and two expansin genes (DcEXPA1 and DcEXPA2) are associated with petal growth and development during carnation flower opening. PMID:20959626

  20. Molecular cloning, overexpression, and enzymatic characterization of glycosyl hydrolase family 16 β-Agarase from marine bacterium Saccharophagus sp. AG21 in Escherichia coli.

    Science.gov (United States)

    Lee, Youngdeuk; Oh, Chulhong; De Zoysa, Mahanama; Kim, Hyowon; Wickramaarachchi, Wickramaarachchige Don Niroshana; Whang, Ilson; Kang, Do-Hyung; Lee, Jehee

    2013-01-01

    An agar-degrading bacterium was isolated from red seaweed (Gelidium amansii) on a natural seawater agar plate, and identified as Saccharophagus sp. AG21. The β-agarase gene from Saccharophagus sp. AG21 (agy1) was screened by long and accurate (LA)-PCR. The predicted sequence has a 1,908 bp open reading frame encoding 636 amino acids (aa), and includes a glycosyl hydrolase family 16 (GH16) β-agarase module and two carbohydrate binding modules of family 6 (CBM6). The deduced aa sequence showed 93.7% and 84.9% similarity to β-agarase of Saccharophagus degradans and Microbulbifer agarilyticus, respectively. The mature agy1 was cloned and overexpressed as a His-tagged recombinant β-agarase (rAgy1) in Escherichia coli, and had a predicted molecular mass of 69 kDa and an isoelectric point of 4.5. rAgy1 showed optimum activity at 55oC and pH 7.6, and had a specific activity of 85 U/mg. The rAgy1 activity was enhanced by FeSO4 (40%), KCl (34%), and NaCl (34%), compared with the control. The newly identified rAgy1 is a β-agarase, which acts to degrade agarose to neoagarotetraose (NA4) and neoagarohexaose (NA6) and may be useful for applications in the cosmetics, food, bioethanol, and reagent industries.

  1. Crystal Structure of a Hidden Protein, YcaC, a Putative Cysteine Hydrolase from Pseudomonas aeruginosa, with and without an Acrylamide Adduct

    Directory of Open Access Journals (Sweden)

    Morten K. Grøftehauge

    2015-07-01

    Full Text Available As part of the ongoing effort to functionally and structurally characterize virulence factors in the opportunistic pathogen Pseudomonas aeruginosa, we determined the crystal structure of YcaC co-purified with the target protein at resolutions of 2.34 and 2.56 Å without a priori knowledge of the protein identity or experimental phases. The three-dimensional structure of YcaC adopts a well-known cysteine hydrolase fold with the putative active site residues conserved. The active site cysteine is covalently bound to propionamide in one crystal form, whereas the second form contains an S-mercaptocysteine. The precise biological function of YcaC is unknown; however, related prokaryotic proteins have functions in antibacterial resistance, siderophore production and NADH biosynthesis. Here, we show that YcaC is exceptionally well conserved across both bacterial and fungal species despite being non-ubiquitous. This suggests that whilst YcaC may not be part of an integral pathway, the function could confer a significant evolutionary advantage to microbial life.

  2. Glycoside Hydrolase MoGls2 Controls Asexual/Sexual Development, Cell Wall Integrity and Infectious Growth in the Rice Blast Fungus.

    Directory of Open Access Journals (Sweden)

    Mengying Li

    Full Text Available N-linked glycosylation is a way of glycosylation for newly synthesized protein, which plays a key role in the maturation and transport of proteins. Glycoside hydrolases (GHs are essential in this process, and are involved in processing of N-linked glycoproteins or degradation of carbohydrate structures. Here, we identified and characterized MoGls2 in Magnaporthe oryzae, which is a yeast glucosidase II homolog Gls2 and is required for trimming the final glucose in N-linked glycans and normal cell wall synthesis. Target deletion of MoGLS2 in M. oryzae resulted in a reduced mycelial growth, an increased conidial production, delayed conidial germination and loss the ability of sexual reproduction. Pathogenicity assays revealed that the ΔMogls2 mutant showed significantly decreased in virulence and infectious growth. Further studies showed that the mutant was less sensitive to salt and osmotic stress, and increased sensitivity to cell wall stresses. Additionally, the ΔMogls2 mutant showed a defect in cell wall integrity. Our results indicate that MoGls2 is a key protein for the growth and development of M. oryzae, involving in the regulation of asexual/sexual development, stress response, cell wall integrity and infectious growth.

  3. Bile salt tolerance of Lactococcus lactis is enhanced by expression of bile salt hydrolase thereby producing less bile acid in the cells.

    Science.gov (United States)

    Bi, Jie; Liu, Song; Du, Guocheng; Chen, Jian

    2016-04-01

    Changes of bile salt tolerance, morphology and amount of bile acid within cells were studied to evaluate the exact effects of bile salt hydrolase (BSH) on bile salt tolerance of microorganism. The effect of BSHs on the bile salt tolerance of Lactococcus lactis was examined by expressing two BSHs (BSH1 and BSH2). Growth of L. lactis expressing BSH1 or BSH2 was better under bile salt stress compared to wild-type L. lactis. As indicated by transmission electron microscopy, bile acids released by the action of BSH induced the formation of micelles around the membrane surface of cells subject to conjugated bile salt stress. A similar micelle containing bile acid was observed in the cytoplasm by liquid chromatography-mass spectrometry. BSH1 produced fewer bile acid micelles in the cytoplasm and achieved better cell growth of L. lactis compared to BSH2. Expression of BSH improved bile salt tolerance of L. lactis but excessive production by BSH of bile acid micelles in the cytoplasm inhibited cell growth.

  4. Association of ubiquitin carboxy-terminal hydrolase-L1 in cerebrospinal fluid with clinical severity in a cohort of patients with Guillain-Barré syndrome.

    Science.gov (United States)

    Nagamine, Satoshi; Fujiwara, Yuuki; Shimizu, Toshio; Kawata, Akihiro; Wada, Keiji; Isozaki, Eiji; Kabuta, Tomohiro

    2015-06-01

    Guillain-Barré syndrome (GBS) is an acute immune-mediated polyneuropathy. Although its pathogenic mechanism has been revealed and various therapeutic trials have been performed, a proportion of patients experience the severe sequelae associated with GBS. In this paper, we investigated whether the amount of the neuron-specific protein, ubiquitin carboxy-terminal hydrolase-L1 (UCH-L1), in the cerebrospinal fluid of patients with GBS was correlated with the clinical course of the disease. UCH-L1 protein levels were greater in patients with GBS than in controls. The patients with GBS whose UCH-L1 protein levels were higher than those of the controls presented with more severe symptoms at peak. UCH-L1 protein levels tended to become elevated as the total protein levels were increased; however, elevated UCH-L1 without an increase in total protein might be correlated with severe disease course (bedridden or ventilator supported). These results suggest that UCH-L1 could be a biomarker associated with the severity of the disease at the acute phase of GBS.

  5. In vitro analysis of protection of the enzyme bile salt hydrolase against enteric conditions by whey protein-gum arabic microencapsulation.

    Science.gov (United States)

    Lambert, J M; Weinbreck, F; Kleerebezem, M

    2008-09-24

    The interest in efficient intestinal delivery of health-promoting substances is increasing. However, the delivery of vulnerable substances such as enzymes requires specific attention. The transit through the stomach, where the pH is very low, can be detrimental to the enzymatic activity of the protein to be delivered. Here, we describe the microencapsulation of the model enzyme bile salt hydrolase (Bsh) using whey protein-gum arabic microencapsulates for food-grade and targeted enzyme delivery in the proximal region of the small intestine. Furthermore, the efficacy of enteric coating microencapsulates for site-specific enzyme delivery was compared in vitro with living Lactobacillus plantarum WCFS1 bacteria that endogenously produce the Bsh enzyme. Microencapsulates allowed highly effective protection of the enzyme under gastric conditions. Moreover, Bsh release under intestinal conditions appeared to be very efficient, although in the presence of pancreatin, the Bsh activity decreased in time due to proteolytic degradation. In comparison, L. plantarum appeared to be capable to withstand gastric conditions as well as pancreatin challenge. Delivery using encapsulates and live bacteria each have different (dis)advantages that are discussed. In conclusion, live bacteria and food-grade microencapsulates provide alternatives for dedicated enteric delivery of specific enzymes, and the choice of enzyme to be delivered may determine which mode of delivery is most suitable.

  6. Evaluation of the glycoside hydrolase activity of a Brettanomyces strain on glycosides from sour cherry (Prunus cerasus L.) used in the production of special fruit beers.

    Science.gov (United States)

    Daenen, Luk; Sterckx, Femke; Delvaux, Freddy R; Verachtert, Hubert; Derdelinckx, Guy

    2008-11-01

    The glycoside hydrolase activity of Saccharomyces cerevisiae and Brettanomyces custersii was examined on sour cherry (Prunus cerasus L.) glycosides with bound volatile compounds. Refermentations by the beta-glucosidase-negative S. cerevisiae strains LD25 and LD40 of sour cherry juice-supplemented beer demonstrated only a moderate increase of volatiles. In contrast, the beta-glucosidase-positive B. custersii strain LD72 showed a more pronounced activity towards glycosides with aliphatic alcohols, aromatic compounds and terpenoid alcohols. Important contributors to sour cherry aroma such as benzaldehyde, linalool and eugenol were released during refermentation as shown by analytical tools. A gradually increasing release was observed during refermentations by B. custersii when whole sour cherries, sour cherry pulp or juice were supplemented in the beer. Refermentations with whole sour cherries and with sour cherry stones demonstrated an increased formation of benzyl compounds. Thus, amygdalin was partially hydrolysed, and a large part of the benzaldehyde formed was mainly reduced to benzyl alcohol and some further esterified to benzyl acetate. These findings demonstrate the importance and interesting role of certain Brettanomyces species in the production of fruit lambic beers such as 'Kriek'.

  7. Insights into the structure and function of fungal β-mannosidases from glycoside hydrolase family 2 based on multiple crystal structures of the Trichoderma harzianum enzyme.

    Science.gov (United States)

    Nascimento, Alessandro S; Muniz, Joao Renato C; Aparício, Ricardo; Golubev, Alexander M; Polikarpov, Igor

    2014-09-01

    Hemicellulose is an important part of the plant cell wall biomass, and is relevant to cellulosic ethanol technologies. β-Mannosidases are enzymes capable of cleaving nonreducing residues of β-d-mannose from β-d-mannosides and hemicellulose mannose-containing polysaccharides, such as mannans and galactomannans. β-Mannosidases are distributed between glycoside hydrolase (GH) families 1, 2, and 5, and only a handful of the enzymes have been structurally characterized to date. The only published X-ray structure of a GH family 2 mannosidase is that of the bacterial Bacteroides thetaiotaomicron enzyme. No structures of eukaryotic mannosidases of this family are currently available. To fill this gap, we set out to solve the structure of Trichoderma harzianum GH family 2 β-mannosidase and to refine it to 1.9-Å resolution. Structural comparisons of the T. harzianum GH2 β-mannosidase highlight similarities in its structural architecture with other members of GH family 2, reveal the molecular mechanism of β-mannoside binding and recognition, and shed light on its putative galactomannan-binding site. Coordinates and observed structure factor amplitudes have been deposited with the Protein Data Bank (4CVU and 4UOJ). The T. harzianum β-mannosidase 2A nucleotide sequence has GenBank accession number BankIt1712036 GeneMark.hmm KJ624918. © 2014 FEBS.

  8. Epoxide hydrolase Lsd19 for polyether formation in the biosynthesis of lasalocid A: direct experimental evidence on polyene-polyepoxide hypothesis in polyether biosynthesis.

    Science.gov (United States)

    Shichijo, Yoshihiro; Migita, Akira; Oguri, Hiroki; Watanabe, Mami; Tokiwano, Tetsuo; Watanabe, Kenji; Oikawa, Hideaki

    2008-09-17

    Polyether metabolites are an important class of natural products. Although their biosynthesis, especially construction of polyether skeletons, attracted organic chemists for many years, no experimental data on the enzymatic polyether formation has been obtained. In this study, a putative epoxide hydrolase gene lsd19 found on the biosynthetic gene cluster of an ionophore polyether lasalocid was cloned and successfully overexpressed in Escherichia coli. Using the purified Lsd19, a proposed substrate, bisepoxyprelasalocid, and its synthesized analogue were successfully converted into lasalocid A and its derivative via a 6-endo-tet cyclization mode. On the other hand, treatment of the bisepoxide with trichloroacetic acid gave isolasalocid A via a 5-exo-tet cyclization mode. Therefore, the enzymatic conversion observed in this study unambiguously showed that the bisepoxyprelasalocid is an intermediate of the lasalocid biosynthesis and that Lsd19 catalyzes the sequential cyclic ether formations involving an energetically disfavored 6-endo-tet cyclization. This is the first example of the enzymatic epoxide-opening reactions leading to a polyether natural product.

  9. Restored Plasma Anandamide and Endometrial Expression of Fatty Acid Amide Hydrolase in Women With Polycystic Ovary Syndrome by the Combination Use of Diane-35 and Metformin.

    Science.gov (United States)

    Cui, Na; Feng, Xiaoye; Zhao, Zhiming; Zhang, Jie; Xu, Yueming; Wang, Luning; Hao, Guimin

    2017-04-01

    Polycystic ovary syndrome (PCOS) is a metabolic and endocrinal disorder affecting a number of women of reproductive age. We aimed to reveal the correlation between the endocannabinoid system and PCOS, which may provide a new therapeutic target for PCOS treatment. Serum levels of anandamide and 2-arachidonoylglycerol andexpression of cannabinoid receptors and fatty acid amide hydrolase (FAAH) in the endometrium were compared between women with PCOS and infertile women without PCOS, as well as women with PCOS before and after treatment with Diane-35 and metformin. Cannabinoid receptors and FAAH in the endometrium were stained using the immunohistochemical method. Results were analyzed by calculating integrated optical density. Plasma anandamide was increased significantly in women with PCOS compared with infertile women without PCOS. Treatment with Diane-35 and metformin reversed this increase in women with PCOS. No significant difference in 2-arachidonoylglycerol was observed between the infertile women with or without PCOS. The women with PCOS had lower endometrial expression of FAAH compared with infertile women without PCOS, whereas no significant difference in endometrial expression of cannabinoid receptors was observed between the women with PCOS and infertile women without PCOS. We found that after treatment with Diane-35 and metformin, FAAH expression tended toward a significant increase compared with women before the treatment. Endocannabinoid system may be involved in the progression of PCOS, and serum anandamide could serve as a potential biomarker of clinical diagnosis of PCOS. Copyright © 2017 Elsevier HS Journals, Inc. All rights reserved.

  10. In silico investigation of cycloartane triterpene derivatives from Cimicifuga dahurica (Turcz.) Maxim. roots for the development of potent soluble epoxide hydrolase inhibitors.

    Science.gov (United States)

    Thao, Nguyen Phuong; Kim, Jang Hoon; Thuy Luyen, Bui Thi; Dat, Nguyen Tien; Kim, Young Ho

    2017-05-01

    In our search for natural soluble epoxide hydrolase (sEH) inhibitors from plants, we found that an ethanolic extract of the roots of Cimicifuga dahurica (Turcz.) Maxim. significantly inhibits sEH in vitro. A phytochemical study on the dichloromethane fraction of C. dahurica resulted in the isolation of two new cycloartane triterpenoids (1 and 6), together with 13 known cycloartane analogues (2-5 and 7-15). The structures of compounds were determined by spectroscopic methods. All of the triterpenoid derivatives inhibited sEH enzymatic activity in a concentration-dependent manner, and 13 of the tested compounds showed significant activity. Among them, compounds 1, 3, 5, 7, 9, and 12 showed the highest levels of inhibitory activity, with IC 50 values of about 5μM or less. Kinetic analysis of compounds 1, 3, 5-9, 11, 12, and 14 revealed that compounds 3, 6, 7, 11, and 14 were non-competitive; 1, 5, 9, and 12 were mixed-type; and 8 was a competitive inhibitor. Furthermore, in silico molecular docking indicated that compounds 3, 6-9, 11, 12, and 14 bound to sEH in a similar manner and had stable binding energies, as calculated by AutoDock 4.2 and processed in a 10,000-ps molecular dynamics simulation to assess the binding stability of compounds 5, 7, and 9. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Possible Correlation Between Bile Salt Hydrolysis and AHL Deamidation: Staphylococcus epidermidis RM1, a Potent Quorum Quencher and Bile Salt Hydrolase Producer.

    Science.gov (United States)

    Mukherji, Ruchira; Prabhune, Asmita

    2015-05-01

    The aim of the present work was to isolate a bile salt hydrolase (BSH) producer from fermented soy curd and explore the ability of the BSH produced to cleave bacterial quorum sensing signals. Bacterial isolates with possible ability to deconjugate bile salts were enriched and isolated on De Man, Rogosa and Sharpe (MRS) medium containing 0.2% bile salts. BSH-producing positive isolate with orange-pink-pigmented colonies was isolated and was identified as a strain of Staphylococcus epidermidis using biochemical and phylogenetic tools. S. epidermidis RM1 was shown to possess both potent BSH and N-acyl homoserine lactone (AHL) cleavage activity. Genetic basis of this dual-enzyme activity was explored by means of specific primers designed using S. epidermidis ATCC 12228 genome as template. It was observed that a single enzyme was not responsible for both the activity. Two different genetic elements corresponding to each of the enzymatic activity were successfully amplified from the genomic DNA of the isolate.

  12. Purification, crystallization and preliminary X-ray analysis of a thermostable glycoside hydrolase family 43 β-xylosidase from Geobacillus thermoleovorans IT-08

    International Nuclear Information System (INIS)

    Rohman, Ali; Oosterwijk, Niels van; Kralj, Slavko; Dijkhuizen, Lubbert; Dijkstra, Bauke W.; Puspaningsih, Ni Nyoman Tri

    2007-01-01

    The β-xylosidase was crystallized using PEG 6000 as precipitant. 5% PEG 6000 yielded bipyramid-shaped tetragonal crystals diffracting to 1.55 Å resolution, and 13% PEG 6000 gave rectangular monoclinic crystals diffracting to 1.80 Å resolution. The main enzymes involved in xylan-backbone hydrolysis are endo-1,4-β-xylanase and β-xylosidase. β-Xylosidase converts the xylo-oligosaccharides produced by endo-1,4-β-xylanase into xylose monomers. The β-xylosidase from the thermophilic Geobacillus thermoleovorans IT-08, a member of glycoside hydrolase family 43, was crystallized at room temperature using the hanging-drop vapour-diffusion method. Two crystal forms were observed. Bipyramid-shaped crystals belonging to space group P4 3 2 1 2, with unit-cell parameters a = b = 62.53, c = 277.4 Å diffracted to 1.55 Å resolution. The rectangular crystals belonged to space group P2 1 , with unit-cell parameters a = 57.94, b = 142.1, c = 153.9 Å, β = 90.5°, and diffracted to 1.80 Å resolution

  13. Cholesterol-Lowering Potentials of Lactic Acid Bacteria Based on Bile-Salt Hydrolase Activity and Effect of Potent Strains on Cholesterol Metabolism In Vitro and In Vivo

    Directory of Open Access Journals (Sweden)

    Cheng-Chih Tsai

    2014-01-01

    Full Text Available This study collected different probiotic isolates from animal and plant sources to evaluate the bile-salt hydrolase activity of probiotics in vitro. The deconjugation potential of bile acid was determined using high-performance liquid chromatography. HepG2 cells were cultured with probiotic strains with high BSH activity. The triglyceride (TG and apolipoprotein B (apo B secretion by HepG2 cells were evaluated. Our results show that the BSH activity and bile-acid deconjugation abilities of Pediococcus acidilactici NBHK002, Bifidobacterium adolescentis NBHK006, Lactobacillus rhamnosus NBHK007, and Lactobacillus acidophilus NBHK008 were higher than those of the other probiotic strains. The cholesterol concentration in cholesterol micelles was reduced within 24 h. NBHK007 reduced the TG secretion by 100% after 48 h of incubation. NBHK002, NBHK006, and NBHK007 could reduce apo B secretion by 33%, 38%, and 39%, respectively, after 24 h of incubation. The product PROBIO S-23 produced a greater decrease in the total concentration of cholesterol, low-density lipoprotein, TG, and thiobarbituric acid reactive substance in the serum or livers of hamsters with hypercholesterolemia compared with that of hamsters fed with a high-fat and high-cholesterol diet. These results show that the three probiotic strains of lactic acid bacteria are better candidates for reducing the risk of cardiovascular disease.

  14. Development of a Novel Optical Biosensor for Detection of Organophoshorus Pesticides Based on Methyl Parathion Hydrolase Immobilized by Metal-Chelate Affinity

    Directory of Open Access Journals (Sweden)

    Wensheng Lan

    2012-06-01

    Full Text Available We have developed a novel optical biosensor device using recombinant methyl parathion hydrolase (MPH enzyme immobilized on agarose by metal-chelate affinity to detect organophosphorus (OP compounds with a nitrophenyl group. The biosensor principle is based on the optical measurement of the product of OP catalysis by MPH (p-nitrophenol. Briefly, MPH containing six sequential histidines (6× His tag at its N-terminal was bound to nitrilotriacetic acid (NTA agarose with Ni ions, resulting in the flexible immobilization of the bio-reaction platform. The optical biosensing system consisted of two light-emitting diodes (LEDs and one photodiode. The LED that emitted light at the wavelength of the maximum absorption for p-nitrophenol served as the signal light, while the other LED that showed no absorbance served as the reference light. The optical sensing system detected absorbance that was linearly correlated to methyl parathion (MP concentration and the detection limit was estimated to be 4 μM. Sensor hysteresis was investigated and the results showed that at lower concentration range of MP the difference got from the opposite process curves was very small. With its easy immobilization of enzymes and simple design in structure, the system has the potential for development into a practical portable detector for field applications.

  15. Purification and Characterization of Tannin Acyl Hydrolase Produced by Mixed Solid State Fermentation of Wheat Bran and Marigold Flower by Penicillium notatum NCIM 923

    Directory of Open Access Journals (Sweden)

    Saswati Gayen

    2013-01-01

    Full Text Available Tannin acyl hydrolase produced extracellularly by the fungal strain Penicillium notatum NCIM 923 in mixed solid state fermentation of wheat bran and marigold flower in the ratio 4 : 1 was purified from the cell-free extract broth by ammonium sulphate fractionation followed by diethylaminoethyl-cellulose column chromatography. Tannase was purified by 19.89-fold with yield of 11.77%. The specific activity of crude tannase was found to be 1.31 U/mg protein while that of purified tannase was 22.48 U/mg protein. SDS-PAGE analysis indicated that the enzyme is dimeric with one major band of molecular mass 97 kDa and a very light band of molecular mass 43 kDa. Temperature of 35 to 40°C and pH 5 were optimum for tannase activity. The enzyme retained more than 60% of its stability at 60°C and 40% stability at pH 3 and 8, respectively. Km was found to be 0.33×10-2 M and Vmax=40 U/mg. Since the enzyme is active over a wide range of pH and temperature, it could find potential use in the food processing industry.

  16. Purification and characterization of tannin acyl hydrolase produced by mixed solid state fermentation of wheat bran and marigold flower by Penicillium notatum NCIM 923.

    Science.gov (United States)

    Gayen, Saswati; Ghosh, Uma

    2013-01-01

    Tannin acyl hydrolase produced extracellularly by the fungal strain Penicillium notatum NCIM 923 in mixed solid state fermentation of wheat bran and marigold flower in the ratio 4 : 1 was purified from the cell-free extract broth by ammonium sulphate fractionation followed by diethylaminoethyl-cellulose column chromatography. Tannase was purified by 19.89-fold with yield of 11.77%. The specific activity of crude tannase was found to be 1.31 U/mg protein while that of purified tannase was 22.48 U/mg protein. SDS-PAGE analysis indicated that the enzyme is dimeric with one major band of molecular mass 97 kDa and a very light band of molecular mass 43 kDa. Temperature of 35 to 40°C and pH 5 were optimum for tannase activity. The enzyme retained more than 60% of its stability at 60°C and 40% stability at pH 3 and 8, respectively. K m was found to be 0.33 × 10(-2) M and V max = 40 U/mg. Since the enzyme is active over a wide range of pH and temperature, it could find potential use in the food processing industry.

  17. A metagenome-derived thermostable β-glucanase with an unusual module architecture which defines the new glycoside hydrolase family GH148.

    Science.gov (United States)

    Angelov, Angel; Pham, Vu Thuy Trang; Übelacker, Maria; Brady, Silja; Leis, Benedikt; Pill, Nicole; Brolle, Judith; Mechelke, Matthias; Moerch, Matthias; Henrissat, Bernard; Liebl, Wolfgang

    2017-12-11

    The discovery of novel and robust enzymes for the breakdown of plant biomass bears tremendous potential for the development of sustainable production processes in the rapidly evolving new bioeconomy. By functional screening of a metagenomic library from a volcano soil sample a novel thermostable endo-β-glucanase (EngU) which is unusual with regard to its module architecture and cleavage specificity was identified. Various recombinant EngU variants were characterized. Assignment of EngU to an existing glycoside hydrolase (GH) family was not possible. Two regions of EngU showed weak sequence similarity to proteins of the GH clan GH-A, and acidic residues crucial for catalytic activity of EngU were identified by mutation. Unusual, a carbohydrate-binding module (CBM4) which displayed binding affinity for β-glucan, lichenin and carboxymethyl-cellulose was found as an insertion between these two regions. EngU hydrolyzed β-1,4 linkages in carboxymethyl-cellulose, but displayed its highest activity with mixed linkage (β-1,3-/β-1,4-) glucans such as barley β-glucan and lichenin, where in contrast to characterized lichenases cleavage occurred predominantly at the β-1,3 linkages of C4-substituted glucose residues. EngU and numerous related enzymes with previously unknown function represent a new GH family of biomass-degrading enzymes within the GH-A clan. The name assigned to the new GH family is GH148.

  18. Development of a Novel Optical Biosensor for Detection of Organophoshorus Pesticides Based on Methyl Parathion Hydrolase Immobilized by Metal-Chelate Affinity

    Science.gov (United States)

    Lan, Wensheng; Chen, Guoping; Cui, Feng; Tan, Feng; Liu, Ran; Yushupujiang, Maolidan

    2012-01-01

    We have developed a novel optical biosensor device using recombinant methyl parathion hydrolase (MPH) enzyme immobilized on agarose by metal-chelate affinity to detect organophosphorus (OP) compounds with a nitrophenyl group. The biosensor principle is based on the optical measurement of the product of OP catalysis by MPH (p-nitrophenol). Briefly, MPH containing six sequential histidines (6× His tag) at its N-terminal was bound to nitrilotriacetic acid (NTA) agarose with Ni ions, resulting in the flexible immobilization of the bio-reaction platform. The optical biosensing system consisted of two light-emitting diodes (LEDs) and one photodiode. The LED that emitted light at the wavelength of the maximum absorption for p-nitrophenol served as the signal light, while the other LED that showed no absorbance served as the reference light. The optical sensing system detected absorbance that was linearly correlated to methyl parathion (MP) concentration and the detection limit was estimated to be 4 μM. Sensor hysteresis was investigated and the results showed that at lower concentration range of MP the difference got from the opposite process curves was very small. With its easy immobilization of enzymes and simple design in structure, the system has the potential for development into a practical portable detector for field applications. PMID:23012501

  19. Isolation and characterization of 9-lipoxygenase and epoxide hydrolase 2 genes: Insight into lactone biosynthesis in mango fruit (Mangifera indica L.).

    Science.gov (United States)

    Deshpande, Ashish B; Chidley, Hemangi G; Oak, Pranjali S; Pujari, Keshav H; Giri, Ashok P; Gupta, Vidya S

    2017-06-01

    Uniqueness and diversity of mango flavour across various cultivars are well known. Among various flavour metabolites lactones form an important class of aroma volatiles in certain mango varieties due to their ripening specific appearance and lower odour detection threshold. In spite of their biological and biochemical importance, lactone biosynthetic pathway in plants remains elusive. Present study encompasses quantitative real-time analysis of 9-lipoxygenase (Mi9LOX), epoxide hydrolase 2 (MiEH2), peroxygenase, hydroperoxide lyase and acyl-CoA-oxidase genes during various developmental and ripening stages in fruit of Alphonso, Pairi and Kent cultivars with high, low and no lactone content and explains their variable lactone content. Study also covers isolation, recombinant protein characterization and transient over-expression of Mi9LOX and MiEH2 genes in mango fruits. Recombinant Mi9LOX utilized linoleic and linolenic acids, while MiEH2 utilized aromatic and fatty acid epoxides as their respective substrates depicting their role in fatty acid metabolism. Significant increase in concentration of δ-valerolactone and δ-decalactone upon Mi9LOX over-expression and that of δ-valerolactone, γ-hexalactone and δ-hexalactone upon MiEH2 over-expression further suggested probable involvement of these genes in lactone biosynthesis in mango. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Structure of a bacterial glycoside hydrolase family 63 enzyme in complex with its glycosynthase product, and insights into the substrate specificity.

    Science.gov (United States)

    Miyazaki, Takatsugu; Ichikawa, Megumi; Yokoi, Gaku; Kitaoka, Motomitsu; Mori, Haruhide; Kitano, Yoshikazu; Nishikawa, Atsushi; Tonozuka, Takashi

    2013-09-01

    Proteins belonging to glycoside hydrolase family 63 (GH63) are found in bacteria, archaea and eukaryotes. Although the eukaryotic GH63 proteins have been identified as processing α-glucosidase I, the substrate specificities of the bacterial and archaeal GH63 proteins are not clear. Here, we converted a bacterial GH63 enzyme, Escherichia coli YgjK, to a glycosynthase to probe its substrate specificity. Two mutants of YgjK (E727A and D324N) were constructed, and both mutants showed glycosynthase activity. The reactions of E727A with β-D-glucosyl fluoride and monosaccharides showed that the largest amount of glycosynthase product accumulated when galactose was employed as an acceptor molecule. The crystal structure of E727A complexed with the reaction product indicated that the disaccharide bound at the active site was 2-O-α-D-glucopyranosyl-α-D-galactopyranose (Glc12Gal). A comparison of the structures of E727A-Glc12Gal and D324N-melibiose showed that there were two main types of conformation: the open and closed forms. The structure of YgjK adopted the closed form when subsite -1 was occupied by glucose. These results suggest that sugars containing the Glc12Gal structure are the most likely candidates for natural substrates of YgjK. © 2013 FEBS.

  1. Efficient Degradation of Malathion in the Presence of Detergents Using an Engineered Organophosphorus Hydrolase Highly Expressed by Pichia pastoris without Methanol Induction.

    Science.gov (United States)

    Bai, Yun-Peng; Luo, Xiao-Jing; Zhao, Yu-Lian; Li, Chun-Xiu; Xu, Dian-Sheng; Xu, Jian-He

    2017-10-18

    The biodegradation of pesticides by organophosphorus hydrolases (OPHs) requires an efficient enzyme production technology in industry. Herein, a Pichia pastoris strain was constructed for the extracellular expression of PoOPH M9 , an engineered malathion-degrading enzyme. After optimization, the maximum titer and yield of fermentation reached 50.8 kU/L and 4.1 g protein /L after 3 days, with the highest space-time yield (STY) reported so far, 640 U L -1 h -1 . PoOPH M9 displayed its high activity and stability in the presence of 0.1% (w/w) plant-derived detergent. Only 0.04 mg/mL enzyme could completely remove 0.15 mM malathion in aqueous solution within 20 min. Furthermore, 12 μmol malathion on apples and cucumbers surfaces was completely removed by 0.05 mg/mL PoOPH M9 in tap water after 35 min washing. The efficient production of the highly active PoOPH M9 has cleared a major barrier to biodegradation of pesticide residues in food industry.

  2. In Vitro Drug Metabolism by Human Carboxylesterase 1

    DEFF Research Database (Denmark)

    Thomsen, Ragnar; Rasmussen, Henrik B; Linnet, Kristian

    2014-01-01

    Carboxylesterase 1 (CES1) is the major hydrolase in human liver. The enzyme is involved in the metabolism of several important therapeutic agents, drugs of abuse, and endogenous compounds. However, no studies have described the role of human CES1 in the activation of two commonly prescribed...... a panel of therapeutic drugs and drugs of abuse to assess their inhibition of the hydrolysis of p-nitrophenyl acetate by recombinant CES1 and human liver microsomes. The screening assay confirmed several known inhibitors of CES1 and identified two previously unreported inhibitors: the dihydropyridine...... calcium antagonist, isradipine, and the immunosuppressive agent, tacrolimus. CES1 plays a role in the metabolism of several drugs used in the treatment of common conditions, including hypertension, congestive heart failure, and diabetes mellitus; thus, there is a potential for clinically relevant drug-drug...

  3. Expression of Membrane-Bound Human AminopeptidaseP as a Soluble Enzyme and an Investigation into Its Efficacy Towards Offering Protection Against the Toxicity of Chemical Warfare Nerve Agents

    Science.gov (United States)

    2016-09-01

    APP appears to be the cleavage of an N-terminal amino acid residue from peptides exhibiting a proline at P-1 residue such as bradykinin [11]. In...amino acid residue 658, replacing the Trp codon (TGG) immediately upstream of the hydrophobic peptide acting as the GPI-anchoring signal. A 6...hydrolase activity between human and chimeric recombinant mammalian paraoxonase-1 enzymes. Biochemistry, 2009. 48(43): p. 10416-22. 19. Aleti, V., et al

  4. Gene cloning and characterization of a cold-adapted β-glucosidase belonging to glycosyl hydrolase family 1 from a psychrotolerant bacterium Micrococcus antarcticus.

    Science.gov (United States)

    Fan, Hong-Xia; Miao, Li-Li; Liu, Ying; Liu, Hong-Can; Liu, Zhi-Pei

    2011-06-10

    The gene bglU encoding a cold-adapted β-glucosidase (BglU) was cloned from Micrococcus antarcticus. Sequence analysis revealed that the bglU contained an open reading frame of 1419 bp and encoded a protein of 472 amino acid residues. Based on its putative catalytic domains, BglU was classified as a member of the glycosyl hydrolase family 1 (GH1). BglU possessed lower arginine content and Arg/(Arg+Lys) ratio than mesophilic GH1 β-glucosidases. Recombinant BglU was purified with Ni2+ affinity chromatography and subjected to enzymatic characterization. SDS-PAGE and native staining showed that it was a monomeric protein with an apparent molecular mass of 48 kDa. BglU was particularly thermolabile since its half-life time was only 30 min at 30°C and it exhibited maximal activity at 25°C and pH 6.5. Recombinant BglU could hydrolyze a wide range of aryl-β-glucosides and β-linked oligosaccharides with highest activity towards cellobiose and then p-nitrophenyl-β-d-glucopyranoside (pNPG). Under the optimal conditions with pNPG as substrate, the K(m) and k(cat) were 7 mmol/L and 7.85 × 103/s, respectively. This is the first report of cloning and characterization of a cold-adapted β-glucosidase belonging to GH1 from a psychrotolerant bacterium. Copyright © 2011 Elsevier Inc. All rights reserved.

  5. Molecular cloning and sequence analysis of complementary DNA encoding rat mammary gland medium-chain S-acyl fatty acid synthetase thio ester hydrolase

    International Nuclear Information System (INIS)

    Safford, R.; de Silva, J.; Lucas, C.

    1987-01-01

    Poly(A) + RNA from pregnant rat mammary glands was size-fractionated by sucrose gradient centrifugation, and fractions enriched in medium-chain S-acyl fatty acid synthetase thio ester hydrolase (MCH) were identified by in vitro translation and immunoprecipitation. A cDNA library was constructed, in pBR322, from enriched poly(A) + RNA and screened with two oligonucleotide probes deduced from rat MCH amino acid sequence data. Cross-hybridizing clones were isolated and found to contain cDNA inserts ranging from ∼ 1100 to 1550 base pairs (bp). A 1550-bp cDNA insert, from clone 43H09, was confirmed to encode MCH by hybrid-select translation/immunoprecipitation studies and by comparison of the amino acid sequence deduced from the DNA sequence of the clone to the amino acid sequence of the MCH peptides. Northern blot analysis revealed the size of the MCH mRNA to be 1500 nucleotides, and it is therefore concluded that the 1550-bp insert (including G x C tails) of clone 43H09 represents a full- or near-full-length copy of the MCH gene. The rat MCH sequence is the first reported sequence of a thioesterase from a mammalian source, but comparison of the deduced amino acid sequences of MCH and the recently published mallard duck medium-chain S-acyl fatty acid synthetase thioesterase reveals significant homology. In particular, a seven amino acid sequence containing the proposed active serine of the duck thioesterase is found to be perfectly conserved in rat MCH

  6. Getting ready for host invasion: elevated expression and action of xyloglucan endotransglucosylases/hydrolases in developing haustoria of the holoparasitic angiosperm Cuscuta.

    Science.gov (United States)

    Olsen, Stian; Striberny, Bernd; Hollmann, Julien; Schwacke, Rainer; Popper, Zoë; Krause, Kirsten

    2016-02-01

    Changes in cell walls have been previously observed in the mature infection organ, or haustorium, of the parasitic angiosperm Cuscuta, but are not equally well charted in young haustoria. In this study, we focused on the molecular processes in the early stages of developing haustoria; that is, before the parasite engages in a physiological contact with its host. We describe first the identification of differentially expressed genes in young haustoria whose development was induced by far-red light and tactile stimuli in the absence of a host plant by suppression subtractive hybridization. To improve sequence information and to aid in the identification of the obtained candidates, reference transcriptomes derived from two species of Cuscuta, C. gronovii and C. reflexa, were generated. Subsequent quantitative gene expression analysis with different tissues of C. reflexa revealed that among the genes that were up-regulated in young haustoria, two xyloglucan endotransglucosylase/hydrolase (XTH) genes were highly expressed almost exclusively at the onset of haustorium development. The same expression pattern was also found for the closest XTH homologues from C. gronovii. In situ assays for XTH-specific action suggested that xyloglucan endotransglucosylation was most pronounced in the cell walls of the swelling area of the haustorium facing the host plant, but was also detectable in later stages of haustoriogenesis. We propose that xyloglucan remodelling by Cuscuta XTHs prepares the parasite for host infection and possibly aids the invasive growth of the haustorium. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  7. Co-silencing of tomato S-adenosylhomocysteine hydrolase genes confers increased immunity against Pseudomonas syringae pv. tomato DC3000 and enhanced tolerance to drought stress

    Directory of Open Access Journals (Sweden)

    Li Xiao Hui

    2015-09-01

    Full Text Available S-adenosylhomocysteine hydrolase (SAHH, catalyzing the reversible hydrolysis of S-adenosylhomocysteine to adenosine and homocysteine, is a key enzyme that maintain the cellular methylation potential in all organisms. We report here the biological functions of tomato SlSAHHs in stress response. The tomato genome contains three SlSAHH genes that encode SlSAHH proteins with high level of sequence identity. qRT-PCR analysis revealed that SlSAHHs responded with distinct expression induction patterns to Pseudomonas syringae pv. tomato (Pst DC3000 and Botrytis cinerea as well as to defense signaling hormones such as salicylic acid, jasmonic acid and a precursor of ethylene. Virus-induced gene silencing-based knockdown of individual SlSAHH gene did not affect the growth performance and the response to Pst DC3000. However, co-silencing of three SlSAHH genes using a conserved sequence led to significant inhibition of vegetable growth. The SlSAHH-co-silenced plants displayed increased resistance to Pst DC3000 but did not alter the resistance to B. cinerea. Co-silencing of SlSAHHs resulted in constitutively activated defense responses including elevated SA level, upregulated expression of defense-related and PAMP-triggered immunity marker genes and increased callose deposition and H2O2 accumulation. Furthermore, the SlSAHH-co-silenced plants also exhibited enhanced drought stress tolerance although they had relatively small roots. These data demonstrate that, in addition to the functions in growth and development, SAHHs also play important roles in regulating biotic and abiotic stress responses in plants.

  8. Hepatitis B spliced protein (HBSP) promotes the carcinogenic effects of benzo [alpha] pyrene by interacting with microsomal epoxide hydrolase and enhancing its hydrolysis activity

    International Nuclear Information System (INIS)

    Chen, Jin-Yan; Chen, Wan-Nan; Jiao, Bo-Yan; Lin, Wan-Song; Wu, Yun-Li; Liu, Ling-Ling; Lin, Xu

    2014-01-01

    The risk of hepatocellular carcinoma (HCC) increases in chronic hepatitis B surface antigen (HBsAg) carriers who often have concomitant increase in the levels of benzo[alpha]pyrene-7,8-diol-9,10-epoxide(±) (BPDE)-DNA adduct in liver tissues, suggesting a possible co-carcinogenesis of Hepatitis B virus (HBV) and benzo[alpha]pyrene in HCC; however the exact mechanisms involved are unclear. The interaction between hepatitis B spliced protein (HBSP) and microsomal epoxide hydrolase (mEH) was confirmed using GST pull-down, co-immunoprecipitation and mammalian two-hybrid assay; the effects of HBSP on mEH-mediated B[alpha]P metabolism was examined by high performance liquid chromatography (HPLC); and the influences of HBSP on B[alpha]P carcinogenicity were evaluated by bromodeoxyuridine cell proliferation, anchorage-independent growth and tumor xenograft. HBSP could interact with mEH in vitro and in vivo, and this interaction was mediated by the N terminal 47 amino acid residues of HBSP. HBSP could greatly enhance the hydrolysis activity of mEH in cell-free mouse liver microsomes, thus accelerating the metabolism of benzo[alpha]pyrene to produce more ultimate carcinnogen, BPDE, and this effect of HBSP requires the intact HBSP molecule. Expression of HBSP significantly increased the formation of BPDE-DNA adduct in benzo[alpha]pyrene-treated Huh-7 hepatoma cells, and this enhancement was blocked by knockdown of mEH. HBSP could enhance the cell proliferation, accelerate the G1/S transition, and promote cell transformation and tumorigenesis of B[alpha]P-treated Huh-7 hepatoma cells. Our results demonstrated that HBSP could promote carcinogenic effects of B[alpha]P by interacting with mEH and enhancing its hydrolysis activity

  9. A comparative metagenome survey of the fecal microbiota of a breast- and a plant-fed Asian elephant reveals an unexpectedly high diversity of glycoside hydrolase family enzymes.

    Directory of Open Access Journals (Sweden)

    Nele Ilmberger

    Full Text Available A phylogenetic and metagenomic study of elephant feces samples (derived from a three-weeks-old and a six-years-old Asian elephant was conducted in order to describe the microbiota inhabiting this large land-living animal. The microbial diversity was examined via 16S rRNA gene analysis. We generated more than 44,000 GS-FLX+454 reads for each animal. For the baby elephant, 380 operational taxonomic units (OTUs were identified at 97% sequence identity level; in the six-years-old animal, close to 3,000 OTUs were identified, suggesting high microbial diversity in the older animal. In both animals most OTUs belonged to Bacteroidetes and Firmicutes. Additionally, for the baby elephant a high number of Proteobacteria was detected. A metagenomic sequencing approach using Illumina technology resulted in the generation of 1.1 Gbp assembled DNA in contigs with a maximum size of 0.6 Mbp. A KEGG pathway analysis suggested high metabolic diversity regarding the use of polymers and aromatic and non-aromatic compounds. In line with the high phylogenetic diversity, a surprising and not previously described biodiversity of glycoside hydrolase (GH genes was found. Enzymes of 84 GH families were detected. Polysaccharide utilization loci (PULs, which are found in Bacteroidetes, were highly abundant in the dataset; some of these comprised cellulase genes. Furthermore the highest coverage for GH5 and GH9 family enzymes was detected for Bacteroidetes, suggesting that bacteria of this phylum are mainly responsible for the degradation of cellulose in the Asian elephant. Altogether, this study delivers insight into the biomass conversion by one of the largest plant-fed and land-living animals.

  10. A comparative study on the metabolism of Epimedium koreanum Nakai-prenylated flavonoids in rats by an intestinal enzyme (lactase phlorizin hydrolase) and intestinal flora.

    Science.gov (United States)

    Zhou, Jing; Chen, Yan; Wang, Ying; Gao, Xia; Qu, Ding; Liu, Congyan

    2013-12-24

    The aim of this study was to compare the significance of the intestinal hydrolysis of prenylated flavonoids in Herba Epimedii by an intestinal enzyme and flora. Flavonoids were incubated at 37 °C with rat intestinal enzyme and intestinal flora. HPLC-UV was used to calculate the metabolic rates of the parent drug in the incubation and LC/MS/MS was used to determine the chemical structures of metabolites generated by different flavonoid glycosides. Rates of flavonoid metabolism by rat intestinal enzyme were quicker than those of intestinal flora. The sequence of intestinal flora metabolic rates was icariin>epimedin B>epimedin A>epimedin C>baohuoside I, whereas the order of intestinal enzyme metabolic rates was icariin>epimedin A>epimedin C>epimedin B>baohuoside I. Meanwhile, the LC/MS/MS graphs showed that icariin produced three products, epimedin A/B/C had four and baohuoside I yielded one product in incubations of both intestinal enzyme and flora, which were more than the results of HPLC-UV due to the fact LC/MS/MS has lower detectability and higher sensitivity. Moreover, the outcomes indicated that the rate of metabolization of flavonoids by intestinal enzyme were faster than those of intestinal flora, which was consistent with the HPLC-UV results. In conclusion, the metabolic pathways of the same components by intestinal flora and enzyme were the same. What's more, an intestinal enzyme such as lactase phlorizin hydrolase exhibited a more significant metabolic role in prenylated flavonoids of Herba Epimedi compared with intestinal flora.

  11. Full Fatty Acid Amide Hydrolase Inhibition Combined with Partial Monoacylglycerol Lipase Inhibition: Augmented and Sustained Antinociceptive Effects with Reduced Cannabimimetic Side Effects in Mice.

    Science.gov (United States)

    Ghosh, Sudeshna; Kinsey, Steven G; Liu, Qing-Song; Hruba, Lenka; McMahon, Lance R; Grim, Travis W; Merritt, Christina R; Wise, Laura E; Abdullah, Rehab A; Selley, Dana E; Sim-Selley, Laura J; Cravatt, Benjamin F; Lichtman, Aron H

    2015-08-01

    Inhibition of fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL), the primary hydrolytic enzymes for the respective endocannabinoids N-arachidonoylethanolamine (AEA) and 2-arachidonylglycerol (2-AG), produces antinociception but with minimal cannabimimetic side effects. Although selective inhibitors of either enzyme often show partial efficacy in various nociceptive models, their combined blockade elicits augmented antinociceptive effects, but side effects emerge. Moreover, complete and prolonged MAGL blockade leads to cannabinoid receptor type 1 (CB1) receptor functional tolerance, which represents another challenge in this potential therapeutic strategy. Therefore, the present study tested whether full FAAH inhibition combined with partial MAGL inhibition would produce sustained antinociceptive effects with minimal cannabimimetic side effects. Accordingly, we tested a high dose of the FAAH inhibitor PF-3845 (N-​3-​pyridinyl-​4-​[[3-​[[5-​(trifluoromethyl)-​2-​pyridinyl]oxy]phenyl]methyl]-​1-​piperidinecarboxamide; 10 mg/kg) given in combination with a low dose of the MAGL inhibitor JZL184 [4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate] (4 mg/kg) in mouse models of inflammatory and neuropathic pain. This combination of inhibitors elicited profound increases in brain AEA levels (>10-fold) but only 2- to 3-fold increases in brain 2-AG levels. This combination produced significantly greater antinociceptive effects than single enzyme inhibition and did not elicit common cannabimimetic effects (e.g., catalepsy, hypomotility, hypothermia, and substitution for Δ(9)-tetrahydrocannabinol in the drug-discrimination assay), although these side effects emerged with high-dose JZL184 (i.e., 100 mg/kg). Finally, repeated administration of this combination did not lead to tolerance to its antiallodynic actions in the carrageenan assay or CB1 receptor functional tolerance. Thus, full FAAH inhibition

  12. Biochemical Characterization of the Lactobacillus reuteri Glycoside Hydrolase Family 70 GTFB Type of 4,6-α-Glucanotransferase Enzymes That Synthesize Soluble Dietary Starch Fibers.

    Science.gov (United States)

    Bai, Yuxiang; van der Kaaij, Rachel Maria; Leemhuis, Hans; Pijning, Tjaard; van Leeuwen, Sander Sebastiaan; Jin, Zhengyu; Dijkhuizen, Lubbert

    2015-10-01

    4,6-α-Glucanotransferase (4,6-α-GTase) enzymes, such as GTFB and GTFW of Lactobacillus reuteri strains, constitute a new reaction specificity in glycoside hydrolase family 70 (GH70) and are novel enzymes that convert starch or starch hydrolysates into isomalto/maltopolysaccharides (IMMPs). These IMMPs still have linear chains with some α1→4 linkages but mostly (relatively long) linear chains with α1→6 linkages and are soluble dietary starch fibers. 4,6-α-GTase enzymes and their products have significant potential for industrial applications. Here we report that an N-terminal truncation (amino acids 1 to 733) strongly enhances the soluble expression level of fully active GTFB-ΔN (approximately 75-fold compared to full-length wild type GTFB) in Escherichia coli. In addition, quantitative assays based on amylose V as the substrate are described; these assays allow accurate determination of both hydrolysis (minor) activity (glucose release, reducing power) and total activity (iodine staining) and calculation of the transferase (major) activity of these 4,6-α-GTase enzymes. The data show that GTFB-ΔN is clearly less hydrolytic than GTFW, which is also supported by nuclear magnetic resonance (NMR) analysis of their final products. From these assays, the biochemical properties of GTFB-ΔN were characterized in detail, including determination of kinetic parameters and acceptor substrate specificity. The GTFB enzyme displayed high conversion yields at relatively high substrate concentrations, a promising feature for industrial application. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  13. Differential metabolism of acrylonitrile to cyanide is responsible for the greater sensitivity of male vs female mice: role of CYP2E1 and epoxide hydrolases

    International Nuclear Information System (INIS)

    Chanas, Brian; Wang, Hongbing; Ghanayem, Burhan I.

    2003-01-01

    Acrylonitrile (AN) is a potent toxicant and a known rodent carcinogen. AN epoxidation to cyanoethylene oxide (CEO) via CYP2E1 and its subsequent metabolism via epoxide hydrolases (EH) to yield cyanide is thought to be responsible for the acute toxicity and mortality of AN. Recent reports showed that male mice are more sensitive than females to the acute toxicity/mortality of AN. The present work was undertaken to assess the metabolic and enzymatic basis for the greater sensitivity of male vs female mice to AN toxicity. Male and female wild-type and CYP2E1-null mice received AN at 0, 2.5, 10, 20, or 40 mg/kg by gavage. Cyanide concentrations were measured at 1 or 3 h after dosing. Current data demonstrated that cyanide levels in blood and tissues of AN-treated wild-type mice of both sexes were significantly greater than in vehicle-treated controls and increased in a dose-dependent manner. In contrast, cyanide levels in AN-treated CYP2E1-null mice were not statistically different from those measured in vehicle-treated controls. Furthermore, higher levels of cyanide were detected in male wild-type mice vs females in association with greater sensitivity of males to the acute toxicity/mortality of this chemical. Using Western blot analysis, negligible difference in CYP2E1 expression with higher levels of soluble and microsomal EH (sEH and mEH) was detected in the liver of male vs female mice. In kidneys, male mice exhibited higher expression of both renal CYP2E1 and sEH than did female mice. In conclusion, higher blood and tissue cyanide levels are responsible for the greater sensitivity of male vs female mice to AN. Further, higher expression of CYP2E1 and EH in male mice may contribute to greater formation of CEO and its subsequent metabolism to yield cyanide, respectively

  14. Oxidation of the diphosphoinositol polyphosphate phosphohydrolase-like Nudix hydrolase Aps from Drosophila melanogaster induces thermolability--A possible regulatory switch?

    Science.gov (United States)

    Winward, Lucinda; Whitfield, William G F; McLennan, Alexander G; Safrany, Stephen T

    2010-07-01

    Unlike mammalian cells, Drosophila melanogaster contains only a single member of the diphosphoinositol polyphosphate phosphohydrolase subfamily of the Nudix hydrolases, suggesting that functional specialisation has not occurred in this organism. In order to evaluate its function, Aps was cloned and characterized. It hydrolyses a range of (di)nucleoside polyphosphates, the most efficient being guanosine 5'-tetraphosphate (K(m)=11 microM, k(cat)=0.79 s(-1)). However, it shows a 5-fold preference for the hydrolysis of diphosphoinositol pentakisphosphate (PP-InsP(5), K(m)=0.07 microM, k(cat)=0.024 s(-1)). Assayed at 26 degrees C, Aps had an alkaline pH optimum and required a divalent ion: Mg(2+) (10-20 mM) or Mn(2+) (1 mM) were preferred for nucleotide hydrolysis and Mg(2+) (0.5-1 mM) or Co(2+) (1-100 microM) for PP-InsP(5) hydrolysis. GFP-fusions showed that Aps was predominantly cytoplasmic, with some nuclear localization. In the absence of dithiothreitol Aps was heat labile, rapidly losing activity even at 36 degrees C, while in the presence of dithiothreitol, Aps was heat stable, surviving for 5 min at 76 degrees C. Heat lability was restored by H(2)O(2) and mass spectrometric analysis suggested that this was due to reversible dimerisation involving two inter-molecular disulphides between Cys23 and Cys25. Aps expression was highest in embryos and declined throughout development. The ratio of PP-InsP(5) to inositol hexakisphosphate also decreased throughout development, with the highest level of PP-InsP(5) found in embryos. These data suggest that the redox state of Aps may play a role in controlling its activity by altering its stability, something that could be important for regulating PP-InsP(5) during development. Copyright 2010 Elsevier Ltd. All rights reserved.

  15. Expression of S-adenosylmethionine Hydrolase in Tissues Synthesizing Secondary Cell Walls Alters Specific Methylated Cell Wall Fractions and Improves Biomass Digestibility

    Directory of Open Access Journals (Sweden)

    Aymerick Eudes

    2016-07-01

    Full Text Available Plant biomass is a large source of fermentable sugars for the synthesis of bioproducts using engineered microbes. These sugars are stored as cell wall polymers, mainly cellulose and hemicellulose, and are embedded with lignin, which makes their enzymatic hydrolysis challenging. One of the strategies to reduce cell wall recalcitrance is the modification of lignin content and composition. Lignin is a phenolic polymer of methylated aromatic alcohols and its synthesis in tissues developing secondary cell walls is a significant sink for the consumption of the methyl donor S-adenosylmethionine (AdoMet. In this study, we demonstrate in Arabidopsis stems that targeted expression of S-adenosylmethionine hydrolase (AdoMetase, E.C. 3.3.1.2 in secondary cell-wall synthesizing tissues reduces the AdoMet pool and impacts lignin content and composition. In particular, both NMR analysis and pyrolysis gas chromatography mass spectrometry of lignin in engineered biomass showed relative enrichment of non-methylated p-hydroxycinnamyl (H units and a reduction of dimethylated syringyl (S units. This indicates a lower degree of methylation compared to that in wild-type lignin. Quantification of cell wall-bound hydroxycinnamates revealed a reduction of ferulate in AdoMetase transgenic lines. Biomass from transgenic lines, in contrast to that in control plants, exhibits an enrichment of glucose content and a reduction in the degree of hemicellulose glucuronoxylan methylation. We also show that these modifications resulted in a reduction of cell wall recalcitrance, because sugar yield generated by enzymatic biomass saccharification was greater than that of wild type plants. Considering that transgenic plants show no important diminution of biomass yields, and that heterologous expression of AdoMetase protein can be spatiotemporally optimized, this novel approach provides a valuable option for the improvement of lignocellulosic biomass feedstock.

  16. Characterization of Bile Salt Hydrolase from Lactobacillus gasseri FR4 and Demonstration of Its Substrate Specificity and Inhibitory Mechanism Using Molecular Docking Analysis

    Directory of Open Access Journals (Sweden)

    Rizwana Parveen Rani

    2017-05-01

    Full Text Available Probiotic bacteria are beneficial to the health of poultry animals, thus are used as alternative candidates for antibiotics used as growth promoters (AGPs. However, they also reduce the body weight gain due to innate bile salt hydrolase (BSH activity. Hence, the addition of a suitable BSH inhibitor along with the probiotic feed can decrease the BSH activity. In this study, a BSH gene (981 bp encoding 326-amino acids was identified from the genome of Lactobacillus gasseri FR4 (LgBSH. The LgBSH-encoding gene was cloned and purified using an Escherichia coli BL21 (DE3 expression system, and its molecular weight (37 kDa was confirmed by SDS–PAGE and a Western blot analysis. LgBSH exhibited greater hydrolysis toward glyco-conjugated bile salts compared to tauro-conjugated bile salts. LgBSH displayed optimal activity at 52°C at a pH of 5.5, and activity was further increased by several reducing agents (DTT, surfactants (Triton X-100 and Tween 80, and organic solvents (isopropanol, butanol, and acetone. Riboflavin and penicillin V, respectively, inhibited LgBSH activity by 98.31 and 97.84%. A homology model of LgBSH was predicted using EfBSH (4WL3 as a template. Molecular docking analysis revealed that the glycocholic acid had lowest binding energy of -8.46 kcal/mol; on the other hand, inhibitors, i.e., riboflavin and penicillin V, had relatively higher binding energies of -6.25 and -7.38 kcal/mol, respectively. Our results suggest that L. gasseri FR4 along with riboflavin might be a potential alternative to AGPs for poultry animals.

  17. Cyanuric acid hydrolase from Azorhizobium caulinodans ORS 571: crystal structure and insights into a new class of Ser-Lys dyad proteins.

    Directory of Open Access Journals (Sweden)

    Seunghee Cho

    Full Text Available Cyanuric acid hydrolase (CAH catalyzes the hydrolytic ring-opening of cyanuric acid (2,4,6-trihydroxy-1,3,5-triazine, an intermediate in s-triazine bacterial degradation and a by-product from disinfection with trichloroisocyanuric acid. In the present study, an X-ray crystal structure of the CAH-barbituric acid inhibitor complex from Azorhizobium caulinodans ORS 571 has been determined at 2.7 Å resolution. The CAH protein fold consists of three structurally homologous domains forming a β-barrel-like structure with external α-helices that result in a three-fold symmetry, a dominant feature of the structure and active site that mirrors the three-fold symmetrical shape of the substrate cyanuric acid. The active site structure of CAH is similar to that of the recently determined AtzD with three pairs of active site Ser-Lys dyads. In order to determine the role of each Ser-Lys dyad in catalysis, a mutational study using a highly sensitive, enzyme-coupled assay was conducted. The 10⁹-fold loss of activity by the S226A mutant was at least ten times lower than that of the S79A and S333A mutants. In addition, bioinformatics analysis revealed the Ser226/Lys156 dyad as the only absolutely conserved dyad in the CAH/barbiturase family. These data suggest that Lys156 activates the Ser226 nucleophile which can then attack the substrate carbonyl. Our combination of structural, mutational, and bioinformatics analyses differentiates this study and provides experimental data for mechanistic insights into this unique protein family.

  18. Enantioconvergent hydrolysis of racemic styrene oxide at high concentration by a pair of novel epoxide hydrolases into (R)-phenyl-1,2-ethanediol.

    Science.gov (United States)

    Wang, Rui; Hu, Die; Zong, Xuncheng; Li, Jinping; Ding, Lei; Wu, Minchen; Li, Jianfang

    2017-12-01

    To prepare (R)-phenyl-1,2-ethanediol ((R)-PED) with high enantiomeric excess (ee p ) and yield from racemic styrene oxide (rac-SO) at high concentration by bi-enzymatic catalysis. The bi-enzymatic catalysis was designed for enantioconvergent hydrolysis of rac-SO by a pair of novel epoxide hydrolases (EHs), a Vigna radiata EH3 (VrEH3) and a variant (AuEH2 A250I ) of Aspergillus usamii EH2. The simultaneous addition mode of VrEH3 and AuEH2 A250I , exhibiting the highest average turnover frequency (aTOF) of 0.12 g h -1 g -1 , was selected, by which rac-SO (10 mM) was converted into (R)-PED with 92.6% ee p and 96.3% yield. Under the optimized reaction conditions: dry weight ratio 14:1 of VrEH3-expressing E. coli/vreh3 to AuEH2 A250I -expressing E. coli/Aueh2 A250I and reaction at 20 °C, rac-SO (10 mM) was completely hydrolyzed in 2.3 h, affording (R)-PED with 98% ee p . At the weight ratio 0.8:1 of rac-SO to two mixed dry cells, (R)-PED with 97.4% ee p and 98.7% yield was produced from 200 mM (24 mg/ml) rac-SO in 10.5 h. Enantioconvergent hydrolysis of rac-SO at high concentration catalyzed by both VrEH3 and AuEH2 A250I is an effective method for preparing (R)-PED with high ee p and yield.

  19. Effects of URB597 as an inhibitor of fatty acid amide hydrolase on WIN55, 212-2-induced learning and memory deficits in rats.

    Science.gov (United States)

    Hasanein, Parisa; Teimuri Far, Massoud

    2015-04-01

    Cannabinoid and endocannabinoid systems have been implicated in several physiological functions including modulation of cognition. In this study we evaluated the effects and interaction between fatty-acid amide hydrolase (FAAH) inhibitor URB597 and CB1 receptor agonist WIN55, 212-2 on memory using object recognition and passive avoidance learning (PAL) tests. Learning and memory impairment was induced by WIN 55, 212-2 administration (1mg/kg, i.p.) 30min before the acquisition trial. URB597 (0.1, 0.3 and 1mg/kg, i.p.) or SR141716A (1mg/kg, i.p.) was injected to rats 10min before WIN 55, 212-2 or URB597 respectively. URB597 (0.3 and 1mg/kg) but not 0.1mg/kg induced higher discrimination index (DI) in object recognition test and enhanced memory acquisition in PAL test. The cognitive enhancing effect of URB597 was blocked by a CB1 receptor antagonist, SR141716A which at this dose alone had no effect on cognition. WIN55, 212-2 caused cognition deficits in both tests. URB597 (0.3 and 1mg/kg) treatment could alleviate the negative influence of WIN 55, 212-2 on cognition and memory. These results indicate URB597 potential to protect against memory deficits induced by cannabinoid. Therefore, in combination with URB597 beneficial effects, this study suggests that URB597 has recognition and acquisition memory enhancing effects. It may also constitute a novel approach for the treatment of cannabinoid induced memory deficits and lead to a better understanding of the brain mechanisms underlying cognition. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Diversity of hydrolases from hydrothermal vent sediments of the Levante Bay, Vulcano Island (Aeolian archipelago) identified by activity-based metagenomics and biochemical characterization of new esterases and an arabinopyranosidase.

    Science.gov (United States)

    Placido, Antonio; Hai, Tran; Ferrer, Manuel; Chernikova, Tatyana N; Distaso, Marco; Armstrong, Dale; Yakunin, Alexander F; Toshchakov, Stepan V; Yakimov, Michail M; Kublanov, Ilya V; Golyshina, Olga V; Pesole, Graziano; Ceci, Luigi R; Golyshin, Peter N

    2015-12-01

    A metagenomic fosmid expression library established from environmental DNA (eDNA) from the shallow hot vent sediment sample collected from the Levante Bay, Vulcano Island (Aeolian archipelago) was established in Escherichia coli. Using activity-based screening assays, we have assessed 9600 fosmid clones corresponding to approximately 350 Mbp of the cloned eDNA, for the lipases/esterases/lactamases, haloalkane and haloacid dehalogenases, and glycoside hydrolases. Thirty-four positive fosmid clones were selected from the total of 120 positive hits and sequenced to yield ca. 1360 kbp of high-quality assemblies. Fosmid inserts were attributed to the members of ten bacterial phyla, including Proteobacteria, Bacteroidetes, Acidobateria, Firmicutes, Verrucomicrobia, Chloroflexi, Spirochaetes, Thermotogae, Armatimonadetes, and Planctomycetes. Of ca. 200 proteins with high biotechnological potential identified therein, we have characterized in detail three distinct α/β-hydrolases (LIPESV12_9, LIPESV12_24, LIPESV12_26) and one new α-arabinopyranosidase (GLV12_5). All LIPESV12 enzymes revealed distinct substrate specificities tested against 43 structurally diverse esters and 4 p-nitrophenol carboxyl esters. Of 16 different glycosides tested, the GLV12_5 hydrolysed only p-nitrophenol-α-(L)-arabinopyranose with a high specific activity of about 2.7 kU/mg protein. Most of the α/β-hydrolases were thermophilic and revealed a high tolerance to, and high activities in the presence of, numerous heavy metal ions. Among them, the LIPESV12_24 was the best temperature-adapted, retaining its activity after 40 min of incubation at 90 °C. Furthermore, enzymes were active in organic solvents (e.g., >30% methanol). Both LIPESV12_24 and LIPESV12_26 had the GXSXG pentapeptides and the catalytic triads Ser-Asp-His typical to the representatives of carboxylesterases of EC 3.1.1.1.

  1. Inhibition of SAH-hydrolase during tobacco seeds germination induced by treatment by DHPA leads to mitotically heritable DNA hypomethylation, ectopic expression of floral genes and floral whorl malformations

    Czech Academy of Sciences Publication Activity Database

    Fulneček, Jaroslav; Matyášek, Roman; Kabáthová, E.; Votruba, Ivan; Holý, Antonín; Kovařík, Aleš

    2013-01-01

    Roč. 280, Suppl. 1 (2013), s. 522-522 ISSN 1742-464X. [Congress of the Federation of European Biochemical Societies (FEBS) /38./. 06.07.2013-11.07.2013, Saint Petersburg] R&D Projects: GA ČR GBP501/12/G090; GA ČR GA206/09/1751; GA ČR GA13-10057S Institutional support: RVO:68081707 ; RVO:61388963 Keywords : SAH-hydrolase * DNA hypomethylation * DHPA Subject RIV: CE - Biochemistry

  2. Analysis of the peptidoglycan hydrolase complement of Lactobacillus casei and characterization of the major γ-D-glutamyl-L-lysyl-endopeptidase.

    Science.gov (United States)

    Regulski, Krzysztof; Courtin, Pascal; Meyrand, Mickael; Claes, Ingmar J J; Lebeer, Sarah; Vanderleyden, Jos; Hols, Pascal; Guillot, Alain; Chapot-Chartier, Marie-Pierre

    2012-01-01

    Peptidoglycan (PG) is the major component of Gram positive bacteria cell wall and is essential for bacterial integrity and shape. Bacteria synthesize PG hydrolases (PGHs) which are able to cleave bonds in their own PG and play major roles in PG remodelling required for bacterial growth and division. Our aim was to identify the main PGHs in Lactobacillus casei BL23, a lactic acid bacterium with probiotic properties.The PGH complement was first identified in silico by amino acid sequence similarity searches of the BL23 genome sequence. Thirteen PGHs were detected with different predicted hydrolytic specificities. Transcription of the genes was confirmed by RT-PCR. A proteomic analysis combining the use of SDS-PAGE and LC-MS/MS revealed the main seven PGHs synthesized during growth of L. casei BL23. Among these PGHs, LCABL_02770 (renamed Lc-p75) was identified as the major one. This protein is the homolog of p75 (Msp1) major secreted protein of Lactobacillus rhamnosus GG, which was shown to promote survival and growth of intestinal epithelial cells. We identified its hydrolytic specificity on PG and showed that it is a γ-D-glutamyl-L-lysyl-endopeptidase. It has a marked specificity towards PG tetrapeptide chains versus tripeptide chains and for oligomers rather than monomers. Immunofluorescence experiments demonstrated that Lc-p75 localizes at cell septa in agreement with its role in daughter cell separation. It is also secreted under an active form as detected in zymogram. Comparison of the muropeptide profiles of wild type and Lc-p75-negative mutant revealed a decrease of the amount of disaccharide-dipeptide in the mutant PG in agreement with Lc-p75 activity. As a conclusion, Lc-p75 is the major L. casei BL23 PGH with endopeptidase specificity and a key role in daughter cell separation. Further studies will aim at investigating the role of Lc-p75 in the anti-inflammatory potential of L. casei BL23.

  3. Characterisation of (R-2-(2-Fluorobiphenyl-4-yl-N-(3-Methylpyridin-2-ylPropanamide as a Dual Fatty Acid Amide Hydrolase: Cyclooxygenase Inhibitor.

    Directory of Open Access Journals (Sweden)

    Sandra Gouveia-Figueira

    Full Text Available Increased endocannabinoid tonus by dual-action fatty acid amide hydrolase (FAAH and substrate selective cyclooxygenase (COX-2 inhibitors is a promising approach for pain-relief. One such compound with this profile is 2-(2-fluorobiphenyl-4-yl-N-(3-methylpyridin-2-ylpropanamide (Flu-AM1. These activities are shown by Flu-AM1 racemate, but it is not known whether its two single enantiomers behave differently, as is the case towards COX-2 for the parent flurbiprofen enantiomers. Further, the effects of the compound upon COX-2-derived lipids in intact cells are not known.COX inhibition was determined using an oxygraphic method with arachidonic acid and 2-arachidonoylglycerol (2-AG as substrates. FAAH was assayed in mouse brain homogenates using anandamide (AEA as substrate. Lipidomic analysis was conducted in unstimulated and lipopolysaccharide + interferon γ- stimulated RAW 264.7 macrophage cells. Both enantiomers inhibited COX-2 in a substrate-selective and time-dependent manner, with IC50 values in the absence of a preincubation phase of: (R-Flu-AM1, COX-1 (arachidonic acid 6 μM; COX-2 (arachidonic acid 20 μM; COX-2 (2-AG 1 μM; (S-Flu-AM1, COX-1 (arachidonic acid 3 μM; COX-2 (arachidonic acid 10 μM; COX-2 (2-AG 0.7 μM. The compounds showed no enantiomeric selectivity in their FAAH inhibitory properties. (R-Flu-AM1 (10 μM greatly inhibited the production of prostaglandin D2 and E2 in both unstimulated and lipopolysaccharide + interferon γ- stimulated RAW 264.7 macrophage cells. Levels of 2-AG were not affected either by (R-Flu-AM1 or by 10 μM flurbiprofen, either alone or in combination with the FAAH inhibitor URB597 (1 μM.Both enantiomers of Flu-AM1 are more potent inhibitors of 2-AG compared to arachidonic acid oxygenation by COX-2. Inhibition of COX in lipopolysaccharide + interferon γ- stimulated RAW 264.7 cells is insufficient to affect 2-AG levels despite the large induction of COX-2 produced by this treatment.

  4. Cell wall proteome of sugarcane stems: comparison of a destructive and a non-destructive extraction method showed differences in glycoside hydrolases and peroxidases.

    Science.gov (United States)

    Calderan-Rodrigues, Maria Juliana; Jamet, Elisabeth; Douché, Thibaut; Bonassi, Maria Beatriz Rodrigues; Cataldi, Thaís Regiani; Fonseca, Juliana Guimarães; San Clemente, Hélène; Pont-Lezica, Rafael; Labate, Carlos Alberto

    2016-01-11

    Sugarcane has been used as the main crop for ethanol production for more than 40 years in Brazil. Recently, the production of bioethanol from bagasse and straw, also called second generation (2G) ethanol, became a reality with the first commercial plants started in the USA and Brazil. However, the industrial processes still need to be improved to generate a low cost fuel. One possibility is the remodeling of cell walls, by means of genetic improvement or transgenesis, in order to make the bagasse more accessible to hydrolytic enzymes. We aimed at characterizing the cell wall proteome of young sugarcane culms, to identify proteins involved in cell wall biogenesis. Proteins were extracted from the cell walls of 2-month-old culms using two protocols, non-destructive by vacuum infiltration vs destructive. The proteins were identified by mass spectrometry and bioinformatics. A predicted signal peptide was found in 84 different proteins, called cell wall proteins (CWPs). As expected, the non-destructive method showed a lower percentage of proteins predicted to be intracellular than the destructive one (33% vs 44%). About 19% of CWPs were identified with both methods, whilst the infiltration protocol could lead to the identification of 75% more CWPs. In both cases, the most populated protein functional classes were those of proteins related to lipid metabolism and oxido-reductases. Curiously, a single glycoside hydrolase (GH) was identified using the non-destructive method whereas 10 GHs were found with the destructive one. Quantitative data analysis allowed the identification of the most abundant proteins. The results highlighted the importance of using different protocols to extract proteins from cell walls to expand the coverage of the cell wall proteome. Ten GHs were indicated as possible targets for further studies in order to obtain cell walls less recalcitrant to deconstruction. Therefore, this work contributed to two goals: enlarge the coverage of the sugarcane

  5. The Exiguobacterium sibiricum 255-15 GtfC Enzyme Represents a Novel Glycoside Hydrolase 70 Subfamily of 4,6-α-Glucanotransferase Enzymes.

    Science.gov (United States)

    Gangoiti, Joana; Pijning, Tjaard; Dijkhuizen, Lubbert

    2016-01-15

    The glycoside hydrolase 70 (GH70) family originally was established for glucansucrase enzymes found solely in lactic acid bacteria synthesizing α-glucan polysaccharides from sucrose (e.g., GtfA). In recent years, we have characterized GtfB and related Lactobacillus enzymes as 4,6-α-glucanotransferase enzymes. These GtfB-type enzymes constitute the first GH70 subfamily of enzymes that are unable to act on sucrose as a substrate but are active with maltodextrins and starch, cleave α1→4 linkages, and synthesize linear α1→6-glucan chains. The GtfB disproportionating type of activity results in the conversion of malto-oligosaccharides into isomalto/malto-polysaccharides with a relatively high percentage of α1→6 linkages. This paper reports the identification of the members of a second GH70 subfamily (designated GtfC enzymes) and the characterization of the Exiguobacterium sibiricum 255-15 GtfC enzyme, which is also inactive with sucrose and displays 4,6-α-glucanotransferase activity with malto-oligosaccharides. GtfC differs from GtfB in synthesizing isomalto/malto-oligosaccharides. Biochemically, the GtfB- and GtfC-type enzymes are related, but phylogenetically, they clearly constitute different GH70 subfamilies, displaying only 30% sequence identity. Whereas the GtfB-type enzyme largely has the same domain order as glucansucrases (with α-amylase domains A, B, and C plus domains IV and V), this GtfC-type enzyme differs in the order of these domains and completely lacks domain V. In GtfC, the sequence of conserved regions I to IV of clan GH-H is identical to that in GH13 (I-II-III-IV) but different from that in GH70 (II-III-IV-I because of a circular permutation of the (β/α)8 barrel. The GtfC 4,6-α-glucanotransferase enzymes thus represent structurally and functionally very interesting evolutionary intermediates between α-amylase and glucansucrase enzymes. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  6. Analysis of the peptidoglycan hydrolase complement of Lactobacillus casei and characterization of the major γ-D-glutamyl-L-lysyl-endopeptidase.

    Directory of Open Access Journals (Sweden)

    Krzysztof Regulski

    Full Text Available Peptidoglycan (PG is the major component of Gram positive bacteria cell wall and is essential for bacterial integrity and shape. Bacteria synthesize PG hydrolases (PGHs which are able to cleave bonds in their own PG and play major roles in PG remodelling required for bacterial growth and division. Our aim was to identify the main PGHs in Lactobacillus casei BL23, a lactic acid bacterium with probiotic properties.The PGH complement was first identified in silico by amino acid sequence similarity searches of the BL23 genome sequence. Thirteen PGHs were detected with different predicted hydrolytic specificities. Transcription of the genes was confirmed by RT-PCR. A proteomic analysis combining the use of SDS-PAGE and LC-MS/MS revealed the main seven PGHs synthesized during growth of L. casei BL23. Among these PGHs, LCABL_02770 (renamed Lc-p75 was identified as the major one. This protein is the homolog of p75 (Msp1 major secreted protein of Lactobacillus rhamnosus GG, which was shown to promote survival and growth of intestinal epithelial cells. We identified its hydrolytic specificity on PG and showed that it is a γ-D-glutamyl-L-lysyl-endopeptidase. It has a marked specificity towards PG tetrapeptide chains versus tripeptide chains and for oligomers rather than monomers. Immunofluorescence experiments demonstrated that Lc-p75 localizes at cell septa in agreement with its role in daughter cell separation. It is also secreted under an active form as detected in zymogram. Comparison of the muropeptide profiles of wild type and Lc-p75-negative mutant revealed a decrease of the amount of disaccharide-dipeptide in the mutant PG in agreement with Lc-p75 activity. As a conclusion, Lc-p75 is the major L. casei BL23 PGH with endopeptidase specificity and a key role in daughter cell separation. Further studies will aim at investigating the role of Lc-p75 in the anti-inflammatory potential of L. casei BL23.

  7. Functional characterization of a Nudix hydrolase AtNUDX8 upon pathogen attack indicates a positive role in plant immune responses.

    Directory of Open Access Journals (Sweden)

    Jose Pedro Fonseca

    Full Text Available Nudix hydrolases comprise a large gene family of twenty nine members in Arabidopsis, each containing a conserved motif capable of hydrolyzing specific substrates like ADP-glucose and NADH. Until now only two members of this family, AtNUDX6 and AtNUDX7, have been shown to be involved in plant immunity. RPP4 is a resistance gene from a multigene family that confers resistance to downy mildew. A time course expression profiling after Hyaloperonospora arabidopsidis inoculation in both wild-type (WT and the rpp4 mutant was carried out to identify differentially expressed genes in RPP4-mediated resistance. AtNUDX8 was one of several differentially expressed, downregulated genes identified. A T-DNA knockout mutant (KO-nudx8 was obtained from a Salk T-DNA insertion collection, which exhibited abolished AtNUDX8 expression. The KO-nudx8 mutant was infected separately from the oomycete pathogen Hpa and the bacterial pathogen Pseudomonas syringae pv. maculicola ES4326. The mutant displayed a significantly enhanced disease susceptibility to both pathogens when compared with the WT control. We observed a small, stunted phenotype for KO-nudx8 mutant plants when grown over a 12/12 hour photoperiod but not over a 16/8 hour photoperiod. AtNUDX8 expression peaked at 8 hours after the lights were turned on and this expression was significantly repressed four-fold by salicylic acid (SA. The expression of three pathogen-responsive thioredoxins (TRX-h2, TRX-h3 and TRX-h5 were downregulated at specific time points in the KO-nudx8 mutant when compared with the WT. Furthermore, KO-nudx8 plants like the npr1 mutant, displayed SA hypersensitivity. Expression of a key SA biosynthetic gene ICS1 was repressed at specific time points in the KO-nudx8 mutant suggesting that AtNUDX8 is involved in SA signaling in plants. Similarly, NPR1 and PR1 transcript levels were also downregulated at specific time points in the KO-nudx8 mutant. This study shows that AtNUDX8 is involved in

  8. Comparative metabolism of methacrylonitrile and acrylonitrile to cyanide using cytochrome P4502E1 and microsomal epoxide hydrolase-null mice

    International Nuclear Information System (INIS)

    El Hadri, L.; Chanas, B.; Ghanayem, B.I.

    2005-01-01

    Methacrylonitrile (MAN) and acrylonitrile (AN) are metabolized via glutathione (GSH) conjugation or epoxide formation. We have recently shown that CYP2E1 is essential for AN epoxidation and subsequent cyanide liberation. Current studies were designed to compare the enzymatic basis of MAN vs. AN metabolism to cyanide using wild-type (WT), CYP2E1-, and mEH-null mice. Mice received a single gavage dose of 0.047, 0.095, 0.19, or 0.38 mmol/kg of MAN or AN, and blood cyanide was measured at 1 or 3 h later. Blood cyanide levels in WT mice treated with AN or MAN were dose and time dependent. At equimolar doses, significantly higher levels of cyanide were detected in the blood of MAN- vs. AN-treated mice. Further, while significant reduction in blood cyanide levels occurred in MAN-treated CYP2E1-null vs. WT mice, AN metabolism to cyanide was largely abolished in CYP2E1-null mice. Pretreatment of mice with 1-aminobenzotriazole (ABT, CYP inhibitor) demonstrated that CYPs other than CYP2E1 also contribute to MAN metabolism to cyanide. Blood cyanide levels in mEH-null mice treated with aliphatic nitriles are generally lower than levels in similarly treated WT mice. Western blot analysis showed that expression of sEH was greater in male vs. female mice. The role of various epoxide hydrolases (EHs) in the production of cyanide from aliphatic nitriles is apparently structure and dose dependent. Regardless of genotype, significantly higher levels of cyanide were measured in the blood of male vs. female mice treated with MAN or AN. In conclusion, these data showed that (1) at equimolar doses, higher blood cyanide levels were detected in mice treated with MAN vs. AN; (2) while CYP2E1 is the only enzyme responsible for AN metabolism to cyanide, other CYPs also contribute to MAN metabolism; and (3) significantly higher levels of cyanide were measured in the blood of male vs. female treated with either nitrile. Higher blood cyanide levels in male vs. female mice and in MAN- vs. AN

  9. Cross-protective immunity to Leishmania amazonensis is mediated by CD4+ and CD8+-epitopes of Leishmania donovani Nucleoside Hydrolase terminal domains

    Directory of Open Access Journals (Sweden)

    Dirlei eNico

    2014-05-01

    Full Text Available The Nucleoside hydrolase of Leishmania donovani (NH36 is a phylogenetic marker of high homology among Leishmania parasites. In mice and dog vaccination NH36 induces a CD4+ T cell-driven protective response against Leishmania chagasi infection directed against its C-terminal domain (F3. The C-terminal and N-terminal domain vaccines also decreased the footpad lesion caused by Leishmania amazonensis. We studied the basis of the crossed immune response using recombinant generated peptides covering the whole NH36 sequence and saponin for mice prophylaxis against L. amazonensis. The F1 (amino acids 1-103 and F3 peptide (amino acids 199-314 vaccines enhanced the IgG and IgG2a anti-NH36 antibodies to similar levels. The F3 vaccine induced the strongest DTH response, the highest proportions of NH36-specific CD4+ and CD8+ T cells after challenge and the highest expression of IFN-γ and TNF-α. The F1 vaccine, on the other hand, induced a weaker but significant DTH response and a mild enhancement of IFN-γ and TNF-α levels. The in vivo depletion with anti-CD4 or CD8 monoclonal antibodies disclosed that cross-protection against L. amazonensis infection was mediated by a CD4+ T cell response directed against the C-terminal domain (75% of reduction of the size of footpad lesion followed by a CD8+ T cell response against the N-terminal domain of NH36 (57% of reduction of footpad lesions. Both vaccines were capable of inducing long-term cross-immunity. The amino acid sequence of NH36 showed 93% identity to the sequence of the NH A34480 of L. amazonensis which also showed the presence of completely conserved predicted epitopes for CD4+ and CD8+ T cells in F1 domain, and of CD4+ epitopes differing in a single amino acid, in F1 and F3 domains. The identification of the C-terminal and N-terminal domains as the targets of the immune response to NH36 in the model of L. amazonesis infection represents a basis for the rationale development of a bivalent vaccine

  10. Purification and enzymatic characterization of secretory glycoside hydrolase family 3 (GH3) aryl β-glucosidases screened from Aspergillus oryzae genome.

    Science.gov (United States)

    Kudo, Kanako; Watanabe, Akira; Ujiie, Seiryu; Shintani, Takahiro; Gomi, Katsuya

    2015-12-01

    By a global search of the genome database of Aspergillus oryzae, we found 23 genes encoding putative β-glucosidases, among which 10 genes with a signal peptide belonging to glycoside hydrolase family 3 (GH3) were overexpressed in A. oryzae using the improved glaA gene promoter. Consequently, crude enzyme preparations from three strains, each harboring the genes AO090038000223 (bglA), AO090103000127 (bglF), and AO090003001511 (bglJ), showed a substrate preference toward p-nitrophenyl-β-d-glucopyranoside (pNPGlc) and thus were purified to homogeneity and enzymatically characterized. All the purified enzymes (BglA, BglF, and BglJ) preferentially hydrolyzed aryl β-glycosides, including pNPGlc, rather than cellobiose, and these enzymes were proven to be aryl β-glucosidases. Although the specific activity of BglF toward all the substrates tested was significantly low, BglA and BglJ showed appreciably high activities toward pNPGlc and arbutin. The kinetic parameters of BglA and BglJ for pNPGlc suggested that both the enzymes had relatively higher hydrolytic activity toward pNPGlc among the fungal β-glucosidases reported. The thermal and pH stabilities of BglA were higher than those of BglJ, and BglA was particularly stable in a wide pH range (pH 4.5-10). In contrast, BglJ was the most heat- and alkaline-labile among the three β-glucosidases. Furthermore, BglA was more tolerant to ethanol than BglJ; as a result, it showed much higher hydrolytic activity toward isoflavone glycosides in the presence of ethanol than BglJ. This study suggested that the mining of novel β-glucosidases exhibiting higher activity from microbial genome sequences is of great use for the production of beneficial compounds such as isoflavone aglycones. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  11. The blockade of the transient receptor potential vanilloid type 1 and fatty acid amide hydrolase decreases symptoms and central sequelae in the medial prefrontal cortex of neuropathic rats

    Directory of Open Access Journals (Sweden)

    Di Marzo Vincenzo

    2011-01-01

    Full Text Available Abstract Background Neuropathic pain is a chronic disease resulting from dysfunction within the "pain matrix". The basolateral amygdala (BLA can modulate cortical functions and interactions between this structure and the medial prefrontal cortex (mPFC are important for integrating emotionally salient information. In this study, we have investigated the involvement of the transient receptor potential vanilloid type 1 (TRPV1 and the catabolic enzyme fatty acid amide hydrolase (FAAH in the morphofunctional changes occurring in the pre-limbic/infra-limbic (PL/IL cortex in neuropathic rats. Results The effect of N-arachidonoyl-serotonin (AA-5-HT, a hybrid FAAH inhibitor and TPRV1 channel antagonist, was tested on nociceptive behaviour associated with neuropathic pain as well as on some phenotypic changes occurring on PL/IL cortex pyramidal neurons. Those neurons were identified as belonging to the BLA-mPFC pathway by electrical stimulation of the BLA followed by hind-paw pressoceptive stimulus application. Changes in their spontaneous and evoked activity were studied in sham or spared nerve injury (SNI rats before or after repeated treatment with AA-5-HT. Consistently with the SNI-induced changes in PL/IL cortex neurons which underwent profound phenotypic reorganization, suggesting a profound imbalance between excitatory and inhibitory responses in the mPFC neurons, we found an increase in extracellular glutamate levels, as well as the up-regulation of FAAH and TRPV1 in the PL/IL cortex of SNI rats. Daily treatment with AA-5-HT restored cortical neuronal activity, normalizing the electrophysiological changes associated with the peripheral injury of the sciatic nerve. Finally, a single acute intra-PL/IL cortex microinjection of AA-5-HT transiently decreased allodynia more effectively than URB597 or I-RTX, a selective FAAH inhibitor or a TRPV1 blocker, respectively. Conclusion These data suggest a possible involvement of endovanilloids in the cortical

  12. Friend or foe? Evolutionary history of glycoside hydrolase family 32 genes encoding for sucrolytic activity in fungi and its implications for plant-fungal symbioses

    Directory of Open Access Journals (Sweden)

    James Timothy Y

    2009-06-01

    Full Text Available Abstract Background Many fungi are obligate biotrophs of plants, growing in live plant tissues, gaining direct access to recently photosynthesized carbon. Photosynthate within plants is transported from source to sink tissues as sucrose, which is hydrolyzed by plant glycosyl hydrolase family 32 enzymes (GH32 into its constituent monosaccharides to meet plant cellular demands. A number of plant pathogenic fungi also use GH32 enzymes to access plant-derived sucrose, but less is known about the sucrose utilization ability of mutualistic and commensal plant biotrophic fungi, such as mycorrhizal and endophytic fungi. The aim of this study was to explore the distribution and abundance of GH32 genes in fungi to understand how sucrose utilization is structured within and among major ecological guilds and evolutionary lineages. Using bioinformatic and PCR-based analyses, we tested for GH32 gene presence in all available fungal genomes and an additional 149 species representing a broad phylogenetic and ecological range of biotrophic fungi. Results We detected 9 lineages of GH32 genes in fungi, 4 of which we describe for the first time. GH32 gene number in fungal genomes ranged from 0–12. Ancestral state reconstruction of GH32 gene abundance showed a strong correlation with nutritional mode, and gene family expansion was observed in several clades of pathogenic filamentous Ascomycota species. GH32 gene number was negatively correlated with animal pathogenicity and positively correlated with plant biotrophy, with the notable exception of mycorrhizal taxa. Few mycorrhizal species were found to have GH32 genes as compared to other guilds of plant-associated fungi, such as pathogens, endophytes and lichen-forming fungi. GH32 genes were also more prevalent in the Ascomycota than in the Basidiomycota. Conclusion We found a strong signature of both ecological strategy and phylogeny on GH32 gene number in fungi. These data suggest that plant biotrophic fungi

  13. Comprehensive Identification of Immunodominant Proteins of Brucella abortus and Brucella melitensis Using Antibodies in the Sera from Naturally Infected Hosts

    Directory of Open Access Journals (Sweden)

    Gamal Wareth

    2016-04-01

    Full Text Available Brucellosis is a debilitating zoonotic disease that affects humans and animals. The diagnosis of brucellosis is challenging, as accurate species level identification is not possible with any of the currently available serology-based diagnostic methods. The present study aimed at identifying Brucella (B. species-specific proteins from the closely related species B. abortus and B. melitensis using sera collected from naturally infected host species. Unlike earlier reported investigations with either laboratory-grown species or vaccine strains, in the present study, field strains were utilized for analysis. The label-free quantitative proteomic analysis of the naturally isolated strains of these two closely related species revealed 402 differentially expressed proteins, among which 63 and 103 proteins were found exclusively in the whole cell extracts of B. abortus and B. melitensis field strains, respectively. The sera from four different naturally infected host species, i.e., cattle, buffalo, sheep, and goat were applied to identify the immune-binding protein spots present in the whole protein extracts from the isolated B. abortus and B. melitensis field strains and resolved on two-dimensional gel electrophoresis. Comprehensive analysis revealed that 25 proteins of B. abortus and 20 proteins of B. melitensis were distinctly immunoreactive. Dihydrodipicolinate synthase, glyceraldehyde-3-phosphate dehydrogenase and lactate/malate dehydrogenase from B. abortus, amino acid ABC transporter substrate-binding protein from B. melitensis and fumarylacetoacetate hydrolase from both species were reactive with the sera of all the tested naturally infected host species. The identified proteins could be used for the design of serological assays capable of detecting pan-Brucella, B. abortus- and B. melitensis-specific antibodies.

  14. Comprehensive Identification of Immunodominant Proteins of Brucella abortus and Brucella melitensis Using Antibodies in the Sera from Naturally Infected Hosts.

    Science.gov (United States)

    Wareth, Gamal; Eravci, Murat; Weise, Christoph; Roesler, Uwe; Melzer, Falk; Sprague, Lisa D; Neubauer, Heinrich; Murugaiyan, Jayaseelan

    2016-04-30

    Brucellosis is a debilitating zoonotic disease that affects humans and animals. The diagnosis of brucellosis is challenging, as accurate species level identification is not possible with any of the currently available serology-based diagnostic methods. The present study aimed at identifying Brucella (B.) species-specific proteins from the closely related species B. abortus and B. melitensis using sera collected from naturally infected host species. Unlike earlier reported investigations with either laboratory-grown species or vaccine strains, in the present study, field strains were utilized for analysis. The label-free quantitative proteomic analysis of the naturally isolated strains of these two closely related species revealed 402 differentially expressed proteins, among which 63 and 103 proteins were found exclusively in the whole cell extracts of B. abortus and B. melitensis field strains, respectively. The sera from four different naturally infected host species, i.e., cattle, buffalo, sheep, and goat were applied to identify the immune-binding protein spots present in the whole protein extracts from the isolated B. abortus and B. melitensis field strains and resolved on two-dimensional gel electrophoresis. Comprehensive analysis revealed that 25 proteins of B. abortus and 20 proteins of B. melitensis were distinctly immunoreactive. Dihydrodipicolinate synthase, glyceraldehyde-3-phosphate dehydrogenase and lactate/malate dehydrogenase from B. abortus, amino acid ABC transporter substrate-binding protein from B. melitensis and fumarylacetoacetate hydrolase from both species were reactive with the sera of all the tested naturally infected host species. The identified proteins could be used for the design of serological assays capable of detecting pan-Brucella, B. abortus- and B. melitensis-specific antibodies.

  15. Nomenclature for alleles of the human carboxylesterase 1 gene

    DEFF Research Database (Denmark)

    Rasmussen, Henrik B.; Madsen, Majbritt B.; Bjerre, Ditte

    2017-01-01

    The carboxylesterase 1 gene (CES1) in humans encodes a hydrolase, which is implicated in the metabolism of several commonly used drugs 1. This gene is located on chromosome 16 with a highly homologous pseudogene, CES1P1, in its proximity. A duplicated segment of CES1 replaces most of CES1P1 in some...... appears to be low 8,13. The formation of hybrids consisting of a gene and a related pseudogene has been reported for other genes than CES1. This includes the hybrids of the gene encoding cytochrome P450 2D6 (CYP2D6) and pseudogene CYP2D7, that is, the so-called CYP2D7/D6 hybrids 14......,15. These are categorized as CYP2D6 variants and not as variants of pseudogene CYP2D716....

  16. Effect of sulindac sulfide on metallohydrolases in the human colon cancer cell line HT-29.

    Directory of Open Access Journals (Sweden)

    Hector Guillen-Ahlers

    Full Text Available Matrix metalloproteinase 7 (MMP7, a metallohydrolase involved in the development of several cancers, is downregulated in the Apc(Min/+ colon cancer mouse model following sulindac treatment. To determine whether this effect is relevant to the human condition, HT-29 human colon cancer cells were treated with sulindac and its metabolites, and compared to results obtained from in vivo mouse studies. The expression of MMP7 was monitored. The results demonstrated that sulindac sulfide effectively downregulated both MMP7 expression and activity. Furthermore, activity-based proteomics demonstrated that sulindac sulfide dramatically decreased the activity of leukotriene A4 hydrolase in HT-29 cells as reflected by a decrease in the level of its product, leukotriene B4. This study demonstrates that the effect of sulindac treatment in a mouse model of colon cancer may be relevant to the human counterpart and highlights the effect of sulindac treatment on metallohydrolases.

  17. Functional analysis of the Glucan Degradation Locus (GDL) in Caldicellulosiruptor bescii reveals essential roles of component glycoside hydrolases in plant biomass deconstruction.

    Science.gov (United States)

    Conway, Jonathan M; McKinley, Bennett S; Seals, Nathaniel L; Hernandez, Diana; Khatibi, Piyum A; Poudel, Suresh; Giannone, Richard J; Hettich, Robert L; Williams-Rhaesa, Amanda M; Lipscomb, Gina L; Adams, Michael W W; Kelly, Robert M

    2017-10-06

    The ability to hydrolyze microcrystalline cellulose is an uncommon feature in the microbial world, but one that can be exploited for conversion of lignocellulosic feedstocks into bio-based fuels and chemicals. Understanding the physiological and biochemical mechanisms by which microorganisms deconstruct cellulosic material is key to achieving this objective. The Glucan Degradation Locus (GDL) in the genomes of extremely thermophilic Caldicellulosiruptor species encodes polysaccharide lyases (PLs), unique cellulose binding proteins (tāpirins), and putative post-translational modifying enzymes, in addition to multi-domain, multi-functional glycoside hydrolases (GHs), thereby representing an alternative paradigm for plant biomass degradation, as compared to fungal or cellulosomal systems. To examine the individual and collective in vivo roles of the glycolytic enzymes, the six GHs in the GDL of Caldicellulosiruptor bescii were systematically deleted, and the extent to which the resulting mutant strains could solubilize microcrystalline cellulose (Avicel) and plant biomasses (switchgrass or poplar) was examined. Three of the GDL enzymes, Athe_1867 (CelA) (GH9-CBM3-CBM3-CBM3-GH48), Athe_1859 (GH5-CBM3-CBM3-GH44), and Athe_1857 (GH10-CBM3-CBM3-GH48), acted synergistically in vivo and accounted for 92% of naked microcellulose (Avicel) degradation. However, the relative importance of the GDL GHs varied for the plant biomass substrates tested. Furthermore, mixed cultures of mutant strains showed switchgrass solubilization depended on the secretome-bound enzymes collectively produced by the culture and not on the specific strain from which they came. These results demonstrate that certain GDL GHs are primarily responsible for the degradation of microcrystalline-containing substrates by C. bescii and provide new insights into the workings of a novel microbial mechanism for lignocellulose utilization. Importance The efficient and extensive degradation of complex

  18. 6-Oxocyclohex-1-ene-1-carbonyl-coenzyme A hydrolases from obligately anaerobic bacteria: characterization and identification of its gene as a functional marker for aromatic compounds degrading anaerobes.

    Science.gov (United States)

    Kuntze, Kevin; Shinoda, Yoshifumi; Moutakki, Housna; McInerney, Michael J; Vogt, Carsten; Richnow, Hans-Hermann; Boll, Matthias

    2008-06-01

    In anaerobic bacteria, most aromatic growth substrates are channelled into the benzoyl-coenzyme A (CoA) degradation pathway where the aromatic ring is dearomatized and cleaved into an aliphatic thiol ester. The initial step of this pathway is catalysed by dearomatizing benzoyl-CoA reductases yielding the two electron-reduction product, cyclohexa-1,5-diene-1-carbonyl-CoA, to which water is subsequently added by a hydratase. The next two steps have so far only been studied in facultative anaerobes and comprise the oxidation of the 6-hydroxyl-group to 6-oxocyclohex-1-ene-1-carbonyl-CoA (6-OCH-CoA), the addition of water and hydrolytic ring cleavage yielding 3-hydroxypimelyl-CoA. In this work, two benzoate-induced genes from the obligately anaerobic bacteria, Geobacter metallireducens (bamA(Geo)) and Syntrophus aciditrophicus (bamA(Syn)), were heterologously expressed in Escherichia coli, purified and characterized as 6-OCH-CoA hydrolases. Both enzymes consisted of a single 43 kDa subunit. Some properties of the enzymes are presented and compared with homologues from facultative anaerobes. An alignment of the nucleotide sequences of bamA(Geo) and bamA(Syn) with the corresponding genes from facultative anaerobes identified highly conserved DNA regions, which enabled the discrimination of genes coding for 6-OCH-CoA hydrolases from those coding for related enzymes. A degenerate oligonucleotide primer pair was deduced from conserved regions and applied in polymerase chain reaction reactions. Using these primers, the expected DNA fragment of the 6-OCH-CoA hydrolase genes was specifically amplified from the DNA of nearly all known facultative and obligate anaerobes that use aromatic growth substrates. The only exception was the aromatic compound-degrading Rhodopseudomonas palustris, which uniquely uses a modified benzoyl-CoA degradation pathway. Using the oligonucleotide primers, the expected DNA fragment was also amplified in a toluene-degrading and a m

  19. Sex-related difference in the inductions by perfluoro-octanoic acid of peroxisomal beta-oxidation, microsomal 1-acylglycerophosphocholine acyltransferase and cytosolic long-chain acyl-CoA hydrolase in rat liver.

    Science.gov (United States)

    Kawashima, Y; Uy-Yu, N; Kozuka, H

    1989-01-01

    Inductions by perfluoro-octanoic acid (PFOA) of hepatomegaly, peroxisomal beta-oxidation, microsomal 1-acylglycerophosphocholine acyltransferase and cytosolic long-chain acyl-CoA hydrolase were compared in liver between male and female rats. Marked inductions of these four parameters were seen concurrently in liver of male rats, whereas the inductions in liver of female rats were far less pronounced. The sex-related difference in the response of rat liver to PFOA was much more marked than that seen with p-chlorophenoxyisobutyric acid (clofibric acid) or 2,2'-(decamethylenedithio)diethanol (tiadenol). Hormonal manipulations revealed that this sex-related difference in the inductions is strongly dependent on sex hormones, namely that testosterone is necessary for the inductions, whereas oestradiol prevented the inductions by PFOA. PMID:2570571

  20. In Vivo Sub-chronic Treatment with Dichlorvos in Young Rats Promotes Synaptic Plasticity and Learning by a Mechanism that Involves Acylpeptide Hydrolase Instead of Acetylcholinesterase Inhibition. Correlation with Endogenous β-Amyloid Levels

    Directory of Open Access Journals (Sweden)

    Gonzalo García-Rojo

    2017-07-01

    Full Text Available Acylpeptide hydrolase (APEH is a serine hydrolase that displays two catalytic activities, acting both as an exopeptidase toward short N-acylated peptides and as an endopeptidase toward oxidized peptides or proteins. It has been demonstrated that this enzyme can degrade monomers, dimers, and trimers of the Aβ1-40 peptide in the conditioned media of neuroblastoma cells. In a previous report, we showed that the specific inhibition of this enzyme by the organophosphate molecule dichlorvos (DDVP triggers an enhancement of long-term potentiation in rat hippocampal slices. In this study, we demonstrate that the same effect can be accomplished in vivo by sub-chronic treatment of young rats with a low dose of DDVP (0.1 mg/kg. Besides exhibiting a significant enhancement of LTP, the treated animals also showed improvements in parameters of spatial learning and memory. Interestingly, higher doses of DDVP such as 2 mg/kg did not prove to be beneficial for synaptic plasticity or behavior. Due to the fact that at 2 mg/kg we observed inhibition of both APEH and acetylcholinesterase, we interpret that in order to achieve positive effects on the measured parameters only APEH inhibition should be obtained. The treatment with both DDVP doses produced an increase in the endogenous concentration of Aβ1-40, although this was statistically significant only at the dose of 0.1 mg/kg. We propose that APEH represents an interesting pharmacological target for cognitive enhancement, acting through the modulation of the endogenous concentration of Aβ1-40.

  1. Cdx2 modulates proliferation in normal human intestinal epithelial crypt cells

    International Nuclear Information System (INIS)

    Escaffit, Fabrice; Pare, Frederic; Gauthier, Remy; Rivard, Nathalie; Boudreau, Francois; Beaulieu, Jean-Francois

    2006-01-01

    The homeobox gene Cdx2 is involved in the regulation of the expression of intestine specific markers such as sucrase-isomaltase and lactase-phlorizin hydrolase. Previous studies performed with immortalized or transformed intestinal cell lines have provided evidence that Cdx2 can promote morphological and functional differentiation in these experimental models. However, no data exist concerning the implication of this factor in normal human intestinal cell physiology. In the present work, we have investigated the role of Cdx2 in normal human intestinal epithelial crypt (HIEC) cells that lack this transcription factor. The establishment of HIEC cells expressing Cdx2 in an inducible manner shows that forced expression of Cdx2 significantly alters the proliferation of intestinal crypt cells and stimulates dipeptidylpeptidase IV expression but is not sufficient to trigger intestinal terminal differentiation. These observations suggest that Cdx2 requires additional factors to activate the enterocyte differentiation program in normal undifferentiated cells

  2. Inhibition of recombinant human carboxylesterase 1 and 2 and monoacylglycerol lipase by chlorpyrifos oxon, paraoxon and methyl paraoxon

    International Nuclear Information System (INIS)

    Crow, J. Allen; Bittles, Victoria; Herring, Katye L.; Borazjani, Abdolsamad; Potter, Philip M.; Ross, Matthew K.

    2012-01-01

    Oxons are the bioactivated metabolites of organophosphorus insecticides formed via cytochrome P450 monooxygenase-catalyzed desulfuration of the parent compound. Oxons react covalently with the active site serine residue of serine hydrolases, thereby inactivating the enzyme. A number of serine hydrolases other than acetylcholinesterase, the canonical target of oxons, have been reported to react with and be inhibited by oxons. These off-target serine hydrolases include carboxylesterase 1 (CES1), CES2, and monoacylglycerol lipase. Carboxylesterases (CES, EC 3.1.1.1) metabolize a number of xenobiotic and endobiotic compounds containing ester, amide, and thioester bonds and are important in the metabolism of many pharmaceuticals. Monoglyceride lipase (MGL, EC 3.1.1.23) hydrolyzes monoglycerides including the endocannabinoid, 2-arachidonoylglycerol (2-AG). The physiological consequences and toxicity related to the inhibition of off-target serine hydrolases by oxons due to chronic, low level environmental exposures are poorly understood. Here, we determined the potency of inhibition (IC 50 values; 15 min preincubation, enzyme and inhibitor) of recombinant CES1, CES2, and MGL by chlorpyrifos oxon, paraoxon and methyl paraoxon. The order of potency for these three oxons with CES1, CES2, and MGL was chlorpyrifos oxon > paraoxon > methyl paraoxon, although the difference in potency for chlorpyrifos oxon with CES1 and CES2 did not reach statistical significance. We also determined the bimolecular rate constants (k inact /K I ) for the covalent reaction of chlorpyrifos oxon, paraoxon and methyl paraoxon with CES1 and CES2. Consistent with the results for the IC 50 values, the order of reactivity for each of the three oxons with CES1 and CES2 was chlorpyrifos oxon > paraoxon > methyl paraoxon. The bimolecular rate constant for the reaction of chlorpyrifos oxon with MGL was also determined and was less than the values determined for chlorpyrifos oxon with CES1 and CES2

  3. Inhibition of recombinant human carboxylesterase 1 and 2 and monoacylglycerol lipase by chlorpyrifos oxon, paraoxon and methyl paraoxon

    Energy Technology Data Exchange (ETDEWEB)

    Crow, J. Allen; Bittles, Victoria; Herring, Katye L.; Borazjani, Abdolsamad [Center for Environmental Health Sciences, Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS 39762 (United States); Potter, Philip M. [Department of Chemical Biology and Therapeutics, St. Jude Children' s Research Hospital, 332 N. Lauderdale, Memphis, TN 38105 (United States); Ross, Matthew K., E-mail: mross@cvm.msstate.edu [Center for Environmental Health Sciences, Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS 39762 (United States)

    2012-01-01

    Oxons are the bioactivated metabolites of organophosphorus insecticides formed via cytochrome P450 monooxygenase-catalyzed desulfuration of the parent compound. Oxons react covalently with the active site serine residue of serine hydrolases, thereby inactivating the enzyme. A number of serine hydrolases other than acetylcholinesterase, the canonical target of oxons, have been reported to react with and be inhibited by oxons. These off-target serine hydrolases include carboxylesterase 1 (CES1), CES2, and monoacylglycerol lipase. Carboxylesterases (CES, EC 3.1.1.1) metabolize a number of xenobiotic and endobiotic compounds containing ester, amide, and thioester bonds and are important in the metabolism of many pharmaceuticals. Monoglyceride lipase (MGL, EC 3.1.1.23) hydrolyzes monoglycerides including the endocannabinoid, 2-arachidonoylglycerol (2-AG). The physiological consequences and toxicity related to the inhibition of off-target serine hydrolases by oxons due to chronic, low level environmental exposures are poorly understood. Here, we determined the potency of inhibition (IC{sub 50} values; 15 min preincubation, enzyme and inhibitor) of recombinant CES1, CES2, and MGL by chlorpyrifos oxon, paraoxon and methyl paraoxon. The order of potency for these three oxons with CES1, CES2, and MGL was chlorpyrifos oxon > paraoxon > methyl paraoxon, although the difference in potency for chlorpyrifos oxon with CES1 and CES2 did not reach statistical significance. We also determined the bimolecular rate constants (k{sub inact}/K{sub I}) for the covalent reaction of chlorpyrifos oxon, paraoxon and methyl paraoxon with CES1 and CES2. Consistent with the results for the IC{sub 50} values, the order of reactivity for each of the three oxons with CES1 and CES2 was chlorpyrifos oxon > paraoxon > methyl paraoxon. The bimolecular rate constant for the reaction of chlorpyrifos oxon with MGL was also determined and was less than the values determined for chlorpyrifos oxon with CES1

  4. More Human than Human.

    Science.gov (United States)

    Lawrence, David

    2017-07-01

    Within the literature surrounding nonhuman animals on the one hand and cognitively disabled humans on the other, there is much discussion of where beings that do not satisfy the criteria for personhood fit in our moral deliberations. In the future, we may face a different but related problem: that we might create (or cause the creation of) beings that not only satisfy but exceed these criteria. The question becomes whether these are minimal criteria, or hierarchical, such that those who fulfill them to greater degree should be afforded greater consideration. This article questions the validity and necessity of drawing divisions among beings that satisfy the minimum requirements for personhood; considering how future beings-intelligent androids, synthezoids, even alternate-substrate sentiences-might fit alongside the "baseline" human. I ask whether these alternate beings ought to be considered different to us, and why this may or may not matter in terms of a notion of "human community." The film Blade Runner, concerned in large part with humanity and its key synthezoid antagonist Roy Batty, forms a framing touchstone for my discussion. Batty is stronger, faster, more resilient, and more intelligent than Homo sapiens. His exploits, far beyond the capability of normal humans, are contrasted with his frailty and transient lifespan, his aesthetic appreciation of the sights he has seen, and his burgeoning empathy. Not for nothing does his creator within the mythos term him "more human than human."

  5. Stereo-selectivity and regio-selectivity in the metabolism of 7,8-dihydrobenzo[a]pyrene by cytochrome P450, epoxide hydrolase and hepatic microsomes from 3-methylcholanthrene-treated rats.

    Science.gov (United States)

    Adams, J D; Yagi, H; Levin, W; Jerina, D M

    1995-03-30

    The active site of cytochrome P450 1A1 has been probed with the substrate 7,8-dihydrobenzo[a]pyrene using a purified, reconstituted system composed of cytochrome P450 1A1, NADPH-cytochrome c reductase and lipid in the presence or absence of epoxide hydrolase. The turnover of the substrate was found to be 38 nmol/nmol of cytochrome P450/min. The metabolic products that were identified are: a phenolic 7,8-dihydrobenzo[a]pyrene (20-29%); 9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (17-28%); benzo[a]pyrene (12-19%); 7-hydroxy-7,8-dihydrobenzo[a]pyrene (13-16%); 8-hydroxy-7,8-dihydrobenzo[a]pyrene (7-15%); 3-hydroxybenzo[a]pyrene (7-15%); 4,5-epoxy-4,5,7,8-tetrahydrobenzo[a]pyrene (0-4%); and a triol of 7,8,9,10-tetrahydrobenzo[a]pyrene (0-4%). 9,10-Epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene undergoes rapid hydrolysis to cis- and trans-9,10-dihydroxy-dihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (2:1) by benzylic attack of water at C-10. Approximately 71% of the trans diols are derived from (+)-(9S,10R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, indicating that cytochrome P450 1A1 has more than a 2:1 preference for selective epoxidation of an enantiotopic face of 7,8-dihydrobenzo[a]pyrene. This stereo-selectivity agrees with the postulated stereo-selectivity predicted by a previously described active site model for cytochrome P450 1A1. Epoxide hydrolase in pure form or in hepatic microsomes catalyzes the hydrolysis of 9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, which is inhibited by 1,1,1-trichloropropane 2,3-oxide. The (+)-(9S,10R)-isomer of the epoxide is slightly preferred as a substrate over its enantiomer and is cleaved by benzylic and n