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Sample records for a4 hydrolase inhibitors

  1. Peptidyl-urea based inhibitors of soluble epoxide hydrolases

    We prepared a series of amino acid derived cyclohexyl and adamantyl ureas and tested them as inhibitors of the human soluble epoxide hydrolase, and obtained very potent compounds (K(I)=15nM) that are >10-fold more soluble than previously described sEH inhibitors. While our lead compound 2 showed low...

  2. Potent Urea and Carbamate Inhibitors of Soluble Epoxide Hydrolases

    Morisseau, Christophe; Goodrow, Marvin H.; Dowdy, Deanna; Zheng, Jiang; Greene, Jessica F.; Sanborn, James R.; Hammock, Bruce D.

    1999-08-01

    The soluble epoxide hydrolase (sEH) plays a significant role in the biosynthesis of inflammation mediators as well as xenobiotic transformations. Herein, we report the discovery of substituted ureas and carbamates as potent inhibitors of sEH. Some of these selective, competitive tightbinding inhibitors with nanomolar Ki values interacted stoichiometrically with the homogenous recombinant murine and human sEHs. These inhibitors enhance cytotoxicity of trans-stilbene oxide, which is active as the epoxide, but reduce cytotoxicity of leukotoxin, which is activated by epoxide hydrolase to its toxic diol. They also reduce toxicity of leukotoxin in vivo in mice and prevent symptoms suggestive of acute respiratory distress syndrome. These potent inhibitors may be valuable tools for testing hypotheses of involvement of diol and epoxide lipids in chemical mediation in vitro or in vivo systems.

  3. Effect of Bile Salt Hydrolase Inhibitors on a Bile Salt Hydrolase from Lactobacillus acidophilus

    Jun Lin

    2014-12-01

    Full Text Available Bile salt hydrolase (BSH, a widely distributed function of the gut microbiota, has a profound impact on host lipid metabolism and energy harvest. Recent studies suggest that BSH inhibitors are promising alternatives to antibiotic growth promoters (AGP for enhanced animal growth performance and food safety. Using a high-purity BSH from Lactobacillus salivarius strain, we have identified a panel of BSH inhibitors. However, it is still unknown if these inhibitors also effectively inhibit the function of the BSH enzymes from other bacterial species with different sequence and substrate spectrum. In this study, we performed bioinformatics analysis and determined the inhibitory effect of identified BSH inhibitors on a BSH from L. acidophilus. Although the L. acidophilus BSH is phylogenetically distant from the L. salivarius BSH, sequence analysis and structure modeling indicated the two BSH enzymes contain conserved, catalytically important amino residues and domain. His-tagged recombinant BSH from L. acidophilus was further purified and used to determine inhibitory effect of specific compounds. Previously identified BSH inhibitors also exhibited potent inhibitory effects on the L. acidophilus BSH. In conclusion, this study demonstrated that the BSH from L. salivarius is an ideal candidate for screening BSH inhibitors, the promising alternatives to AGP for enhanced feed efficiency, growth performance and profitability of food animals.

  4. Sulfonyl fluoride inhibitors of fatty acid amide hydrolase.

    Alapafuja, Shakiru O; Nikas, Spyros P; Bharathan, Indu T; Shukla, Vidyanand G; Nasr, Mahmoud L; Bowman, Anna L; Zvonok, Nikolai; Li, Jing; Shi, Xiaomeng; Engen, John R; Makriyannis, Alexandros

    2012-11-26

    Sulfonyl fluorides are known to inhibit esterases. Early work from our laboratory has identified hexadecyl sulfonylfluoride (AM374) as a potent in vitro and in vivo inhibitor of fatty acid amide hydrolase (FAAH). We now report on later generation sulfonyl fluoride analogs that exhibit potent and selective inhibition of FAAH. Using recombinant rat and human FAAH, we show that 5-(4-hydroxyphenyl)pentanesulfonyl fluoride (AM3506) has similar inhibitory activity for both the rat and the human enzyme, while rapid dilution assays and mass spectrometry analysis suggest that the compound is a covalent modifier for FAAH and inhibits its action in an irreversible manner. Our SAR results are highlighted by molecular docking of key analogs. PMID:23083016

  5. Discovery of enantioselectivity of urea inhibitors of soluble epoxide hydrolase.

    Manickam, Manoj; Pillaiyar, Thanigaimalai; Boggu, PullaReddy; Venkateswararao, Eeda; Jalani, Hitesh B; Kim, Nam-Doo; Lee, Seul Ki; Jeon, Jang Su; Kim, Sang Kyum; Jung, Sang-Hun

    2016-07-19

    Soluble epoxide hydrolase (sEH) hydrolyzes epoxyeicosatrienoic acids (EETs) in the metabolic pathway of arachidonic acid and has been considered as an important therapeutic target for chronic diseases such as hypertension, diabetes and inflammation. Although many urea derivatives are known as sEH inhibitors, the enantioselectivity of the inhibitors is not highlighted in spite of the stereoselective hydrolysis of EETs by sEH. In an effort to explore the importance of enantioselectivity in the urea scaffold, a series of enantiomers with the stereocenter adjacent to the urea nitrogen atom were prepared. The selectivity of enantiomers of 1-(α-alkyl-α-phenylmethyl)-3-(3-phenylpropyl)ureas showed wide range differences up to 125 fold with the low IC50 value up to 13 nM. The S-configuration with planar phenyl and small alkyl groups at α-position is crucial for the activity and selectivity. However, restriction of the free rotation of two α-groups with indan-1-yl or 1,2,3,4-tetrahydronaphthalen-1-yl moiety abolishes the selectivity between the enantiomers, despite the increase in activity up to 13 nM. The hydrophilic group like sulfonamido group at para position of 3-phenylpropyl motif of 1-(α-alkyl-α-phenylmethyl-3-(3-phenylpropyl)urea improves the activity as well as enantiomeric selectivity. All these ureas are proved to be specific inhibitor of sEH without inhibition against mEH. PMID:27092411

  6. Discovery of triterpenoids as reversible inhibitors of α/β-hydrolase domain containing 12 (ABHD12.

    Teija Parkkari

    Full Text Available BACKGROUND: α/β-Hydrolase domain containing (ABHD12 is a recently discovered serine hydrolase that acts in vivo as a lysophospholipase for lysophosphatidylserine. Dysfunctional ABHD12 has been linked to the rare neurodegenerative disorder called PHARC (polyneuropathy, hearing loss, ataxia, retinosis pigmentosa, cataract. In vitro, ABHD12 has been implicated in the metabolism of the endocannabinoid 2-arachidonoylglycerol (2-AG. Further studies on ABHD12 function are hampered as no selective inhibitor have been identified to date. In contrast to the situation with the other endocannabinoid hydrolases, ABHD12 has remained a challenging target for inhibitor development as no crystal structures are available to facilitate drug design. METHODOLOGY/PRINCIPAL FINDINGS: Here we report the unexpected discovery that certain triterpene-based structures inhibit human ABHD12 hydrolase activity in a reversible manner, the best compounds showing submicromolar potency. Based on structure activity relationship (SAR data collected for 68 natural and synthetic triterpenoid structures, a pharmacophore model has been constructed. A pentacyclic triterpene backbone with carboxyl group at position 17, small hydrophobic substituent at the position 4, hydrogen bond donor or acceptor at position 3 accompanied with four axial methyl substituents was found crucial for ABHD12 inhibitor activity. Although the triterpenoids typically may have multiple protein targets, we witnessed unprecedented selectivity for ABHD12 among the metabolic serine hydrolases, as activity-based protein profiling of mouse brain membrane proteome indicated that the representative ABHD12 inhibitors did not inhibit other serine hydrolases, nor did they target cannabinoid receptors. CONCLUSIONS/SIGNIFICANCE: We have identified reversibly-acting triterpene-based inhibitors that show remarkable selectivity for ABHD12 over other metabolic serine hydrolases. Based on SAR data, we have constructed the first

  7. Discovery of Triterpenoids as Reversible Inhibitors of α/β-hydrolase Domain Containing 12 (ABHD12)

    Parkkari, Teija; Haavikko, Raisa; Laitinen, Tuomo; Navia-Paldanius, Dina; Rytilahti, Roosa; Vaara, Miia; Lehtonen, Marko; Alakurtti, Sami; Yli-Kauhaluoma, Jari; Nevalainen, Tapio; Savinainen, Juha R.; Laitinen, Jarmo T.

    2014-01-01

    Background α/β-hydrolase domain containing (ABHD)12 is a recently discovered serine hydrolase that acts in vivo as a lysophospholipase for lysophosphatidylserine. Dysfunctional ABHD12 has been linked to the rare neurodegenerative disorder called PHARC (polyneuropathy, hearing loss, ataxia, retinosis pigmentosa, cataract). In vitro, ABHD12 has been implicated in the metabolism of the endocannabinoid 2-arachidonoylglycerol (2-AG). Further studies on ABHD12 function are hampered as no selective inhibitor have been identified to date. In contrast to the situation with the other endocannabinoid hydrolases, ABHD12 has remained a challenging target for inhibitor development as no crystal structures are available to facilitate drug design. Methodology/Principal Findings Here we report the unexpected discovery that certain triterpene-based structures inhibit human ABHD12 hydrolase activity in a reversible manner, the best compounds showing submicromolar potency. Based on structure activity relationship (SAR) data collected for 68 natural and synthetic triterpenoid structures, a pharmacophore model has been constructed. A pentacyclic triterpene backbone with carboxyl group at position 17, small hydrophobic substituent at the position 4, hydrogen bond donor or acceptor at position 3 accompanied with four axial methyl substituents was found crucial for ABHD12 inhibitor activity. Although the triterpenoids typically may have multiple protein targets, we witnessed unprecedented selectivity for ABHD12 among the metabolic serine hydrolases, as activity-based protein profiling of mouse brain membrane proteome indicated that the representative ABHD12 inhibitors did not inhibit other serine hydrolases, nor did they target cannabinoid receptors. Conclusions/Significance We have identified reversibly-acting triterpene-based inhibitors that show remarkable selectivity for ABHD12 over other metabolic serine hydrolases. Based on SAR data, we have constructed the first pharmacophore

  8. Carbocyclic pyrimidine nucleosides as inhibitors of S-adenosylhomocysteine hydrolase.

    Mosley, Sylvester L; Bakke, Brian A; Sadler, Joshua M; Sunkara, Naresh K; Dorgan, Kathleen M; Zhou, Zhaohui Sunny; Seley-Radtke, Katherine L

    2006-12-01

    The design, synthesis, and unexpected inhibitory activity against S-adenosyl-homocysteine (SAH) hydrolase (SAHase, EC 3.3.1.1) for a series of truncated carbocyclic pyrimidine nucleoside analogues is presented. Of the four nucleosides obtained, 10 was found to be active with a Ki value of 5.0 microM against SAHase. PMID:16904326

  9. DEVELOPMENT OF METABOLICALLY STABLE INHIBITORS OF MAMMALIAN MICROSOMAL EPOXIDE HYDROLASE

    The microsomal epoxide hydrolase (mEH) plays a significant role in the metabolism of xenobiotics such as polyaromatic toxicants. Additionally, polymorphism studies have underlined a potential role of this enzyme in relation to a number of diseases, such as emphysema, spontaneous abortion, eclampsia ...

  10. In silico modeling of the molecular structure and binding of leukotriene A4 into leukotriene A4 hydrolase

    Paz, Paula B.; Vega-Hissi, Esteban G.; Estrada, Mario R.; Martinez, Juan C. Garro

    2012-01-01

    A combined molecular docking and molecular structure in silico analysis on the substrate and product of leukotriene A4 hydrolase (LTA4H) was performed. The molecular structures of the substrate leukotriene A4 (LTA4) and product leukotirene B4 (LTB4) were studied through Density Functional Theory (DFT) calculations at the B3LYP/6-31+G(d) level of theory in both, gas and condensed phases. The whole LTB4 molecule was divided into three fragments (hydrophobic tail, triene motif, and a polar acidi...

  11. In silico modeling of the molecular structure and binding of leukotriene A4 into leukotriene A4 hydrolase

    Paz, Paula B; Estrada, Mario R; Martinez, Juan C Garro

    2012-01-01

    A combined molecular docking and molecular structure in silico analysis on the substrate and product of leukotriene A4 hydrolase (LTA4H) was performed. The molecular structures of the substrate leukotriene A4 (LTA4) and product leukotirene B4 (LTB4) were studied through Density Functional Theory (DFT) calculations at the B3LYP/6-31+G(d) level of theory in both, gas and condensed phases. The whole LTB4 molecule was divided into three fragments (hydrophobic tail, triene motif, and a polar acidic group) which were subjected to a full conformational study employing the most stable conformations of them to build conformers of the complete molecule and geometry optimize further. LTA4 conformers structures were modeled from the LTB4 minimum energy conformers. Both, protonated and deprotonated species of LTA4 and LTB4, were analyzed according to pKa values founded in the literature. Finally, a binding model of LTA4 with LTA4 hydrolase is proposed according to docking results which show intermolecular interactions tha...

  12. Inhibitors of Fatty Acid Amide Hydrolase and Monoacylglycerol Lipase: New Targets for Future Antidepressants.

    Ogawa, Shintaro; Kunugi, Hiroshi

    2015-01-01

    Cannabis and analogs of Δ9-tetrahydrocannabinol have been used for therapeutic purposes, but their therapeutic use remains limited because of various adverse effects. Endogenous cannabinoids have been discovered, and dysregulation of endocannabinoid signaling is implicated in the pathophysiology of major depressive disorder (MDD). Recently, endocannabinoid hydrolytic enzymes such as fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) have become new therapeutic targets in the treatment of MDD. Several FAAH or MAGL inhibitors are reported to have no cannabimimetic side effects and, therefore, are new potential therapeutic options for patients with MDD who are resistant to first-line antidepressants (selective serotonin and serotonin-norepinephrine reuptake inhibitors). In this review, we focus on the possible relationships between MDD and the endocannabinoid system as well as the inhibitors' therapeutic potential. MAGL inhibitors may reduce inflammatory responses through activation of cannabinoid receptor type 2. In the hypothalamic-pituitary-adrenal axis, repeated FAAH inhibitor administration may be beneficial for reducing circulating glucocorticoid levels. Both FAAH and MAGL inhibitors may contribute to dopaminergic system regulation. Recently, several new inhibitors have been developed with strong potency and selectivity. FAAH inhibitor, MAGL inhibitor, or dual blocker use would be promising new treatments for MDD. Further pre-clinical studies and clinical trials using these inhibitors are warranted. PMID:26630956

  13. Synthesis and structure-activity relationship of piperidine-derived non-urea soluble epoxide hydrolase inhibitors

    Pecic, Stevan; Pakhomova, Svetlana; Newcomer, Marcia E.; Morisseau, Christophe; Hammock, Bruce D.; Zhu, Zhengxiang; Rinderspacher, Alison; Deng, Shi-Xian [UCD; (LSU); (Columbia)

    2013-09-27

    A series of potent amide non-urea inhibitors of soluble epoxide hydrolase (sEH) is disclosed. The inhibition of soluble epoxide hydrolase leads to elevated levels of epoxyeicosatrienoic acids (EETs), and thus inhibitors of sEH represent one of a novel approach to the development of vasodilatory and anti-inflammatory drugs. Structure–activities studies guided optimization of a lead compound, identified through high-throughput screening, gave rise to sub-nanomolar inhibitors of human sEH with stability in human liver microsomal assay suitable for preclinical development.

  14. Discovery of Potent Soluble Epoxide Hydrolase (sEH) Inhibitors by Pharmacophore-Based Virtual Screening.

    Waltenberger, Birgit; Garscha, Ulrike; Temml, Veronika; Liers, Josephine; Werz, Oliver; Schuster, Daniela; Stuppner, Hermann

    2016-04-25

    There is an increasing interest in the development of soluble epoxide hydrolase (sEH) inhibitors, which block the degradation of endogenous anti-inflammatory epoxyeicosatrienoic acids. Within this study, a set of pharmacophore models for sEH inhibitors was developed. The Specs database was virtually screened and a cell-free sEH activity assay was used for the biological investigation of virtual hits. In total, out of 48 tested compounds, 19 were sEH inhibitors with IC50 < 10 μM, representing a prospective true positive hit rate of 40%. Six of these compounds displayed IC50 values in the low nanomolar range. The most potent compound 21, a urea derivative, inhibited sEH with an IC50 = 4.2 nM. The applied approach also enabled the identification of diverse chemical scaffolds, e.g. the pyrimidinone derivative 29 (IC50 = 277 nM). The generated pharmacophore model set therefore represents a valuable tool for the selection of compounds for biological testing. PMID:26882208

  15. Anti-Ulcer Efficacy of Soluble Epoxide Hydrolase Inhibitor TPPU on Diclofenac-Induced Intestinal Ulcers.

    Goswami, Sumanta Kumar; Wan, Debin; Yang, Jun; Trindade da Silva, Carlos A; Morisseau, Christophe; Kodani, Sean D; Yang, Guang-Yu; Inceoglu, Bora; Hammock, Bruce D

    2016-06-01

    Proton pump inhibitors such as omeprazole (OME) reduce the severity of gastrointestinal (GI) ulcers induced by nonsteroidal anti-inflammatory drugs (NSAIDs) but can also increase the chance of dysbiosis. The aim of this study was to test the hypothesis that preventive use of a soluble epoxide hydrolase inhibitor (sEHI) such as TPPU can decrease NSAID-induced ulcers by increasing anti-inflammatory epoxyeicosatrienoic acids (EETs). Dose- [10, 30, and 100 mg/kg, by mouth (PO)] and time-dependent (6 and 18 hours) ulcerative effects of diclofenac sodium (DCF, an NSAID) were studied in the small intestine of Swiss Webster mice. Dose-dependent effects of TPPU (0.001-0.1 mg/kg per day for 7 days, in drinking water) were evaluated in DCF-induced intestinal toxicity and compared with OME (20 mg/kg, PO). In addition, the effect of treatment was studied on levels of Hb in blood, EETs in plasma, inflammatory markers such as myeloperoxidase (MPO) in intestinal tissue homogenates, and tissue necrosis factor-α (TNF-α) in serum. DCF dose dependently induced ulcers that were associated with both a significant (P NSAID-induced ulcers. PMID:26989141

  16. Urea and amide-based inhibitors of the juvenile hormone epoxide hydrolase of the tobacco hornworm (Manduca sexta: Sphingidae).

    Severson, Tonya F; Goodrow, Marvin H; Morisseau, Christophe; Dowdy, Deanna L; Hammock, Bruce D

    2002-12-01

    A new class of inhibitors of juvenile hormone epoxide hydrolase (JHEH) of Manduca sexta and further in vitro characterization of the enzyme are reported. The compounds are based on urea and amide pharmacophores that were previously demonstrated as effective inhibitors of mammalian soluble and microsomal epoxide hydrolases. The best inhibitors against JHEH activity so far within this class are N-[(Z)-9-octadecenyl]-N'-propylurea and N-hexadecyl-N'-propylurea, which inhibited hydrolysis of a surrogate substrate (t-DPPO) with an IC(50) around 90 nM. The importance of substitution number and type was investigated and results indicated that N, N'-disubstitution with asymmetric alkyl groups was favored. Potencies of pharmacophores decreased as follows: amide>urea>carbamate>carbodiimide>thiourea and thiocarbamate for N, N'-disubstituted compounds with symmetric substituents, and urea>amide>carbamate for compounds with asymmetric N, N'-substituents. JHEH hydrolyzes t-DPPO with a K(m) of 65.6 microM and a V(max) of 59 nmol min(-1) mg(-1) and has a substantially lower K(m) of 3.6 microM and higher V(max) of 322 nmol min(-1) mg(-1) for JH III. Although none of these compounds were potent inhibitors of hydrolysis of JH III by JHEH, they are the first leads toward inhibitors of JHEH that are not potentially subject to metabolism through epoxide degradation. PMID:12429126

  17. ADMINISTRATION OF A SUBSTITUTED ADAMANTLY-UREA INHIBITOR OF THE SOLUBLE EPOXIDE HYDROLASE PROTECTS THE KIDNEY FROM DAMAGE IN HYPERTENSIVE GOTO-KAKIZAKI RATS

    Hypertension and type II diabetes are co-morbid diseases that lead to the development of nephropathy. Soluble epoxide hydrolase (sEH) inhibitors are reported to provide protection from renal injury. We hypothesized that the sEH inhibitor 12-(3-adamantan-1-yl-ureido) dodecanoic acid (AUDA) protects ...

  18. Inhibitors of monoacylglycerol lipase, fatty-acid amide hydrolase and endocannabinoid transport differentially suppress capsaicin-induced behavioral sensitization through peripheral endocannabinoid mechanisms

    Spradley, Jessica M.; Guindon, Josée; Hohmann, Andrea G.

    2010-01-01

    Monoacylglycerol lipase (MGL) and fatty acid amide hydrolase (FAAH) degrade the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide (AEA), respectively. Pharmacological inhibition of these enzymes in the periphery may elucidate the role of endocannabinoids in controlling nociceptive transmission. We compared effects of the MGL inhibitor JZL184, the FAAH inhibitor URB597, and the endocannabinoid uptake inhibitor VDM11, administered locally in the paw, on behavioral hypersensitivities...

  19. Physical Nature of Fatty Acid Amide Hydrolase Interactions with Its Inhibitors: Testing a Simple Nonempirical Scoring Model.

    Giedroyć-Piasecka, Wiktoria; Dyguda-Kazimierowicz, Edyta; Beker, Wiktor; Mor, Marco; Lodola, Alessio; Sokalski, W Andrzej

    2014-12-26

    Fatty acid amide hydrolase (FAAH) is an enzyme responsible for the deactivating hydrolysis of fatty acid ethanolamide neuromodulators. FAAH inhibitors have gained considerable interest due to their possible application in the treatment of anxiety, inflammation, and pain. In the context of inhibitor design, the availability of reliable computational tools for predicting binding affinity is still a challenging task, and it is now well understood that empirical scoring functions have several limitations that in principle could be overcome by quantum mechanics. Herein, systematic ab initio analyses of FAAH interactions with a series of inhibitors belonging to the class of the N-alkylcarbamic acid aryl esters have been performed. In contrast to our earlier studies of other classes of enzyme-inhibitor complexes, reasonable correlation with experimental results required us to consider correlation effects along with electrostatic term. Therefore, the simplest comprehensive nonempirical model allowing for qualitative predictions of binding affinities for FAAH ligands consists of electrostatic multipole and second-order dispersion terms. Such a model has been validated against the relative stabilities of the benchmark S66 set of biomolecular complexes. As it does not involve parameters fitted to experimentally derived data, this model offers a unique opportunity for generally applicable inhibitor design and virtual screening. PMID:25420234

  20. Discovery of a Potent, Selective, and Efficacious Class of Reversible α-Ketoheterocycle Inhibitors of Fatty Acid Amide Hydrolase Effective as Analgesicsa

    Boger, Dale L.; Miyauchi, Hiroshi; Du, Wu; Hardouin, Christophe; Fecik, Robert A.; Cheng, Heng; Hwang, Inkyu; Hedrick, Michael P.; Leung, Donmienne; Acevedo, Orlando; Guimarães, Cristiano R. W.; Jorgensen, William L.; Cravatt, Benjamin F.

    2005-01-01

    Fatty acid amide hydrolase (FAAH) degrades neuromodulating fatty acid amides including anandamide (endogenous cannabinoid agonist) and oleamide (sleep-inducing lipid) at their sites of action and is intimately involved in their regulation. Herein we report the discovery of a potent, selective, and efficacious class of reversible FAAH inhibitors that produce analgesia in animal models validating a new therapeutic target for pain intervention. Key to the useful inhibitor discovery was the routi...

  1. The Soluble Epoxide Hydrolase Inhibitor AR9281 Decreases Blood Pressure, Ameliorates Renal Injury and Improves Vascular Function in Hypertension

    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.

  2. Potent natural soluble epoxide hydrolase inhibitors from Pentadiplandra brazzeana baillon: synthesis, quantification, and measurement of biological activities in vitro and in vivo.

    Seiya Kitamura

    Full Text Available We describe here three urea-based soluble epoxide hydrolase (sEH inhibitors from the root of the plant Pentadiplandra brazzeana. The concentration of these ureas in the root was quantified by LC-MS/MS, showing that 1, 3-bis (4-methoxybenzyl urea (MMU is the most abundant (42.3 μg/g dry root weight. All of the ureas were chemically synthesized, and their inhibitory activity toward recombinant human and recombinant rat sEH was measured. The most potent compound, MMU, showed an IC50 of 92 nM via fluorescent assay and a Ki of 54 nM via radioactivity-based assay on human sEH. MMU effectively reduced inflammatory pain in a rat nociceptive pain assay. These compounds are among the most potent sEH inhibitors derived from natural sources. Moreover, inhibition of sEH by these compounds may mechanistically explain some of the therapeutic effects of P. brazzeana.

  3. Structure-Based Inhibitor Design for Evaluation of a CYP3A4 Pharmacophore Model.

    Kaur, Parminder; Chamberlin, A Richard; Poulos, Thomas L; Sevrioukova, Irina F

    2016-05-12

    Human cytochrome P450 3A4 (CYP3A4) is a key xenobiotic-metabolizing enzyme that oxidizes and clears the majority of drugs. CYP3A4 inhibition may lead to drug-drug interactions, toxicity, and other adverse effects but, in some cases, could be beneficial and enhance therapeutic efficiency of coadministered pharmaceuticals that are metabolized by CYP3A4. On the basis of our investigations of analogs of ritonavir, a potent CYP3A4 inactivator and pharmacoenhancer, we have built a pharmacophore model for a CYP3A4-specific inhibitor. This study is the first attempt to test this model using a set of rationally designed compounds. The functional and structural data presented here agree well with the proposed pharmacophore. In particular, we confirmed the importance of a flexible backbone, the H-bond donor/acceptor moiety, and aromaticity of the side group analogous to Phe-2 of ritonavir and demonstrated the leading role of hydrophobic interactions at the sites adjacent to the heme and phenylalanine cluster in the ligand binding process. The X-ray structures of CYP3A4 bound to the rationally designed inhibitors provide deeper insights into the mechanism of the CYP3A4-ligand interaction. Most importantly, two of our compounds (15a and 15b) that are less complex than ritonavir have comparable submicromolar affinity and inhibitory potency for CYP3A4 and, thus, could serve as templates for synthesis of second generation inhibitors for further evaluation and optimization of the pharmacophore model. PMID:26371436

  4. Effects of soluble epoxide hydrolase inhibitor on the expression of fatty acid synthase in peripheral blood mononuclear cell in patients with acute coronary syndrome

    Zhao Xuan

    2013-01-01

    Full Text Available Abstract Background Researches have shown that soluble epoxide hydrolase inhibitors (sEHi can protect against the development of atherosclerosis. Simultaneously, emerging evidences have implicated the association between fatty acid synthase (FAS and acute coronary syndrome (ACS. We tested the hypothesis that sEHi could reduce the occurrence of ACS by regulating FAS. Methods Hospitalized ACS patients were selected as the ACS group (n = 65 while healthy normal subjects as the control group (n = 65. The blood levels of lipoproteins, fasting glucose, myocardial enzyme and high-sensitivity C-reactive protein (hs-CRP were measured within 24 hours after admission. The peripheral blood mononuclear cells (PBMCs were isolated and cultured. Trans-4-[4-(3-Adamantan-1-ylureidocyclohexyloxy] benzoic acid (t-AUCB, a kind of sEHi, was then added to cells in various concentrations (0, 10, 50, 100 μmol/L. The expression of FAS, interleukin-6 (IL-6 mRNA and protein was detected by real-time PCR or Western blot, respectively. Results (1 Compared with the control group, the serum concentration of hs-CRP in the ACS group was increased (PPPPP Conclusions sEH inhibition regulated FAS and inhibited inflammation in cultured PBMCs from ACS patients, a mechanism that might prevent rupture of atherosclerotic lesions and protect against development of ACS.

  5. Novel diadenosine polyphosphate analogs with oxymethylene bridges replacing oxygen in the polyphosphate chain. Potential substrates and/or inhibitors of Ap4A hydrolases

    Guranowski, A.; Starzyňska, E.; Pietrowska-Borek, M.; Rejman, Dominik; Blackburn, G. M.

    2009-01-01

    Roč. 276, č. 6 (2009), s. 1546-1553. ISSN 1742-464X Institutional research plan: CEZ:AV0Z40550506 Keywords : diadenosine polyphosphate * oxomethylene * Ap4A hydrolase Subject RIV: CC - Organic Chemistry Impact factor: 3.042, year: 2009

  6. Comparative studies to determine the selective inhibitors for P-glycoprotein and cytochrome P 4503A4

    Achira, Meguru; Ito, Kiyomi; Suzuki, Hiroshi(Department of Physics, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan); Sugiyama, Yuichi

    1999-01-01

    It has been suggested that cytochrome P450 3A4 (CYP3A4) and MDR1 P-glycoprotein (P-gp) act synergistically to limit the bioavailability of orally administered agents. In order to determine the relative role of these proteins, it is essential to identify a selective inhibitor for either P-gp or CYP3A4. In the present investigation, comparative studies were performed to examine the effect of inhibitors on the function of these proteins. The IC50 of P-gp function, determined by examining the inh...

  7. Advances in the Research of Human Soluble Epoxide Hydrolase Inhibitors%人类可溶性环氧化物水解酶抑制剂的研究进展

    黄少胥; 王勇; 龙亚秋

    2012-01-01

    高血压是现代人的常见疾病.虽然有很多不同机理的降压药在临床使用,但是由于个体的差异性,高血压的治疗越来越倾向于个体化治疗,因此降压药的使用也不仅仅局限于血压的降低.人类可溶性环氧化物水解酶抑制剂最大的优势在于其在降压的同时还具有显著的抗炎作用.详细地阐述了人类可溶性环氧化物水解酶抑制剂从早期的环氧结构类型到第三代脲类结构的发展过程和近期研究进展.%Hypertension is a common illness nowadays. Although a wide variety of antihypertensive agents are available in the market for the treatment of hypertension, the antihypertensive treatment tends to be individualized, and the treatment should exceed the limitation of lowering blood pressure only. Compared to other antihypertensive agents, the advantage of human soluble epoxide hydrolase inhibitors (HsEHIs) is that they can not only reduce the blood pressure, but also have remarkable anti-inflammatory effects. Herein, the development of human soluble epoxide hydrolase inhibitors is reviewed from early epoxide HsEHIs to the third generation of urea HsEHIs.

  8. Selective Inhibitors of Fibroblast Activation Protein (FAP) with a (4-Quinolinoyl)-glycyl-2-cyanopyrrolidine Scaffold.

    Jansen, Koen; Heirbaut, Leen; Cheng, Jonathan D; Joossens, Jurgen; Ryabtsova, Oxana; Cos, Paul; Maes, Louis; Lambeir, Anne-Marie; De Meester, Ingrid; Augustyns, Koen; Van der Veken, Pieter

    2013-05-01

    Fibroblast activation protein (FAP) is a serine protease that is generally accepted to play an important role in tumor growth and other diseases involving tissue remodeling. Currently there are no FAP inhibitors with reported selectivity toward both the closely related dipeptidyl peptidases (DPPs) and prolyl oligopeptidase (PREP). We present the discovery of a new class of FAP inhibitors with a N-(4-quinolinoyl)-Gly-(2-cyanopyrrolidine) scaffold. We have explored the effects of substituting the quinoline ring and varying the position of its sp(2) hybridized nitrogen atom. The most promising inhibitors combined low nanomolar FAP inhibition and high selectivity indices (>10(3)) with respect to both the DPPs and PREP. Preliminary experiments on a representative inhibitor demonstrate that plasma stability, kinetic solubility, and log D of this class of compounds can be expected to be satisfactory. PMID:24900696

  9. Comparative studies to determine the selective inhibitors for P-glycoprotein and cytochrome P4503A4.

    Achira, M; Suzuki, H; Ito, K; Sugiyama, Y

    1999-01-01

    It has been suggested that cytochrome P450 3A4 (CYP3A4) and MDR1 P-glycoprotein (P-gp) act synergistically to limit the bioavailability of orally administered agents. In order to determine the relative role of these proteins, it is essential to identify a selective inhibitor for either P-gp or CYP3A4. In the present investigation, comparative studies were performed to examine the effect of inhibitors on the function of these proteins. The IC50of P-gp function, determined by examining the inhibition of the transcellular transport of vinblastine across Caco-2 monolayers, was in the order PSC833 < ketoconazole, verapamil < N-(2(R)-hydroxy-1(S)-indanyl)-5-(2(S)-(1,1-dimethylethylaminocarbonyl)-4-(furo(2,3-b)pyridin-5-yl)methyl)piperazin-1-yl)-4(S)-hydroxy-2(R)-phenylmethylpentanamide (L-754,394). In contrast, the IC50of CYP3A4 function, determined by examining the inhibition of the metabolism of midazolam by intestinal and liver microsomes, was in the order L-754,384 < ketoconazole < PSC 833 and verapamil. The ratio of IC50for P-gp to that for CYP3A4 was more than 200 for L-754,394, 60 ~ 150 for ketoconazole, 1.5 for verapamil, and 0.05 for PSC 833. Collectively, it was demonstrated that PSC 833 and L-754,394 can be used as selective inhibitors of P-gp and CYP3A4, respectively. PMID:11741214

  10. Discovery of Nanomolar Dengue and West Nile Virus Protease Inhibitors Containing a 4-Benzyloxyphenylglycine Residue.

    Behnam, Mira A M; Graf, Dominik; Bartenschlager, Ralf; Zlotos, Darius P; Klein, Christian D

    2015-12-10

    The dengue virus (DENV) and West Nile Virus (WNV) NS2B-NS3 proteases are attractive targets for the development of dual-acting therapeutics against these arboviral pathogens. We present the synthesis and extensive biological evaluation of inhibitors that contain benzyl ethers of 4-hydroxyphenylglycine as non-natural peptidic building blocks synthesized via a copper-complex intermediate. A three-step optimization strategy, beginning with fragment growth of the C-terminal 4-hydroxyphenylglycine to the benzyloxy ether, followed by C- and N-terminal optimization, and finally fragment merging generated compounds with in vitro affinities in the low nanomolar range. The most promising derivative reached Ki values of 12 nM at the DENV-2 and 39 nM at the WNV proteases. Several of the newly discovered protease inhibitors yielded a significant reduction of dengue and West Nile virus titers in cell-based assays of virus replication, with an EC50 value of 3.4 μM at DENV-2 and 15.5 μM at WNV for the most active analogue. PMID:26562070

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

    Lee, Ju-Kyung; Kim, Keun-Cheol

    2013-09-01

    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. PMID:23933322

  12. Valpromide inhibits human epoxide hydrolase.

    Pacifici, G. M.; Franchi, M; Bencini, C; Rane, A

    1986-01-01

    The effect of antipileptic drug valpromide (VPM) on the activity of epoxide hydrolase was studied in human adult and foetal liver, kidneys, lungs, intestine and in placenta. The activity of the epoxide hydrolase was measured with both styrene oxide and benzo(a)pyrene-4,5-oxide as substrates. VPM inhibited the epoxide hydrolase obtained from all organs studied. The degree of inhibition was independent of the substrate used. A lowering of the epoxide hydrolase activity by 50% was observed when ...

  13. Inhibition of Chronic Pancreatitis and Murine Pancreatic Intraepithelial Neoplasia by a Dual Inhibitor of c-RAF and Soluble Epoxide Hydrolase in LSL-KrasG¹²D/Pdx-1-Cre Mice.

    Liao, Jie; Hwang, Sung Hee; Li, Haonan; Liu, Jun-Yan; Hammock, Bruce D; Yang, Guang-Yu

    2016-01-01

    Mutation of Kirsten rat sarcoma viral oncogene homolog (KRAS) and chronic pancreatitis are the most common pathogenic events involved in human pancreatic carcinogenesis. In the process of long-standing chronic inflammation, aberrant metabolites of arachidonic acid play a crucial role in promoting carcinogenesis, in which the soluble epoxide hydrolase (sEH), as a pro-inflammatory enzyme, generally inactivates anti-inflammatory epoxyeicosatrienoic acids (EETs). Herein, we determined the effect of our newly-synthesized novel compound trans-4-{4-[3-(4-chloro-3-trifluoromethyl-phenyl)-ureido]-cyclohexyloxy}-pyridine-2-carboxylic acid methylamide (t-CUPM), a dual inhibitor of sEH and RAF1 proto-oncogene serine/threonine kinase (c-RAF), on inhibiting the development of pancreatitis and pancreatic intraepithelial neoplasia (mPanIN) in LSL-Kras(G12D)/Pdx1-Cre mice. The results showed that t-CUPM significantly reduced the severity of chronic pancreatitis, as measured by the extent of acini loss, inflammatory cell infiltration and stromal fibrosis. The progression of low-grade mPanIN I to high-grade mPanIN II/III was significantly suppressed. Inhibition of mutant Kras-transmitted phosphorylation of mitogen-activated protein kinase's kinase/extracellular signal-regulated kinases was demonstrated in pancreatic tissues by western blots. Quantitative real-time polymerase chain reaction analysis revealed that t-CUPM treatment significantly reduced the levels of inflammatory cytokines including tumor necrosis facor-α, monocyte chemoattractant protein-1, as well as vascular adhesion molecule-1, and the levels of Sonic hedgehog and Gli transcription factor (Hedgehog pathway). Analysis of the eicosanoid profile revealed a significant increase of the EETs/dihydroxyeicosatrienoic acids ratio, which further confirmed sEH inhibition by t-CUPM. These results indicate that simultaneous inhibition of sEH and c-RAF by t-CUPM is important in preventing chronic pancreatitis and carcinogenesis

  14. Early postnatal treatment with soluble epoxide hydrolase inhibitor or 15-deoxy-Δ(12,14)-prostagandin J2 prevents prenatal dexamethasone and postnatal high saturated fat diet induced programmed hypertension in adult rat offspring.

    Lu, Pei-Chen; Sheen, Jiunn-Ming; Yu, Hong-Ren; Lin, Yu-Ju; Chen, Chih-Cheng; Tiao, Mao-Meng; Tsai, Ching-Chou; Huang, Li-Tung; Tain, You-Lin

    2016-07-01

    Prenatal dexamethasone (DEX) exposure, postnatal high-fat (HF) intake, and arachidonic acid pathway are closely related to hypertension. We tested whether a soluble epoxide hydrolase (SEH) inhibitor, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA) or 15-deoxy-Δ(12,14)-prostagandin J2 (15dPGJ2) therapy can rescue programmed hypertension in the DEX+HF two-hit model. Four groups of Sprague Dawley rats were studied: control, DEX+HF, AUDA, and 15dPGJ2. Dexamethasone (0.1mg/kg body weight) was intraperitoneally administered to pregnant rats from gestational day 16-22. Male offspring received high-fat diet (D12331, Research Diets) from weaning to 4 months of age. In AUDA group, mother rats received 25mg/L in drinking water during lactation. In the 15dPGJ2 group, male offspring received 15dPGJ2 1.5mg/kg BW by subcutaneous injection once daily for 1 week after birth. We found postnatal HF diet aggravated prenatal DEX-induced programmed hypertension, which was similarly prevented by early treatment with AUDA or 15dPGJ2. The beneficial effects of AUDA and 15d-PGJ2 therapy include inhibition of SEH, increases of renal angiotensin converting enzyme-2 (ACE2) and angiotensin II type 2 receptor (AT2R) protein levels, and restoration of nitric oxide bioavailability. Better understanding of the impact of arachidonic acid pathway in the two-hit model will help prevent programmed hypertension in children exposed to corticosteroids and postnatal HF intake. PMID:27210044

  15. The fatty acid amide hydrolase inhibitor URB597 exerts anti-inflammatory effects in hippocampus of aged rats and restores an age-related deficit in long-term potentiation

    Murphy Niamh

    2012-04-01

    Full Text Available Abstract Background Several factors contribute to the deterioration in synaptic plasticity which accompanies age and one of these is neuroinflammation. This is characterized by increased microglial activation associated with increased production of proinflammatory cytokines like interleukin-1β (IL-1β. In aged rats these neuroinflammatory changes are associated with a decreased ability of animals to sustain long-term potentiation (LTP in the dentate gyrus. Importantly, treatment of aged rats with agents which possess anti-inflammatory properties to decrease microglial activation, improves LTP. It is known that endocannabinoids, such as anandamide (AEA, have anti-inflammatory properties and therefore have the potential to decrease the age-related microglial activation. However, endocannabinoids are extremely labile and are hydrolyzed quickly after production. Here we investigated the possibility that inhibiting the degradation of endocannabinoids with the fatty acid amide hydrolase (FAAH inhibitor, URB597, could ameliorate age-related increases in microglial activation and the associated decrease in LTP. Methods Young and aged rats received subcutaneous injections of the FAAH inhibitor URB597 every second day and controls which received subcutaneous injections of 30% DMSO-saline every second day for 28 days. Long-term potentiation was recorded on day 28 and the animals were sacrificed. Brain tissue was analyzed for markers of microglial activation by PCR and for levels of endocannabinoids by liquid chromatography coupled to tandem mass spectrometry. Results The data indicate that expression of markers of microglial activation, MHCII, and CD68 mRNA, were increased in the hippocampus of aged, compared with young, rats and that these changes were associated with increased expression of the proinflammatory cytokines interleukin (IL-1β and tumor necrosis factor-α (TNFα which were attenuated by treatment with URB597. Coupled with these changes, we

  16. Evaluation of Ketoconazole and Its Alternative Clinical CYP3A4/5 Inhibitors as Inhibitors of Drug Transporters: The In Vitro Effects of Ketoconazole, Ritonavir, Clarithromycin, and Itraconazole on 13 Clinically-Relevant Drug Transporters.

    Vermeer, Lydia M M; Isringhausen, Caleb D; Ogilvie, Brian W; Buckley, David B

    2016-03-01

    Ketoconazole is a potent CYP3A4/5 inhibitor and, until recently, recommended by the Food and Drug Administration (FDA) and the European Medicines Agency as a strong CYP3A4/5 inhibitor in clinical drug-drug interaction (DDI) studies. Ketoconazole sporadically causes liver injury or adrenal insufficiency. Because of this, the FDA and European Medicines Agency recommended suspension of ketoconazole use in DDI studies in 2013. The FDA specifically recommended use of clarithromycin or itraconazole as alternative strong CYP3A4/5 inhibitors in clinical DDI studies, but many investigators have also used ritonavir as an alternative. Although the effects of these clinical CYP3A4/5 inhibitors on other CYPs are largely established, reports on the effects on the broad range of drug transporter activities are sparse. In this study, the inhibitory effects of ketoconazole, clarithromycin, ritonavir, and itraconazole (and its CYP3A4-inhibitory metabolites, hydroxy-, keto-, and N-desalkyl itraconazole) toward 13 drug transporters (OATP1B1, OATP1B3, OAT1, OAT3, OCT1, OCT2, MATE1, MATE2-K, P-gp, BCRP, MRP2, MRP3, and BSEP) were systematically assessed in transporter-expressing HEK-293 cell lines or membrane vesicles. In vitro findings were translated into clinical context with the basic static model approaches outlined by the FDA in its 2012 draft guidance on DDIs. The results indicate that, like ketoconazole, the alternative clinical CYP3A4/5 inhibitors ritonavir, clarithromycin, and itraconazole each have unique transporter inhibition profiles. None of the alternatives to ketoconazole provided a clean inhibition profile toward the 13 drug transporters evaluated. The results provide guidance for the selection of clinical CYP3A4/5 inhibitors when transporters are potentially involved in a victim drug's pharmacokinetics. PMID:26668209

  17. Discovery of a Highly Selective CYP3A4 Inhibitor Suitable for Reaction Phenotyping Studies and Differentiation of CYP3A4 and CYP3A5

    Li, Xiaohai; Song, Xinyi; Kamenecka, Theodore M.; Cameron, Michael D.

    2012-01-01

    Current molecular tools lack the ability to differentiate the activity of CYP3A4 and CYP3A5 in biological samples such as human liver microsomes. Kinetic experiments and the CYP3A4 crystal structure indicate that the active sites of both enzymes are large and flexible, and have more than one binding subsite within the active site. 1-(4-Imidazopyridinyl-7phenyl)-3-(4′-cyanobiphenyl) urea (SR-9186) was optimized through several rounds of structural refinement from an initial screening hit to ob...

  18. Inhibitors

    ... wrong place in the body. Immune Tolerance Induction (ITI) Therapy: The goal of ITI therapy is to stop the inhibitor reaction from ... body to accept clotting factor concentrate treatments. With ITI therapy, people receive large amounts of clotting factor ...

  19. Inhibition of Chronic Pancreatitis and Murine Pancreatic Intraepithelial Neoplasia by a Dual Inhibitor of c-RAF and Soluble Epoxide Hydrolase in LSL-KrasG12D/Pdx-1-Cre Mice

    Liao, Jie; Hwang, Sung Hee; Li, Haonan; Liu, Jun-Yan; Hammock, Bruce D.; Yang, Guang-Yu

    2016-01-01

    Mutation of Kirsten rat sarcoma viral oncogene homolog (KRAS) and chronic pancreatitis are the most common pathogenic events involved in human pancreatic carcinogenesis. In the process of long-standing chronic inflammation, aberrant metabolites of arachidonic acid play a crucial role in promoting carcinogenesis, in which the soluble epoxide hydrolase (sEH), as a pro-inflammatory enzyme, generally inactivates anti-inflammatory epoxyeicosatrienoic acids (EETs). Herein, we determined the effect ...

  20. Prenatal serotonin reuptake inhibitor (SRI) antidepressant exposure and serotonin transporter promoter genotype (SLC6A4) influence executive functions at 6 years of age

    Whitney eWeikum; Ursula eBrain; Cecil MY Chau; Ruth Eckstein Grunau; W Thomas Boyce; Adele eDiamond; Oberlander, Tim F.

    2013-01-01

    Prenatal exposure to serotonin reuptake inhibitor (SRI) antidepressants and maternal depression may affect prefrontal cognitive skills (executive functions; EFs) including self-control, working memory and cognitive flexibility. We examined long-term effects of prenatal SRI exposure on EFs to determine whether effects are moderated by maternal mood and/or genetic variations in SLC6A4 (a gene that codes for the serotonin transporter [5-HTT] central to the regulation of synaptic serotonin levels...

  1. Effect of ketoconazole-mediated CYP3A4 inhibition on clinical pharmacokinetics of panobinostat (LBH589), an orally active histone deacetylase inhibitor

    Hamberg, Paul; Woo, Margaret M.; Chen, Lin-Chi; Verweij, Jaap; Porro, M.G.; Zhao, Ling; Li, Weili; van der Biessen, Diane; Sharma, Hari; Hengelage, Thomas; Jonge, Maja

    2011-01-01

    Abstract Purpose Panobinostat is partly metabolized by CYP3A4 in vitro. This study evaluated the effect of a potent CYP3A inhibitor, ketoconazole, on the pharmacokinetics and safety of panobinostat. Methods Patients received a single panobinostat oral dose on day 1, followed by 4 days wash-out period. On days 5?9, ketoconazole was administered. On day 8, a single panobinostat dose was co-administered with ...

  2. Trichostatin A, a histone deacetylase inhibitor stimulate CYP3A4 proximal promoter activity in Hepa-I cells.

    Ahn, Mee Ryung; Kim, Dae-Kee; Sheen, Yhun Yhong

    2004-04-01

    Cytochrome P450 3A4 (CYP3A4) is the most abundant CYPs in human liver, comprising approximately 30% of the total liver CYPs contents and is involved in the metabolism of more than 60% of currently used therapeutic drugs. However, the molecular mechanisms underlying regulation of CYP3A4 gene expression have not been understood. Thus, this study has been carried out to gain the insight of the molecular mechanism of CYP3A4 gene expression, investigating if the histone deacetylation is involved in the regulation of CYP3A4 gene expression by proximal promoter. Also SXR was investigated to see if they were involved in the regulation of CYP3A4 proximal promoter activity. Hepa-I cells were transfected with a plasmid containing approximately 1 kb of the human CYP3A4 proximal promoter region (863 to +64 bp) cloned in front of a reporter gene, luciferase, in the presence or absence of SXR. Transfected cells were treated with CYP3A4 inducers such as rifampicin, PCN and RU 486, in order to examine the regulation of CYP3A4 gene expression in the presence or absence of trichostatin A (TSA). In Hepa-I cells, CYP3A4 inducers increased modestly the luciferase activity when TSA was co-treated, but this increment was not enhanced by SXR cotransfection. Taken together, these results indicated that the inhibition of histone deacetylation was required to SXR-mediated increase in CYP3A4 proximal promoter region when rifampicin, or PCN was treated. Further a trans-activation by SXR may demand other species-specific transcription factors. PMID:15180307

  3. Polyglycine hydrolases secreted by pathogenic fungi

    Pathogens are known to produce proteases that target host defense proteins. Here we describe polyglycine hydrolases, fungal proteases that selectively cleave glycine-glycine peptide bonds within the polyglycine interdomain linker of targeted plant defense chitinases. Polyglycine hydrolases were puri...

  4. Hydrolase activity in Jerusalem artichoke and chicory

    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.

  5. Effect of ketoconazole-mediated CYP3A4 inhibition on clinical pharmacokinetics of panobinostat (LBH589), an orally active histone deacetylase inhibitor

    Hamberg, Paul; Woo, Margaret M.; Chen, Lin-Chi; Verweij, Jaap; Porro, M.G.; Zhao, Ling; Li, Weili; van de Biessen, D; Sharma, Hari; Hengelage, Thomas; Jonge, Maja

    2011-01-01

    textabstractPurpose: Panobinostat is partly metabolized by CYP3A4 in vitro. This study evaluated the effect of a potent CYP3A inhibitor, ketoconazole, on the pharmacokinetics and safety of panobinostat. Methods: Patients received a single panobinostat oral dose on day 1, followed by 4 days wash-out period. On days 5-9, ketoconazole was administered. On day 8, a single panobinostat dose was co-administered with ketoconazole. Panobinostat was administered as single agent three times a week on d...

  6. Metabolic Activation of Pradefovir by CYP3A4 and Its Potential as an Inhibitor or Inducer

    Lin, Chin-Chung; Fang, Che; Benetton, Salete; Xu, Gui-fen; Yeh, Li-Tain

    2006-01-01

    Metabolic activation of pradefovir to 9-(2-phosphonylmethoxyethyl)adenine (PMEA) was evaluated by using cDNA-expressed CYP isozymes in portal vein-cannulated rats following oral administration and in human liver microsomes. The enzyme induction potential of pradefovir was evaluated in rats following multiple oral dosing and in primary cultures of human hepatocytes. The results indicated that CYP3A4 is the only cDNA-expressed CYP isozyme catalyzing the conversion of pradefovir to PMEA. Pradefo...

  7. Content of CYP3A4 inhibitors, naringin, naringenin and bergapten in grapefruit and grapefruit juice products.

    Ho, P C; Saville, D J; Coville, P F; Wanwimolruk, S

    2000-04-01

    The flavonoids, naringin and naringenin and the furanocoumarin, bergapten (5-methoxypsoralen), were detected in some fresh grapefruit and commercial grapefruit juices but were not detected in other fruit juices tested (orange; orange with apple base; dark grape; orange and mango with apple base; orange, peach, passion fruit juice). The contents of these three grapefruit constituents in commercial juice and fresh grapefruit varied from brand to brand and also from lot to lot. Juice was prepared from the fresh fruit via different methods (by hand, squeezer or blender). The naringin content, after hand-squeeze, ranged from 115 to 384 mg/l. With hand-squeeze juice production, bergapten was not detected (less than 0.5 mg/l) in two varieties of grapefruit, and naringenin was usually not in detectable levels (less than 2 mg/l) in three varieties. All three constituents were present in New Zealand grapefruit preparations (including juice by hand-squeeze) and different lots showed variation in content (1.5-, 2.3- and 4.7-fold for naringin, naringenin and bergapten, respectively). Differences in the concentrations of these three constituents, which have potential for drug interaction, may contribute to the variability in pharmacokinetics of CYP3A4 drugs and some contradictory results of drug interaction studies with grapefruit juice. PMID:10812937

  8. Different effects of proton pump inhibitors and famotidine on the clopidogrel metabolic activation by recombinant CYP2B6, CYP2C19 and CYP3A4.

    Ohbuchi, Masato; Noguchi, Kiyoshi; Kawamura, Akio; Usui, Takashi

    2012-07-01

    Inhibitory potential of proton pump inhibitors (PPIs) and famotidine, an H(2) receptor antagonist, on the metabolic activation of clopidogrel was evaluated using recombinant CYP2B6, CYP2C19 and CYP3A4. Formation of the active metabolite from an intermediate metabolite, 2-oxo-clopidogrel, was investigated by liquid chromatography-tandem mass spectrometry and three peaks corresponding to the pharmacologically active metabolite and its stereoisomers were detected. Omeprazole potently inhibited clopidogrel activation by CYP2C19 with an IC(50) of 12.8 μmol/L and more weakly inhibited that by CYP2B6 and CYP3A4. IC(50) of omeprazole for CYP2C19 and CYP3A4 was decreased about two- and three-fold, respectively, by 30-min preincubation with NADPH. Lansoprazole, esomeprazole, pantoprazole, rabeprazole and rabeprazole thioether, a major metabolite, also inhibited metabolic activation by CYP2C19, with an IC(50) of 4.3, 8.9, 48.3, 36.2 and 30.5 μmol/L, respectively. In contrast, famotidine showed no more than 20% inhibition of clopidogrel activation by CYP2B6, CYP2C19 and CYP3A4 at up to 100 μmol/L and had no time-dependent CYP2C19 and CYP3A4 inhibition. These results provide direct evidence that PPIs inhibit clopidogrel metabolic activation and suggest that CYP2C19 inhibition is the main cause of drug-drug interaction between clopidogrel and omeprazole. Famotidine is considered as a safe anti-acid agent for patients taking clopidogrel. PMID:22313038

  9. Detoxification Strategy of Epoxide Hydrolase

    Arand, Michael; Cronin, Annette; Hengstler, Jan G.; Herrero Plana, Maria Elena; Lohmann, Matthias; Oesch, Franz

    2003-01-01

    The human microsomal epoxide hydrolase, a single enzyme, has to detoxify a broad range of structurally diverse, potentially genotoxic epoxides that are formed in the course of xenobiotic metabolism. The enzyme has developed a unique strategy to combine a broad substrate specificity with a high detoxification efficacy, by immediately trapping the reactive compounds as covalent intermediates and by being expressed at high levels for high trapping capacity. Computer simulation and experimental d...

  10. Modeling chemical interaction profiles: I. Spectral data-activity relationship and structure-activity relationship models for inhibitors and non-inhibitors of cytochrome P450 CYP3A4 and CYP2D6 isozymes.

    McPhail, Brooks; Tie, Yunfeng; Hong, Huixiao; Pearce, Bruce A; Schnackenberg, Laura K; Ge, Weigong; Valerio, Luis G; Fuscoe, James C; Tong, Weida; Buzatu, Dan A; Wilkes, Jon G; Fowler, Bruce A; Demchuk, Eugene; Beger, Richard D

    2012-01-01

    An interagency collaboration was established to model chemical interactions that may cause adverse health effects when an exposure to a mixture of chemicals occurs. Many of these chemicals--drugs, pesticides, and environmental pollutants--interact at the level of metabolic biotransformations mediated by cytochrome P450 (CYP) enzymes. In the present work, spectral data-activity relationship (SDAR) and structure-activity relationship (SAR) approaches were used to develop machine-learning classifiers of inhibitors and non-inhibitors of the CYP3A4 and CYP2D6 isozymes. The models were built upon 602 reference pharmaceutical compounds whose interactions have been deduced from clinical data, and 100 additional chemicals that were used to evaluate model performance in an external validation (EV) test. SDAR is an innovative modeling approach that relies on discriminant analysis applied to binned nuclear magnetic resonance (NMR) spectral descriptors. In the present work, both 1D ¹³C and 1D ¹⁵N-NMR spectra were used together in a novel implementation of the SDAR technique. It was found that increasing the binning size of 1D ¹³C-NMR and ¹⁵N-NMR spectra caused an increase in the tenfold cross-validation (CV) performance in terms of both the rate of correct classification and sensitivity. The results of SDAR modeling were verified using SAR. For SAR modeling, a decision forest approach involving from 6 to 17 Mold2 descriptors in a tree was used. Average rates of correct classification of SDAR and SAR models in a hundred CV tests were 60% and 61% for CYP3A4, and 62% and 70% for CYP2D6, respectively. The rates of correct classification of SDAR and SAR models in the EV test were 73% and 86% for CYP3A4, and 76% and 90% for CYP2D6, respectively. Thus, both SDAR and SAR methods demonstrated a comparable performance in modeling a large set of structurally diverse data. Based on unique NMR structural descriptors, the new SDAR modeling method complements the existing SAR

  11. Modeling Chemical Interaction Profiles: I. Spectral Data-Activity Relationship and Structure-Activity Relationship Models for Inhibitors and Non-inhibitors of Cytochrome P450 CYP3A4 and CYP2D6 Isozymes

    Richard D. Beger

    2012-03-01

    Full Text Available An interagency collaboration was established to model chemical interactions that may cause adverse health effects when an exposure to a mixture of chemicals occurs. Many of these chemicals—drugs, pesticides, and environmental pollutants—interact at the level of metabolic biotransformations mediated by cytochrome P450 (CYP enzymes. In the present work, spectral data-activity relationship (SDAR and structure-activity relationship (SAR approaches were used to develop machine-learning classifiers of inhibitors and non-inhibitors of the CYP3A4 and CYP2D6 isozymes. The models were built upon 602 reference pharmaceutical compounds whose interactions have been deduced from clinical data, and 100 additional chemicals that were used to evaluate model performance in an external validation (EV test. SDAR is an innovative modeling approach that relies on discriminant analysis applied to binned nuclear magnetic resonance (NMR spectral descriptors. In the present work, both 1D 13C and 1D 15N-NMR spectra were used together in a novel implementation of the SDAR technique. It was found that increasing the binning size of 1D 13C-NMR and 15N-NMR spectra caused an increase in the tenfold cross-validation (CV performance in terms of both the rate of correct classification and sensitivity. The results of SDAR modeling were verified using SAR. For SAR modeling, a decision forest approach involving from 6 to 17 Mold2 descriptors in a tree was used. Average rates of correct classification of SDAR and SAR models in a hundred CV tests were 60% and 61% for CYP3A4, and 62% and 70% for CYP2D6, respectively. The rates of correct classification of SDAR and SAR models in the EV test were 73% and 86% for CYP3A4, and 76% and 90% for CYP2D6, respectively. Thus, both SDAR and SAR methods demonstrated a comparable performance in modeling a large set of structurally diverse data. Based on unique NMR structural descriptors, the new SDAR modeling method complements the existing SAR

  12. Hierarchical classification of glycoside hydrolases.

    Naumoff, D G

    2011-06-01

    This review deals with structural and functional features of glycoside hydrolases, a widespread group of enzymes present in almost all living organisms. Their catalytic domains are grouped into 120 amino acid sequence-based families in the international classification of the carbohydrate-active enzymes (CAZy database). At a higher hierarchical level some of these families are combined in 14 clans. Enzymes of the same clan have common evolutionary origin of their genes and share the most important functional characteristics such as composition of the active center, anomeric configuration of cleaved glycosidic bonds, and molecular mechanism of the catalyzed reaction (either inverting, or retaining). There are now extensive data in the literature concerning the relationship between glycoside hydrolase families belonging to different clans and/or included in none of them, as well as information on phylogenetic protein relationship within particular families. Summarizing these data allows us to propose a multilevel hierarchical classification of glycoside hydrolases and their homologs. It is shown that almost the whole variety of the enzyme catalytic domains can be brought into six main folds, large groups of proteins having the same three-dimensional structure and the supposed common evolutionary origin. PMID:21639842

  13. Dynamically simulating the interaction of midazolam and the CYP3A4 inhibitor itraconazole using individual coupled whole-body physiologically-based pharmacokinetic (WB-PBPK models

    Jang In-Jin

    2007-03-01

    Full Text Available Abstract Background Drug-drug interactions resulting from the inhibition of an enzymatic process can have serious implications for clinical drug therapy. Quantification of the drugs internal exposure increase upon administration with an inhibitor requires understanding to avoid the drug reaching toxic thresholds. In this study, we aim to predict the effect of the CYP3A4 inhibitors, itraconazole (ITZ and its primary metabolite, hydroxyitraconazole (OH-ITZ on the pharmacokinetics of the anesthetic, midazolam (MDZ and its metabolites, 1' hydroxymidazolam (1OH-MDZ and 1' hydroxymidazolam glucuronide (1OH-MDZ-Glu using mechanistic whole body physiologically-based pharmacokinetic simulation models. The model is build on MDZ, 1OH-MDZ and 1OH-MDZ-Glu plasma concentration time data experimentally determined in 19 CYP3A5 genotyped adult male individuals, who received MDZ intravenously in a basal state. The model is then used to predict MDZ, 1OH-MDZ and 1OH-MDZ-Glu concentrations in an CYP3A-inhibited state following ITZ administration. Results For the basal state model, three linked WB-PBPK models (MDZ, 1OH-MDZ, 1OH-MDZ-Glu for each individual were elimination optimized that resulted in MDZ and metabolite plasma concentration time curves that matched individual observed clinical data. In vivo Km and Vmax optimized values for MDZ hydroxylation were similar to literature based in vitro measures. With the addition of the ITZ/OH-ITZ model to each individual coupled MDZ + metabolite model, the plasma concentration time curves were predicted to greatly increase the exposure of MDZ as well as to both increase exposure and significantly alter the plasma concentration time curves of the MDZ metabolites in comparison to the basal state curves. As compared to the observed clinical data, the inhibited state curves were generally well described although the simulated concentrations tended to exceed the experimental data between approximately 6 to 12 hours following

  14. Prenatal serotonin reuptake inhibitor (SRI antidepressant exposure and serotonin transporter promoter genotype (SLC6A4 influence executive functions at 6 years of age

    Whitney eWeikum

    2013-10-01

    Full Text Available Prenatal exposure to serotonin reuptake inhibitor (SRI antidepressants and maternal depression may affect prefrontal cognitive skills (executive functions; EFs including self-control, working memory and cognitive flexibility. We examined long-term effects of prenatal SRI exposure on EFs to determine whether effects are moderated by maternal mood and/or genetic variations in SLC6A4 (a gene that codes for the serotonin transporter [5-HTT] central to the regulation of synaptic serotonin levels and behavior. Children who were exposed to SRIs prenatally (SRI-exposed N=26 and non-exposed (N=38 were studied at age 6 years (M=6.3 SD=0.5 using the Hearts & Flowers task (H&F to assess EFs. Maternal mood was measured during pregnancy (3rd trimester and when the child was age 6 years (Hamilton Depression Scale. Parent reports of child behavior were also obtained (MacArthur Health & Behavior Questionnaire. Parents of prenatally SRI-exposed children reported fewer child externalizing and inattentive (ADHD behaviors. Generalized estimate equation modeling showed a significant 3-way interaction between prenatal SRI exposure, SLC6A4 variant, and maternal mood at the 6-year time-point on H&F accuracy. For prenatally SRI-exposed children, regardless of maternal mood, the H&F accuracy of children with reduced 5HTT expression (a short [S] allele remained stable. Even with increasing maternal depressive symptoms (though all below clinical threshold, EFs of children with at least one short allele were comparable to children with the same genotype whose mothers reported few if any depressive symptoms – in this sense they showed resilience. Children with two long (L alleles were more sensitive to context. When their mothers had few depressive symptoms, LL children showed extremely good EF performance – better than any other group. When their mothers reported more depressive symptoms, LL children’s EF performance was worse than that of any other group.

  15. Gene evolution of epoxide hydrolases and recommended nomenclature

    Beetham, J K; Grant, D; Arand, M; Garbarino, J; Kiyosue, T; Pinot, F; Oesch, F; Belknap, W R; Shinozaki, K.; Hammock, B. D.

    1995-01-01

    We have analyzed amino acid sequence relationships among soluble and microsomal epoxide hydrolases, haloacid dehalogenases, and a haloalkane dehalogenase. The amino-terminal residues (1-229) of mammalian soluble epoxide hydrolase are homologous to a haloacid dehalogenase. The carboxy-terminal residues (230-554) of mammalian soluble epoxide hydrolase are homologous to haloalkane dehalogenase, to plant soluble epoxide hydrolase, and to microsomal epoxide hydrolase. The shared identity between t...

  16. Modeling Chemical Interaction Profiles: II. Molecular Docking, Spectral Data-Activity Relationship, and Structure-Activity Relationship Models for Potent and Weak Inhibitors of Cytochrome P450 CYP3A4 Isozyme

    Eugene Demchuk

    2012-03-01

    Full Text Available Polypharmacy increasingly has become a topic of public health concern, particularly as the U.S. population ages. Drug labels often contain insufficient information to enable the clinician to safely use multiple drugs. Because many of the drugs are bio-transformed by cytochrome P450 (CYP enzymes, inhibition of CYP activity has long been associated with potentially adverse health effects. In an attempt to reduce the uncertainty pertaining to CYP-mediated drug-drug/chemical interactions, an interagency collaborative group developed a consensus approach to prioritizing information concerning CYP inhibition. The consensus involved computational molecular docking, spectral data-activity relationship (SDAR, and structure-activity relationship (SAR models that addressed the clinical potency of CYP inhibition. The models were built upon chemicals that were categorized as either potent or weak inhibitors of the CYP3A4 isozyme. The categorization was carried out using information from clinical trials because currently available in vitro high-throughput screening data were not fully representative of the in vivo potency of inhibition. During categorization it was found that compounds, which break the Lipinski rule of five by molecular weight, were about twice more likely to be inhibitors of CYP3A4 compared to those, which obey the rule. Similarly, among inhibitors that break the rule, potent inhibitors were 2–3 times more frequent. The molecular docking classification relied on logistic regression, by which the docking scores from different docking algorithms, CYP3A4 three-dimensional structures, and binding sites on them were combined in a unified probabilistic model. The SDAR models employed a multiple linear regression approach applied to binned 1D 13C-NMR and 1D 15N-NMR spectral descriptors. Structure-based and physical-chemical descriptors were used as the basis for developing SAR models by the decision forest method. Thirty-three potent inhibitors

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

    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.…

  18. The role of fatty acid amide hydrolase inhibition in nicotine reward and dependence

    Muldoon, Pretal P.; Lichtman, Aron H.; Parsons, Loren H.; Damaj, M Imad

    2012-01-01

    The endogenous cannabinoid anandamide (AEA) exerts the majority of its effects at CB1 and CB2 receptors and is degraded by fatty acid amide hydrolase (FAAH). FAAH KO mice and animals treated with FAAH inhibitors are impaired in their ability to hydrolyze AEA and other non-cannabinoid lipid signaling molecules, such as oleoylethanolamide (OEA) and palmitoylethanolamide (PEA). AEA and these other substrates activate non- cannabinoid receptor systems, including TRPV1 and PPAR-α receptors. In thi...

  19. Cellular viability effects of fatty acid amide hydrolase inhibition on cerebellar neurons

    Lueneberg Kathia; Domínguez Guadalupe; Arias-Carrión Oscar; Palomero-Rivero Marcela; Millán-Aldaco Diana; Morán. Julio; Drucker-Colín René; Murillo-Rodríguez Eric

    2011-01-01

    Abstract The endocannabinoid anandamide (ANA) participates in the control of cell death inducing the formation of apoptotic bodies and DNA fragmentation. The aim of this study was to evaluate whether the ANA degrading enzyme, the fatty acid amide hydrolase (FAAH), would induce cellular death. Experiments were performed in cerebellar granule neurons cultured with the FAAH inhibitor, URB597 (25, 50 or 100 nM) as well as endogenous lipids such as oleoylethanolamide (OEA) or palmitoylethanolamide...

  20. Hemodynamic profile, responsiveness to anandamide, and baroreflex sensitivity of mice lacking fatty acid amide hydrolase

    Pacher, Pál; Bátkai, Sándor; Osei-Hyiaman, Douglas; Offertáler, László; Liu, Jie; Harvey-White, Judy; Brassai, Attila; Járai, Zoltán; Cravatt, Benjamin F.; Kunos, George

    2005-01-01

    The endocannabinoid anandamide exerts neurobehavioral, cardiovascular, and immune-regulatory effects through cannabinoid receptors (CB). Fatty acid amide hydrolase (FAAH) is an enzyme responsible for the in vivo degradation of anandamide. Recent experimental studies have suggested that targeting the endocannabinergic system by FAAH inhibitors is a promising novel approach for the treatment of anxiety, inflammation, and hypertension. In this study, we compared the cardiac performance of FAAH k...

  1. Fatty acid amide hydrolase (FAAH) inhibition enhances memory acquisition through activation of PPAR-α nuclear receptors

    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 CB1-receptor ligand) and oleoylethanolamide and palmitoylethanolamide (OEA and PEA, ligands for α-type peroxisome proliferator-activated nuclear receptors, PPAR-α) when and where they are naturally released in the brain. Using a passive-avoidance task in rats, we found that memory acquisition was enhanced by the FAAH inhibitor URB597 or by the PPAR-α agonist WY14643, and these enhancements ...

  2. Discovery of a Novel Microsomal Epoxide Hydrolase-Catalyzed Hydration of a Spiro Oxetane.

    Li, Xue-Qing; Hayes, Martin A; Grönberg, Gunnar; Berggren, Kristina; Castagnoli, Neal; Weidolf, Lars

    2016-08-01

    Oxetane moieties are increasingly being used by the pharmaceutical industry as building blocks in drug candidates because of their pronounced ability to improve physicochemical parameters and metabolic stability of drug candidates. The enzymes that catalyze the biotransformation of the oxetane moiety are, however, not well studied. The in vitro metabolism of a spiro oxetane-containing compound AZD1979 [(3-(4-(2-oxa-6-azaspiro[3.3]heptan-6-ylmethyl)phenoxy)azetidin-1-yl)(5-(4-ethoxyphenyl)-1,3,4-oxadiazol-2-yl)methanone] was studied and one of its metabolites, M1, attracted our interest because its formation was NAD(P)H independent. The focus of this work was to elucidate the structure of M1 and to understand the mechanism(s) of its formation. We established that M1 was formed via hydration and ring opening of the oxetanyl moiety of AZD1979. Incubations of AZD1979 using various human liver subcellular fractions revealed that the hydration reaction leading to M1 occurred mainly in the microsomal fraction. The underlying mechanism as a hydration, rather than an oxidation reaction, was supported by the incorporation of (18)O from H2 (18)O into M1. Enzyme kinetics were performed probing the formation of M1 in human liver microsomes. The formation of M1 was substantially inhibited by progabide, a microsomal epoxide hydrolase inhibitor, but not by trans-4-[4-(1-adamantylcarbamoylamino)cyclohexyloxy]benzoic acid, a soluble epoxide hydrolase inhibitor. On the basis of these results, we propose that microsomal epoxide hydrolase catalyzes the formation of M1. The substrate specificity of microsomal epoxide hydrolase should therefore be expanded to include not only epoxides but also the oxetanyl ring system present in AZD1979. PMID:27256986

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

    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

  4. Optimization of a novel series of N-phenylindoline-5-sulfonamide-based acyl CoA:monoacylglycerol acyltransferase-2 inhibitors: Mitigation of CYP3A4 time-dependent inhibition and phototoxic liabilities.

    Sato, Kenjiro; Takahagi, Hiroki; Kubo, Osamu; Hidaka, Kousuke; Yoshikawa, Takeshi; Kamaura, Masahiro; Nakakariya, Masanori; Amano, Nobuyuki; Adachi, Ryutaro; Maki, Toshiyuki; Take, Kazumi; Takekawa, Shiro; Kitazaki, Tomoyuki; Maekawa, Tsuyoshi

    2015-08-01

    Acyl CoA:monoacylglycerol acyltransferase-2 (MGAT2) has emerged as a potential peripheral target for the treatment of obesity and metabolic disorders. We previously identified a novel series of N-phenylindoline-5-sulfonamide derivatives exemplified by 2 as potent and orally bioavailable MGAT2 inhibitors. Despite its attractive potency, further assessment revealed that this compound exhibited time-dependent inhibition (TDI) of cytochrome P450 3A4 (CYP3A4). To remove the undesirable CYP3A4 TDI activity, structural modification was focused on the 2,4-difluoroaniline moiety on the basis of the assumption that this moiety would be involved in mechanism-based inhibition of CYP3A4 via oxidative metabolism. This led to the finding that the introduction of 4-chloro-2,6-difluoroaniline significantly improved CYP3A4 TDI risk. Further optimization resulted in the discovery of N-(4-chloro-2,6-difluorophenyl)-1-{5-[1-methyl-3-(trifluoromethyl)-1H-pyrazol-5-yl]pyrimidin-2-yl}-7-(2-oxopyrrolidin-1-yl)-2,3-dihydro-1H-indole-5-sulfonamide (27c) with potent MGAT2 inhibitory activity (IC50=7.8 nM) and excellent ADME-Tox profiles including metabolic stability, oral bioavailability, and CYP3A4 TDI. In a mouse oral fat tolerance test, compound 27c effectively and dose-dependently suppressed the elevation of plasma triacylglycerol levels after oral administration at doses of 1 and 3mg/kg. We also discuss mitigation of the phototoxic liability of biaryl derivatives on the basis of the HOMO-LUMO gap hypothesis during the course of optimization efforts. PMID:26100443

  5. Aspergillus niger DLFCC-90 Rhamnoside Hydrolase, a New Type of Flavonoid Glycoside Hydrolase

    Liu, Tingqiang; Yu, Hongshan; Zhang, Chunzhi; Lu, Mingchun; Piao, Yongzhe; Ohba, Masashi; Tang, Minqian; Yuan, Xiaodong; Wei, Shenghua; Wang, Kan; Ma, Anzhou; Feng, Xue; Qin, Siqing; Mukai, Chisato; Tsuji, Akira

    2012-01-01

    A novel rutin-α-l-rhamnosidase hydrolyzing α-l-rhamnoside of rutin, naringin, and hesperidin was purified and characterized from Aspergillus niger DLFCC-90, and the gene encoding this enzyme, which is highly homologous to the α-amylase gene, was cloned and expressed in Pichia pastoris GS115. The novel enzyme was classified in glycoside-hydrolase (GH) family 13.

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

    Liu, Jun-Yan; 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 anti-microbial 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 mode...

  7. Re-characterization of mono-2-ethylhexyl phthalate hydrolase belonging to the serine hydrolase family.

    Iwata, Makoto; Imaoka, Takuya; Nishiyama, Takashi; Fujii, Takao

    2016-08-01

    A novel bacterium assimilating di-2-ethylhexyl phthalate as a sole carbon source was isolated, and identified as a Rhodococcus species and the strain was named EG-5. The strain has a mono-2-ethylhexyl phthalate (MEHP) hydrolase (EG-5 MehpH), which exhibits some different enzymatic features when compared with the previously reported MEHP hydrolase (P8219 MehpH) from Gordonia sp. These differences include different pH optimum activity, maximal reaction temperature and heat stability. The Km and Vmax values of EG-5 MehpH were significantly higher than those of P8219 MehpH. The primary structure of EG-5 MehpH showed the highest sequence identity to that of P8219 MehpH (39%) among hydrolases. The phylogenetic tree suggested that EG-5 MehpH and P8219 MehpH were categorized in different groups of the novel MEHP hydrolase family. Mutation of a conserved R(109) residue of EG-5 MehpH to a hydrophobic residue resulted in a dramatic reduction in the Vmax value towards MEHP without affecting the Km value. These results indicate that this residue may neutralize the negative charge of a carboxylate anion of MEHP, and thus inhibit the catalytic nucleophile from attacking the ester bond. In other words, the R residue blocks inhibition from the carboxylate anion of MEHP. Recently, registered hypothetical proteins exhibiting 98% or 99% identities for EG-5 MehpH or for P8219 MehpH were found from some pathogens belonging to Actinomycetes. The protein may have other activities besides MEHP hydrolysis and function in other physiological reactions in some Actinomycetes. PMID:26868518

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

    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 IC50 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

  9. Novel microbial epoxide hydrolases for biohydrolysis of glycidyl derivatives

    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

  10. Further characterization of intestinal lactase/phlorizin hydrolase

    Skovbjerg, H; Norén, O; Sjöström, H;

    1982-01-01

    enzyme were shown to have a considerable activity against cellotriose and cellotetraose, and a low but significant activity against cellulose. The lactase/phlorizin hydrolase isolated from pigs in which the pancreatic ducts had been disconnected 3 days before death and from Ca2+-precipitated enterocyte...... 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....

  11. Blood acylpeptide hydrolase activity is a sensitive marker for exposure to some organophosphate toxicants.

    Quistad, Gary B; Klintenberg, Rebecka; Casida, John E

    2005-08-01

    Acylpeptide hydrolase (APH) unblocks N-acetyl peptides. It is a major serine hydrolase in rat blood, brain, and liver detected by derivatization with (3)H-diisopropyl fluorophosphate (DFP) or a biotinylated fluorophosphonate. Although APH does not appear to be a primary target of acute poisoning by organophosphorus (OP) compounds, the inhibitor specificity of this secondary target is largely unknown. This study fills the gap and emphasizes blood APH as a potential marker of OP exposure. The most potent in vitro inhibitors for human erythrocyte and mouse brain APH are DFP (IC(50) 11-17 nM), chlorpyrifos oxon (IC(50) 21-71 nM), dichlorvos (IC(50) 230-560 nM), naled (IC(50) 370-870 nM), and their analogs with modified alkyl substituents. (3)H-diisopropyl fluorophosphate is a potent inhibitor of mouse blood and brain APH in vivo (ED(50) 0.09-0.2 mg/kg and 0.02-0.03 mg/l for ip and vapor exposure, respectively). Mouse blood and brain APH and blood butyrylcholinesterase (BChE) are of similar sensitivity to DFP in vitro and in vivo (ip and vapor exposure), but APH inhibition is much more persistent in vivo (still >80% inhibition after 4 days). The inhibitory potency of OP pesticides in vivo in mice varies from APH selective (dichlorvos, naled, and trichlorfon), to APH and BChE selective (profenofos and tribufos), to ChE selective or nonselective (many commercial insecticides). Sarin administered ip at a lethal dose to guinea pigs inhibits blood acetylcholinesterase and BChE completely but erythrocyte APH only partially. Blood APH activity is therefore a sensitive marker for exposure to some but not all OP pesticides and chemical warfare agents. PMID:15888665

  12. Molecular Cloning of a Novel cDNA From Mus Muscular BALB/c Mice Encoding Glycosyl Hydrolase Family 1: A Homolog of HumanLactase-Phlorizin Hydrolase

    WEI HE; ZHEN-YU JI; CHENG-YU HUANG

    2006-01-01

    Objective To study the mechanism of lactose intolerance (LI) by cloning the mouse lactase cDNA and recombining a vector. Methods Total murine RNA was isolated from the small intestine of a 4-week-old BALB/c mouse (♂).Gene-specific primers were designed and synthesized according to the cDNA sequences of lactase-phlorizin hydrolase (LPH) in human, rat, and rabbit. A coding sequence (CDS) fragment was obtained using RT-PCR, and inserted into a clone vector pNEB-193, then the cDNA was sequenced and analyzed using bioinformatics. Results The cDNA from the BALB/c mouse with 912 bp encoding 303 amino acid residues. Analysis of the deduced amino acid sequence using bioinformatics revealed that this cDNA shared extensive sequence homology with human LPH containing a conserved glycosy1 hydrolase family 1 motif important for regulating lactase intolerance. Conclusion BALB/c mouse LPH cDNA (GenBank accession No: AY751548) provides a necessary foundation for study of the biological function and regulatory mechanism of the lactose intolerance in mice.

  13. Pharmacological blockade of the fatty acid amide hydrolase (FAAH) alters neural proliferation, apoptosis and gliosis in the rat hippocampus, hypothalamus and striatum in a negative energy context

    Laura Bindila; Antoni Pastor; Margarita Pérez-Martín; Rafael De La Torre; Juan Suarez

    2015-01-01

    Endocannabinoids participate in the control of neurogenesis, neural cell death and gliosis. The pharmacological effect of the fatty acid amide hydrolase (FAAH) inhibitor URB597, which limits the endocannabinoid degradation, was investigated in the present study. Cell proliferation (phospho-H3+ or BrdU+ cells) of the main adult neurogenic zones as well as apoptosis (cleaved caspase-3+), astroglia (GFAP+), and microglia (Iba1+ cells) were analyzed in the hippocampus, hypothalamus and striatum o...

  14. Pharmacological blockade of the fatty acid amide hydrolase (FAAH) alters neural proliferation, apoptosis and gliosis in the rat hippocampus, hypothalamus and striatum in a negative energy context.

    Rivera, Patricia; Bindila, Laura; Pastor, Antoni; Pérez-Martín, Margarita; Pavón, Francisco J.; Serrano, Antonia; de la Torre, Rafael; Lutz, Beat; Rodríguez de Fonseca, Fernando; Suárez, Juan

    2015-01-01

    Endocannabinoids participate in the control of neurogenesis, neural cell death and gliosis. The pharmacological effect of the fatty acid amide hydrolase (FAAH) inhibitor URB597, which limits the endocannabinoid degradation, was investigated in the present study. Cell proliferation (phospho-H3(+) or BrdU(+) cells) of the main adult neurogenic zones as well as apoptosis (cleaved caspase-3(+)), astroglia (GFAP(+)), and microglia (Iba1(+) cells) were analyzed in the hippocampus, hypothalamus and ...

  15. AIG1 and ADTRP are atypical integral membrane hydrolases that degrade bioactive FAHFAs.

    Parsons, William H; Kolar, Matthew J; Kamat, Siddhesh S; Iii, Armand B Cognetta; Hulce, Jonathan J; Saez, Enrique; Kahn, Barbara B; Saghatelian, Alan; Cravatt, Benjamin F

    2016-05-01

    Enzyme classes may contain outlier members that share mechanistic, but not sequence or structural, relatedness with more common representatives. The functional annotation of such exceptional proteins can be challenging. Here, we use activity-based profiling to discover that the poorly characterized multipass transmembrane proteins AIG1 and ADTRP are atypical hydrolytic enzymes that depend on conserved threonine and histidine residues for catalysis. Both AIG1 and ADTRP hydrolyze bioactive fatty acid esters of hydroxy fatty acids (FAHFAs) but not other major classes of lipids. We identify multiple cell-active, covalent inhibitors of AIG1 and show that these agents block FAHFA hydrolysis in mammalian cells. These results indicate that AIG1 and ADTRP are founding members of an evolutionarily conserved class of transmembrane threonine hydrolases involved in bioactive lipid metabolism. More generally, our findings demonstrate how chemical proteomics can excavate potential cases of convergent or parallel protein evolution that defy conventional sequence- and structure-based predictions. PMID:27018888

  16. Engineered bacterial polyester hydrolases efficiently degrade polyethylene terephthalate due to relieved product inhibition.

    Wei, Ren; Oeser, Thorsten; Schmidt, Juliane; Meier, René; Barth, Markus; Then, Johannes; Zimmermann, Wolfgang

    2016-08-01

    Recent studies on the enzymatic degradation of synthetic polyesters have shown the potential of polyester hydrolases from thermophilic actinomycetes for modifying or degrading polyethylene terephthalate (PET). TfCut2 from Thermobifida fusca KW3 and LC-cutinase (LCC) isolated from a compost metagenome are remarkably active polyester hydrolases with high sequence and structural similarity. Both enzymes exhibit an exposed active site in a substrate binding groove located at the protein surface. By exchanging selected amino acid residues of TfCut2 involved in substrate binding with those present in LCC, enzyme variants with increased PET hydrolytic activity at 65°C were obtained. The highest activity in hydrolyzing PET films and fibers were detected with the single variant G62A and the double variant G62A/I213S. Both variants caused a weight loss of PET films of more than 42% after 50 h of hydrolysis, corresponding to a 2.7-fold increase compared to the wild type enzyme. Kinetic analysis based on the released PET hydrolysis products confirmed the superior hydrolytic activity of G62A with a fourfold higher hydrolysis rate constant and a 1.5-fold lower substrate binding constant than those of the wild type enzyme. Mono-(2-hydroxyethyl) terephthalate is a strong inhibitor of TfCut2. A determination of the Rosetta binding energy suggested a reduced interaction of G62A with 2PET, a dimer of the PET monomer ethylene terephthalate. Indeed, G62A revealed a 5.5-fold lower binding constant to the inhibitor than the wild type enzyme indicating that its increased PET hydrolysis activity is the result of a relieved product inhibition by mono-(2-hydroxyethyl) terephthalate. Biotechnol. Bioeng. 2016;113: 1658-1665. © 2016 Wiley Periodicals, Inc. PMID:26804057

  17. Sex-Specific Association of Depression and a Haplotype in Leukotriene A4 Hydrolase Gene

    Depression is genetically determined and inflammation has been implicated. Women are twice as likely to develop depression as men. Whether genetic variants involved in inflammation play a role in the sex difference in depression is unclear. We examined the association, separately in men and women, ...

  18. Diversity and biocatalytic potential of epoxide hydrolases identified by genome analysis

    van der Loo, B; Kingma, J.; Arand, M; Wubbolts, Marcel; Janssen, D B

    2006-01-01

    Epoxide hydrolases play an important role in the biodegradation of organic compounds and are potentially useful in enantioselective biocatalysis. An analysis of various genomic databases revealed that about 20% of sequenced organisms contain one or more putative epoxide hydrolase genes. They were found in all domains of life, and many fungi and actinobacteria contain several putative epoxide hydrolase-encoding genes. Multiple sequence alignments of epoxide hydrolases with other known and puta...

  19. Structure of the Ubiquitin Hydrolase UCH-L3 Complexed with a Suicide Substrate

    Misaghi, S.; Galardy, P.J.; Meester, W.J.; Ovaa, H.; Ploegh, H.L.; Gaudet, R. (Harvard)

    2009-03-24

    Ubiquitin C-terminal hydrolases (UCHs) comprise a family of small ubiquitin-specific proteases of uncertain function. Although no cellular substrates have been identified for UCHs, their highly tissue-specific expression patterns and the association of UCH-L1 mutations with human disease strongly suggest a critical role. The structure of the yeast UCH Yuh1-ubiquitin aldehyde complex identified an active site crossover loop predicted to limit the size of suitable substrates. We report the 1.45 {angstrom} resolution crystal structure of human UCH-L3 in complex with the inhibitor ubiquitin vinylmethylester, an inhibitor that forms a covalent adduct with the active site cysteine of ubiquitin-specific proteases. This structure confirms the predicted mechanism of the inhibitor and allows the direct comparison of a UCH family enzyme in the free and ligand-bound state. We also show the efficient hydrolysis by human UCH-L3 of a 13-residue peptide in isopeptide linkage with ubiquitin, consistent with considerable flexibility in UCH substrate size. We propose a model for the catalytic cycle of UCH family members which accounts for the hydrolysis of larger ubiquitin conjugates.

  20. Human Lung Hydrolases Delineate Mycobacterium tuberculosis–Macrophage Interactions and the Capacity To Control Infection

    Arcos, Jesus; Sasindran, Smitha J.; Fujiwara, Nagatoshi; Turner, Joanne; Schlesinger, Larry S; Torrelles, Jordi B.

    2011-01-01

    Pulmonary surfactant contains homeostatic and antimicrobial hydrolases. When Mycobacterium tuberculosis is initially deposited in the terminal bronchioles and alveoli, as well as following release from lysed macrophages, bacilli are in intimate contact with these lung surfactant hydrolases. We identified and measured several hydrolases in human alveolar lining fluid and lung tissue that, at their physiological concentrations, dramatically modified the M. tuberculosis cell envelope. Independen...

  1. Limonene-1,2-Epoxide Hydrolase from Rhodococcus erythropolis DCL14 Belongs to a Novel Class of Epoxide Hydrolases

    van der Werf, Mariët J.; Overkamp, Karin M; de Bont, Jan A. M.

    1998-01-01

    An epoxide hydrolase from Rhodococcus erythropolis DCL14 catalyzes the hydrolysis of limonene-1,2-epoxide to limonene-1,2-diol. The enzyme is induced when R. erythropolis is grown on monoterpenes, reflecting its role in the limonene degradation pathway of this microorganism. Limonene-1,2-epoxide hydrolase was purified to homogeneity. It is a monomeric cytoplasmic enzyme of 17 kDa, and its N-terminal amino acid sequence was determined. No cofactor was required for activity of this colorless en...

  2. Monoclonal Antibodies Specific for Hippurate Hydrolase of Campylobacter jejuni

    Steele, Marina; Gyles, Carlton; Chan, Voon Loong; Odumeru, Joseph

    2002-01-01

    Eleven monoclonal antibodies raised against recombinant Campylobacter jejuni hippurate hydrolase were tested for binding to lysates from 19 C. jejuni strains, 12 other Campylobacter strains, and 21 non-Campylobacter strains. Several monoclonal antibodies bound to C. jejuni but not to other Campylobacter species and may be useful in a species-specific immunoassay.

  3. Bile salt hydrolase of Bifidobacterium longum - Biochemical and genetic characterization

    Tanaka, H; Hashiba, Honoo; Kok, Jan; Mierau, Igor

    2000-01-01

    A bile salt hydrolase (BSH) was isolated from Bifidobacterium longum SBT2928, purified, and characterized, Furthermore, we describe for the first time cloning and analysis of the gene encoding BSII (bsh) in a member of the genus Bifidobacterium. The enzyme has a native molecular weight of 125,000 to

  4. Properties of epoxide hydrolase from the yeast Rhodotorula glutinis

    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 the juvenile hormone in some insec

  5. Curation of characterized glycoside hydrolases of fungal origin.

    Murphy, Caitlin; Powlowski, Justin; Wu, Min; Butler, Greg; Tsang, Adrian

    2011-01-01

    Fungi produce a wide range of extracellular enzymes to break down plant cell walls, which are composed mainly of cellulose, lignin and hemicellulose. Among them are the glycoside hydrolases (GH), the largest and most diverse family of enzymes active on these substrates. To facilitate research and development of enzymes for the conversion of cell-wall polysaccharides into fermentable sugars, we have manually curated a comprehensive set of characterized fungal glycoside hydrolases. Characterized glycoside hydrolases were retrieved from protein and enzyme databases, as well as literature repositories. A total of 453 characterized glycoside hydrolases have been cataloged. They come from 131 different fungal species, most of which belong to the phylum Ascomycota. These enzymes represent 46 different GH activities and cover 44 of the 115 CAZy GH families. In addition to enzyme source and enzyme family, available biochemical properties such as temperature and pH optima, specific activity, kinetic parameters and substrate specificities were recorded. To simplify comparative studies, enzyme and species abbreviations have been standardized, Gene Ontology terms assigned and reference to supporting evidence provided. The annotated genes have been organized in a searchable, online database called mycoCLAP (Characterized Lignocellulose-Active Proteins of fungal origin). It is anticipated that this manually curated collection of biochemically characterized fungal proteins will be used to enhance functional annotation of novel GH genes. Database URL: http://mycoCLAP.fungalgenomics.ca/. PMID:21622642

  6. Effect of leukocyte hydrolases on bacteria

    Leukocyte extracts, trypsin, and lysozyme are all capable of releasing the bulk of the LPS from S. typhi, S. typhimurium, and E. coli. Bacteria which have been killed by heat, ultraviolet irradiation, or by a variety of metabolic inhibitors and antibiotics which affect protein, DNA, RNA, and cell wall synthesis no longer yield soluble LPS following treatment with the releasing agents. On the other hand, bacteria which are resistant to certain of the antibiotics yield nearly the full amount of soluble LPS following treatment, suggesting that certain heatabile endogenous metabolic pathways collaborate with the releasing agents in the release of LPS from the bacteria. It is suggested that some of the beneficial effects of antibiotics on infections with gram-negative bacteria may be the prevention of massive release of endotoxin by leukocyte enzymes in inflammatory sites

  7. Rationale, design and baseline characteristics of a 4-year (208-week) phase III trial of empagliflozin, an SGLT2 inhibitor, versus glimepiride as add-on to metformin in patients with type 2 diabetes mellitus with insufficient glycemic control

    Ridderstråle, Martin; Svaerd, Robbyna; Zeller, Cordula; Kim, Gabriel; Woerle, Hans J.; Broedl, Uli C.

    2013-01-01

    Background Sulfonylureas (SUs) are commonly used in the treatment of type 2 diabetes (T2DM), usually as second-line treatment after the failure of metformin. However, SUs are associated with poor durability, hypoglycemia and weight gain. Empagliflozin is a sodium glucose cotransporter 2 (SGLT2) inhibitor in development for the treatment of T2DM. In Phase II/III trials, empagliflozin reduced hyperglycemia, body weight and blood pressure, with a low incidence of hypoglycemia. The aim of this Ph...

  8. Optimization of 1,2,5-Thiadiazole Carbamates as Potent and Selective ABHD6 Inhibitors #

    Patel, Jayendra Z.; Nevalainen, Tapio J.; Savinainen, Juha R.; Adams, Yahaya; Laitinen, Tuomo; Runyon, Robert S.; Vaara, Miia; Ahenkorah, Stephen; Kaczor, Agnieszka A.; Navia-Paldanius, Dina; Gynther, Mikko; Aaltonen, Niina; Joharapurkar, Amit A.; Jain, Mukul R.; Haka, Abigail S.; Maxfield, Frederick R.; Laitinen, Jarmo T.; Parkkari, Teija

    2015-01-01

    At present, inhibitors of α/β-hydrolase domain 6 (ABHD6) are viewed as a promising approach to treat inflammation and metabolic disorders. This article describes the optimization of 1,2,5-thiadiazole carbamates as ABHD6 inhibitors. Altogether, 34 compounds were synthesized and their inhibitory activity was tested using lysates of HEK293 cells transiently expressing human ABHD6 (hABHD6). Among the compound series, 4-morpholino-1,2,5-thiadiazol-3-yl cyclooctyl(methyl)carbamate (JZP-430, 55) potently and irreversibly inhibited hABHD6 (IC50 44 nM) and showed good selectivity (∼230 fold) over fatty acid amide hydrolase (FAAH) and lysosomal acid lipase (LAL), the main off-targets of related compounds. Additionally, activity-based protein profiling (ABPP) indicated that compound 55 (JZP-430) displayed good selectivity among the serine hydrolases of mouse brain membrane proteome. PMID:25504894

  9. Inhibition of soluble epoxide hydrolase activity by compounds isolated from the aerial parts of Glycosmis stenocarpa.

    Kim, Jang Hoon; Morgan, Abubaker M A; Tai, Bui Huu; Van, Doan Thi; Cuong, Nguyen Manh; Kim, Young Ho

    2016-08-01

    The aim of this study is to search for soluble epoxide hydrolase (sEH) inhibitors from natural plants, bioassay-guided fractionation of lipophilic n-hexane and chloroform layers of an extract of the aerial parts of Glycosmis stenocarpa led to the isolation of 12 compounds (1-12) including murrayafoline-A (1), isomahanine (2), bisisomahanine (3), saropeptate (4), (24 S)-ergost-4-en-3,6-dione (5), stigmasta-4-en-3,6-dion (6), stigmast-4-en-3-one (7), β-sitosterol (8), 24-methylpollinastanol (9), trans-phytol (10), neosarmentol III (11) and (+)-epiloliolide (12). Their structures were elucidated on the basis of spectroscopic data. Among them, neosarmentol III (11) was isolated from nature for the first time. All the isolated compounds were evaluated for their inhibitory activity against sEH. Among isolated carbazole-type compounds, isomahanine (2) and bisisomahanine (3) were identified as a potent inhibitor of sEH, with IC50 values of 22.5 ± 1.7 and 7.7 ± 1.2 µM, respectively. Moreover, the inhibitory action of 2 and 3 represented mixed-type enzyme inhibition. PMID:26444316

  10. [GH10 Family of Glycoside Hydrolases: Structure and Evolutionary Connections].

    Naumoff, D G

    2016-01-01

    Evolutionary connections were analyzed for endo-β-xylanases, which possess the GH10 family catalytic domains. A homology search yielded thrice as many proteins as are available from the Carbohydrate-Active Enzymes (CAZy) database. Lateral gene transfer was shown to play an important role in evolution of bacterial proteins of the family, especially in the phyla Acidobacteria, Cyanobacteria, Planctomycetes, Spirochaetes, and Verrucomicrobia. In the case of Verrucomicrobia, 23 lateral transfers from organisms of other phyla were detected. Evolutionary relationships were observed between the GH10 family domains and domains with the TIM-barrel tertiary structure from several other glycosidase families. The GH39 family of glycoside hydrolases showed the closest relationship. Unclassified homologs were grouped into 12 novel families of putative glycoside hydrolases (GHL51-GHL62). PMID:27028821

  11. Conformational Variability of Organophosphorous Hydrolase upon Soman and Paraoxon Binding

    Gomes, Diego E.B.; Lins, Roberto D.; Pascutti, Pedro G.; Lei, Chenghong; Soares, Thereza A.

    2011-01-01

    The bacterial enzyme organophosphorous hydrolase (OPH) exhibits both catalytic and substrate promiscuity. It hydrolyzes bonds in a variety of phosphotriester (P-O), phosphonothioate (P-S), phosphofluoridate (P-F) and phosphonocyanate (F-CN) compounds. However, its catalytic efficiency varies markedly for different substrates, limiting the broad-range application of OPH as catalyst in the bioremediation of pesticides and chemical war agents. In the present study, pKa calculations and multiple ...

  12. Discovery of Potent and Reversible Monoacylglycerol Lipase Inhibitors

    King, Alvin R.; Dotsey, Emmanuel Y.; Lodola, Alessio; Jung, Kwang Mook; Ghomian, Azar; Qiu, Yan; Fu, Jin; Mor, Marco; Piomelli, Daniele

    2009-01-01

    Monoacylglycerol lipase (MGL) is a serine hydrolase involved in the biological deactivation of the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG). Previous efforts to design MGL inhibitors have focused on chemical scaffolds that irreversibly block the activity of this enzyme. Here, we describe two naturally occurring terpenoids, pristimerin and euphol, which inhibit MGL activity with high potency (median effective concentration, IC50 = 93 nM and 315 nM, respectively) through a reversible m...

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

    Tyler, Ludmila [United States Department of Agriculture (USDA), Western Regional Research Center (WRRC), Albany; Bragg, Jennifer [United States Department of Agriculture (USDA), Western Regional Research Center (WRRC), Albany; Wu, Jiajie [United States Department of Agriculture (USDA), Western Regional Research Center (WRRC), Albany; Yang, Xiaohan [ORNL; Tuskan, Gerald A [ORNL; Vogel, John [United States Department of Agriculture (USDA), Western Regional Research Center (WRRC), Albany

    2010-01-01

    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, both at the whole-genome level and at the level of 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. Examination of individual glycoside hydrolase familes (GH5, GH13, GH18, GH19, GH28, and GH51) revealed both similarities and distinctions between monocots and dicots, as well as between species. Shared evolutionary histories appear to be modified by lineage-specific expansions or deletions. Within 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 the stage for Brachypodium to be a monocot model for investigations of these enzymes and their diverse roles in planta. Insights

  14. Activity based protein profiling to detect serine hydrolase alterations in virus infected cells

    MdShahiduzzaman; KevinM.Coombs

    2012-01-01

    Activity-based protein profiling (ABPP) is a newly emerging technique that uses active site-directed probes to monitor the functional status of enzymes. Serine hydrolases are one of the largest families of enzymes in mammals. More than 200 serine hydrolases have been identified, but little is known about their specific roles. Serine hydrolases are involved in a variety of physiological functions, including digestion, immune response, blood coagulation, and reproduction. ABPP has been used rec...

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

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

    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 w...

  16. Inactivation of epoxide hydrolase by catalysis-induced formation of isoaspartate

    van Loo, Bert; Permentier, Hjalmar P.; Kingma, Jaap; Baldascini, Helen; Janssen, Dick B.

    2008-01-01

    Epoxide hydrolases catalyze hydrolytic epoxide ring-opening, most often via formation of a covalent hydroxyalkyl-enzyme intermediate. A mutant of Agrobacterium radiobacter epoxide hydrolase, in which the phenylalanine residue that flanks the invariant catalytic aspartate nucleophile is replaced by a threonine, exhibited inactivation during conversion when the (R)-enantiomer of para-nitrostyrene epoxide was used as substrate. HPLC analysis of tryptic fragments of the epoxide hydrolase, followe...

  17. X-ray structure of potato epoxide hydrolase sheds light on substrate specificity in plant enzymes

    Mowbray, Sherry L.; Elfström, Lisa T.; Ahlgren, Kerstin M; Andersson, C. Evalena; Widersten, Mikael

    2006-01-01

    Epoxide hydrolases catalyze the conversion of epoxides to diols. The known functions of such enzymes include detoxification of xenobiotics, drug metabolism, synthesis of signaling compounds, and intermediary metabolism. In plants, epoxide hydrolases are thought to participate in general defense systems. In the present study, we report the first structure of a plant epoxide hydrolase, one of the four homologous enzymes found in potato. The structure was solved by molecular replacement and refi...

  18. Identification of N-acylethanolamines in Dictyostelium discoideum and confirmation of their hydrolysis by fatty acid amide hydrolase[S

    Hayes, Alexander C.; Stupak, Jacek; Li, Jianjun; Cox, Andrew D.

    2013-01-01

    N-acylethanolamines (NAEs) are endogenous lipid-based signaling molecules best known for their role in the endocannabinoid system in mammals, but they are also known to play roles in signaling pathways in plants. The regulation of NAEs in vivo is partly accomplished by the enzyme fatty acid amide hydrolase (FAAH), which hydrolyses NAEs to ethanolamine and their corresponding fatty acid. Inhibition of FAAH has been shown to increase the levels of NAEs in vivo and to produce desirable phenotype...

  19. α/β-Hydrolase Domain 6 Deletion Induces Adipose Browning and Prevents Obesity and Type 2 Diabetes.

    Zhao, Shangang; Mugabo, Yves; Ballentine, Gwynne; Attane, Camille; Iglesias, Jose; Poursharifi, Pegah; Zhang, Dongwei; Nguyen, Thuy Anne; Erb, Heidi; Prentki, Raphael; Peyot, Marie-Line; Joly, Erik; Tobin, Stephanie; Fulton, Stephanie; Brown, J Mark; Madiraju, S R Murthy; Prentki, Marc

    2016-03-29

    Suppression of α/β-domain hydrolase-6 (ABHD6), a monoacylglycerol (MAG) hydrolase, promotes glucose-stimulated insulin secretion by pancreatic β cells. We report here that high-fat-diet-fed ABHD6-KO mice show modestly reduced food intake, decreased body weight gain and glycemia, improved glucose tolerance and insulin sensitivity, and enhanced locomotor activity. ABHD6-KO mice also show increased energy expenditure, cold-induced thermogenesis, brown adipose UCP1 expression, fatty acid oxidation, and white adipose browning. Adipose browning and cold-induced thermogenesis are replicated by the ABHD6 inhibitor WWL70 and by antisense oligonucleotides targeting ABHD6. Our evidence suggests that one mechanism by which the lipolysis derived 1-MAG signals intrinsic and cell-autonomous adipose browning is via PPARα and PPARγ activation, and that ABHD6 regulates adipose browning by controlling signal competent 1-MAG levels. Thus, ABHD6 regulates energy homeostasis, brown adipose function, and white adipose browning and is a potential therapeutic target for obesity and type 2 diabetes. PMID:26997277

  20. 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

    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

  1. Ubiquitin C-terminal hydrolase-L1 increases cancer cell invasion by modulating hydrogen peroxide generated via NADPH oxidase 4

    Kim, Hyun Jung; Magesh, Venkataraman; Lee, Jae-Jin; Kim, Sun; Knaus, Ulla G.; Lee, Kong-Joo

    2015-01-01

    This study explored the role of ubiquitin C-terminal hydrolase-L1 (UCH-L1) in the production of ROS and tumor invasion. UCH-L1 was found to increase cellular ROS levels and promote cell invasion. Silencing UCH-L1, as well as inhibition of H2O2 generation by catalase or by DPI, a NOX inhibitor, suppressed the migration potential of B16F10 cells, indicating that UCH-L1 promotes cell migration by up-regulating H2O2 generation. Silencing NOX4, which generates H2O2, with siRNA eliminated the effec...

  2. CREST - a large and diverse superfamily of putative transmembrane hydrolases

    Olson Eric N

    2011-07-01

    Full Text Available Abstract Background A number of membrane-spanning proteins possess enzymatic activity and catalyze important reactions involving proteins, lipids or other substrates located within or near lipid bilayers. Alkaline ceramidases are seven-transmembrane proteins that hydrolyze the amide bond in ceramide to form sphingosine. Recently, a group of putative transmembrane receptors called progestin and adipoQ receptors (PAQRs were found to be distantly related to alkaline ceramidases, raising the possibility that they may also function as membrane enzymes. Results Using sensitive similarity search methods, we identified statistically significant sequence similarities among several transmembrane protein families including alkaline ceramidases and PAQRs. They were unified into a large and diverse superfamily of putative membrane-bound hydrolases called CREST (alkaline ceramidase, PAQR receptor, Per1, SID-1 and TMEM8. The CREST superfamily embraces a plethora of cellular functions and biochemical activities, including putative lipid-modifying enzymes such as ceramidases and the Per1 family of putative phospholipases involved in lipid remodeling of GPI-anchored proteins, putative hormone receptors, bacterial hemolysins, the TMEM8 family of putative tumor suppressors, and the SID-1 family of putative double-stranded RNA transporters involved in RNA interference. Extensive similarity searches and clustering analysis also revealed several groups of proteins with unknown function in the CREST superfamily. Members of the CREST superfamily share seven predicted core transmembrane segments with several conserved sequence motifs. Conclusions Universal conservation of a set of histidine and aspartate residues across all groups in the CREST superfamily, coupled with independent discoveries of hydrolase activities in alkaline ceramidases and the Per1 family as well as results from previous mutational studies of Per1, suggests that the majority of CREST members are

  3. Active site and laminarin binding in glycoside hydrolase family 55.

    Bianchetti, Christopher M; Takasuka, Taichi E; Deutsch, Sam; Udell, Hannah S; Yik, Eric J; Bergeman, Lai F; Fox, Brian G

    2015-05-01

    The Carbohydrate Active Enzyme (CAZy) database indicates that glycoside hydrolase family 55 (GH55) contains both endo- and exo-β-1,3-glucanases. The founding structure in the GH55 is PcLam55A from the white rot fungus Phanerochaete chrysosporium (Ishida, T., Fushinobu, S., Kawai, R., Kitaoka, M., Igarashi, K., and Samejima, M. (2009) Crystal structure of glycoside hydrolase family 55 β-1,3-glucanase from the basidiomycete Phanerochaete chrysosporium. J. Biol. Chem. 284, 10100-10109). Here, we present high resolution crystal structures of bacterial SacteLam55A from the highly cellulolytic Streptomyces sp. SirexAA-E with bound substrates and product. These structures, along with mutagenesis and kinetic studies, implicate Glu-502 as the catalytic acid (as proposed earlier for Glu-663 in PcLam55A) and a proton relay network of four residues in activating water as the nucleophile. Further, a set of conserved aromatic residues that define the active site apparently enforce an exo-glucanase reactivity as demonstrated by exhaustive hydrolysis reactions with purified laminarioligosaccharides. Two additional aromatic residues that line the substrate-binding channel show substrate-dependent conformational flexibility that may promote processive reactivity of the bound oligosaccharide in the bacterial enzymes. Gene synthesis carried out on ∼30% of the GH55 family gave 34 active enzymes (19% functional coverage of the nonredundant members of GH55). These active enzymes reacted with only laminarin from a panel of 10 different soluble and insoluble polysaccharides and displayed a broad range of specific activities and optima for pH and temperature. Application of this experimental method provides a new, systematic way to annotate glycoside hydrolase phylogenetic space for functional properties. PMID:25752603

  4. Analysis of the key active subsites of glycoside hydrolase 13 family members.

    Kumar, Vikash

    2010-05-01

    alpha-Amylase, pullulanase, neopullulanase, cyclomaltodextrinase (CDase), cyclomaltodextin glucanotransferase (CGTase), etc. are some of the amylolytic enzymes that act on polysaccharides. These enzymes differ from each other with respect to substrate and linkage specificities. These enzymes have been grouped into the GH13 (GH, Glycoside Hydrolase) family in the CAZy database on the basis of similarity in amino acid sequence. Members of this family share three domains viz., A, B, and C, which have several binding subsites to accommodate monomeric units of the polysaccharide substrate. Among these subsites, -2, -1, +1, and +2 subsites are the most critical subsites for catalytic activity. In the present study, the substrate analog-, inhibitor-, or product-bound 3-D structures of 24 members of GH13 family have been analyzed to identify the features of the -2, -1, +1, and +2 subsites shared by all the members for recognition of the common substrate. It is found that neither the number nor the nature of the potential hydrogen bond-forming residues is conserved with the exception of the presence of tyrosine as a stacking residue in the -1 subsite. The relative spatial disposition of the conserved subsite residues are conserved as judged by distance matrices. The backbone of the -2, -1, +1, and +2 subsites does not undergo conformational change for the recognition of the substrate. This analysis suggests that these enzymes recognize their substrate on the basis of shape of the substrate rather than on the basis of specific interactions within the binding site. PMID:20227065

  5. A Novel Saponin Hydrolase from Neocosmospora vasinfecta var. vasinfecta

    Watanabe, Manabu; Sumida, Naomi; Yanai, Koji; Murakami, Takeshi

    2004-01-01

    We isolated a soybean saponin hydrolase from Neocosmospora vasinfecta var. vasinfecta PF1225, a filamentous fungus that can degrade soybean saponin and generate soyasapogenol B. This enzyme was found to be a monomer with a molecular mass of about 77 kDa and a glycoprotein. Nucleotide sequence analysis of the corresponding gene (sdn1) indicated that this enzyme consisted of 612 amino acids and had a molecular mass of 65,724 Da, in close agreement with that of the apoenzyme after the removal of...

  6. Carboxylic ester hydrolases in mitochondria from rat skeletal muscle

    Kirkeby, S; Moe, D; Zelander, T

    1990-01-01

    organophosphate and organomercury. The activity of the indoxyl acetate esterases was enhanced by the non-ionic detergents Tween-40 and Lubrol. After freezing, thawing and high speed centrifugation most of the alpha-naphthyl acetate splitting enzymes were found in the supernatant, indicating that the enzymes are......A mitochondrial pellet, prepared from rat skeletal muscle, contained a number of carboxylic ester hydrolase isoenzymes. The esterases which split alpha-naphthyl acetate were organophosphate sensitive, whereas two out of three indoxyl acetate hydrolysing enzymes were resistant to both...

  7. Gene deficiency and pharmacological inhibition of soluble epoxide hydrolase confers resilience to repeated social defeat stress

    Ren, Qian; Ma, Min; Ishima, Tamaki; Morisseau, Christophe; Yang, Jun; Wagner, Karen M.; Zhang, Ji-chun; Yang, Chun; Yao, Wei; Dong, Chao; Han, Mei; Hammock, Bruce D.; Hashimoto, Kenji

    2016-01-01

    Depression is a severe and chronic psychiatric disease, affecting 350 million subjects worldwide. Although multiple antidepressants have been used in the treatment of depressive symptoms, their beneficial effects are limited. The soluble epoxide hydrolase (sEH) plays a key role in the inflammation that is involved in depression. Thus, we examined here the role of sEH in depression. In both inflammation and social defeat stress models of depression, a potent sEH inhibitor, TPPU, displayed rapid antidepressant effects. Expression of sEH protein in the brain from chronically stressed (susceptible) mice was higher than of control mice. Furthermore, expression of sEH protein in postmortem brain samples of patients with psychiatric diseases, including depression, bipolar disorder, and schizophrenia, was higher than controls. This finding suggests that increased sEH levels might be involved in the pathogenesis of certain psychiatric diseases. In support of this hypothesis, pretreatment with TPPU prevented the onset of depression-like behaviors after inflammation or repeated social defeat stress. Moreover, sEH KO mice did not show depression-like behavior after repeated social defeat stress, suggesting stress resilience. The sEH KO mice showed increased brain-derived neurotrophic factor (BDNF) and phosphorylation of its receptor TrkB in the prefrontal cortex, hippocampus, but not nucleus accumbens, suggesting that increased BDNF-TrkB signaling in the prefrontal cortex and hippocampus confer stress resilience. All of these findings suggest that sEH plays a key role in the pathophysiology of depression, and that epoxy fatty acids, their mimics, as well as sEH inhibitors could be potential therapeutic or prophylactic drugs for depression. PMID:26976569

  8. Gene deficiency and pharmacological inhibition of soluble epoxide hydrolase confers resilience to repeated social defeat stress.

    Ren, Qian; Ma, Min; Ishima, Tamaki; Morisseau, Christophe; Yang, Jun; Wagner, Karen M; Zhang, Ji-Chun; Yang, Chun; Yao, Wei; Dong, Chao; Han, Mei; Hammock, Bruce D; Hashimoto, Kenji

    2016-03-29

    Depression is a severe and chronic psychiatric disease, affecting 350 million subjects worldwide. Although multiple antidepressants have been used in the treatment of depressive symptoms, their beneficial effects are limited. The soluble epoxide hydrolase (sEH) plays a key role in the inflammation that is involved in depression. Thus, we examined here the role of sEH in depression. In both inflammation and social defeat stress models of depression, a potent sEH inhibitor, TPPU, displayed rapid antidepressant effects. Expression of sEH protein in the brain from chronically stressed (susceptible) mice was higher than of control mice. Furthermore, expression of sEH protein in postmortem brain samples of patients with psychiatric diseases, including depression, bipolar disorder, and schizophrenia, was higher than controls. This finding suggests that increased sEH levels might be involved in the pathogenesis of certain psychiatric diseases. In support of this hypothesis, pretreatment with TPPU prevented the onset of depression-like behaviors after inflammation or repeated social defeat stress. Moreover, sEH KO mice did not show depression-like behavior after repeated social defeat stress, suggesting stress resilience. The sEH KO mice showed increased brain-derived neurotrophic factor (BDNF) and phosphorylation of its receptor TrkB in the prefrontal cortex, hippocampus, but not nucleus accumbens, suggesting that increased BDNF-TrkB signaling in the prefrontal cortex and hippocampus confer stress resilience. All of these findings suggest that sEH plays a key role in the pathophysiology of depression, and that epoxy fatty acids, their mimics, as well as sEH inhibitors could be potential therapeutic or prophylactic drugs for depression. PMID:26976569

  9. Inhibition of acid hydrolase activity in human granulocytes by PUVA treatment

    The activity of acid hydrolases in peripheral blood granulocytes was determined. No significant differences could be revealed between healthy and psoriatic donors. During PUVA therapy of psoriasis patients the activity of acid hydrolases in granulocytes was moderately decreased in most cases, but the differences were not significant (high individual variability). Isolated granulocytes were treated in vitro with doses of 8-methoxypsoralen and UVA light as can be achieved in situ in the epidermis during PUVA therapy. A reduced acid hydrolases activity was found in the cells after the treatment, which was not due to secretion of the enzyme or cytotoxic damage. The presence of reduced glutathione prevented this effect. Free extracellular acid hydrolases were not inactivated by PUVA. PUVA-treated granulocytes showed an unimpaired superoxide generation after phagocytic stimulation. These results show that an intracellular inactivation of acid hydrolases and possibly other lysosomal enzymes in granulocytes infiltrating the psoriatic epidermis contribute to the antipsoriatic effects of PUVA therapy. (author)

  10. Activity based protein profiling to detect serine hydrolase alterations in virus infected cells

    MdShahiduzzaman

    2012-08-01

    Full Text Available Activity based protein profiling (ABPP is a newly emerging technique that uses active site-directed probes to monitor the functional status of enzymes. Serine hydrolases are one of the largest families of enzymes in mammals. More than 200 serine hydrolases have been identified but little is known about their specific roles. Serine hydrolases are involved in a variety of physiological functions, including digestion, immune response, blood coagulation and reproduction. ABPP has been used recently to investigate host-virus interactions and to understand the molecular pathogenesis of virus infections. Monitoring the altered serine hydrolases during viral infection gives insight into the catalytic activity of these enzymes that will help to identify novel targets for diagnostic and therapeutic application. This review presents the usefulness of ABPP in detecting and analyzing functional annotation of host cell serine hydrolases as a result of host-virus interaction.

  11. Marine Extremophiles: A Source of Hydrolases for Biotechnological Applications

    Gabriel Zamith Leal Dalmaso

    2015-04-01

    Full Text Available The marine environment covers almost three quarters of the planet and is where evolution took its first steps. Extremophile microorganisms are found in several extreme marine environments, such as hydrothermal vents, hot springs, salty lakes and deep-sea floors. The ability of these microorganisms to support extremes of temperature, salinity and pressure demonstrates their great potential for biotechnological processes. Hydrolases including amylases, cellulases, peptidases and lipases from hyperthermophiles, psychrophiles, halophiles and piezophiles have been investigated for these reasons. Extremozymes are adapted to work in harsh physical-chemical conditions and their use in various industrial applications such as the biofuel, pharmaceutical, fine chemicals and food industries has increased. The understanding of the specific factors that confer the ability to withstand extreme habitats on such enzymes has become a priority for their biotechnological use. The most studied marine extremophiles are prokaryotes and in this review, we present the most studied archaea and bacteria extremophiles and their hydrolases, and discuss their use for industrial applications.

  12. Acetylcarnitine hydrolase activity in bovine caudal epididymal spermatozoa

    Recently, the authors identified mM concentrations of acetylcarnitine in epidiymal fluids and have investigated the metabolism of acetylcarnitine by bovine and hamster caudal epididymal spermatozoa. [1-14C]acetyl-L-carnitine is oxidized to 14CO2 by washed, intact hamster and bovine sperm at maximal rates of 8.4 and 15.2 nmol/hr/107 cells respectively. Conversely, the carnitine moiety of acetyl-L-[3H-methyl]carnitine is not accumulated by sperm under similar conditions. Hydrolysis of [3H]acetyl-L-carnitine and competition of uptake of [3H]acetate by unlabeled acetate was demonstrated in incubations of intact cells of both species. The amount of [3H]acetate accumulated in the incubation medium is time-dependent and also depends on the concentration of unlabeled acetate. A partial solubilization of acetylcarnitine hydrolase activity from washed, intact bovine caudal epididymal spermatozoa in buffer or 0.01% Triton X-100 is observed. There is an enrichment of acetylcarnitine hydrolase activity in purified plasma membranes from bovine caudal epididymal spermatozoa when compared to the activity present in broken cell preparations or other cellular fractions. The results suggest that acetylcarnitine is a substrate for spermatozoa as they traverse the epididymis

  13. Mechanistic investigations of unsaturated glucuronyl hydrolase from Clostridium perfringens.

    Jongkees, Seino A K; Yoo, Hayoung; Withers, Stephen G

    2014-04-18

    Experiments were carried out to probe the details of the hydration-initiated hydrolysis catalyzed by the Clostridium perfringens unsaturated glucuronyl hydrolase of glycoside hydrolase family 88 in the CAZy classification system. Direct (1)H NMR monitoring of the enzymatic reaction detected no accumulated reaction intermediates in solution, suggesting that rearrangement of the initial hydration product occurs on-enzyme. An attempt at mechanism-based trapping of on-enzyme intermediates using a 1,1-difluoro-substrate was unsuccessful because the probe was too deactivated to be turned over by the enzyme. Kinetic isotope effects arising from deuterium-for-hydrogen substitution at carbons 1 and 4 provide evidence for separate first-irreversible and overall rate-determining steps in the hydration reaction, with two potential mechanisms proposed to explain these results. Based on the positioning of catalytic residues in the enzyme active site, the lack of efficient turnover of a 2-deoxy-2-fluoro-substrate, and several unsuccessful attempts at confirmation of a simpler mechanism involving a covalent glycosyl-enzyme intermediate, the most plausible mechanism is one involving an intermediate bearing an epoxide on carbons 1 and 2. PMID:24573682

  14. Acetylcarnitine hydrolase activity in bovine caudal epididymal spermatozoa

    Bruns, K.; Foster, R.A.; Casillas, E.R.

    1986-05-01

    Recently, the authors identified mM concentrations of acetylcarnitine in epidiymal fluids and have investigated the metabolism of acetylcarnitine by bovine and hamster caudal epididymal spermatozoa. (1-/sup 14/C)acetyl-L-carnitine is oxidized to /sup 14/CO/sub 2/ by washed, intact hamster and bovine sperm at maximal rates of 8.4 and 15.2 nmol/hr/10/sup 7/ cells respectively. Conversely, the carnitine moiety of acetyl-L-(/sup 3/H-methyl)carnitine is not accumulated by sperm under similar conditions. Hydrolysis of (/sup 3/H)acetyl-L-carnitine and competition of uptake of (/sup 3/H)acetate by unlabeled acetate was demonstrated in incubations of intact cells of both species. The amount of (/sup 3/H)acetate accumulated in the incubation medium is time-dependent and also depends on the concentration of unlabeled acetate. A partial solubilization of acetylcarnitine hydrolase activity from washed, intact bovine caudal epididymal spermatozoa in buffer or 0.01% Triton X-100 is observed. There is an enrichment of acetylcarnitine hydrolase activity in purified plasma membranes from bovine caudal epididymal spermatozoa when compared to the activity present in broken cell preparations or other cellular fractions. The results suggest that acetylcarnitine is a substrate for spermatozoa as they traverse the epididymis.

  15. Protective mechanisms against homocysteine toxicity: the role of bleomycin hydrolase.

    Zimny, Jaroslaw; Sikora, Marta; Guranowski, Andrzej; Jakubowski, Hieronim

    2006-08-11

    Homocysteine (Hcy) editing by methionyl-tRNA synthetase results in the formation of Hcy-thiolactone and initiates a pathway that has been implicated in human disease. In addition to being cleared from the circulation by urinary excretion, Hcy-thiolactone is detoxified by the serum Hcy-thiolactonase/paraoxonase carried on high density lipoprotein. Whether Hcy-thiolactone is detoxified inside cells was unknown. Here we show that Hcy-thiolactone is hydrolyzed by an intracellular enzyme, which we have purified to homogeneity from human placenta and identified by proteomic analyses as human bleomycin hydrolase (hBLH). We have also purified an Hcy-thiolactonase from the yeast Saccharomyces cerevisiae and identified it as yeast bleomycin hydrolase (yBLH). BLH belongs to a family of evolutionarily conserved cysteine aminopeptidases, and its only known biologically relevant function was deamidation of the anticancer drug bleomycin. Recombinant hBLH or yBLH, expressed in Escherichia coli, exhibits Hcy-thiolactonase activity similar to that of the native enzymes. Active site mutations, C73A for hBLH and H369A for yBLH, inactivate Hcy-thiolactonase activities. Yeast blh1 mutants are deficient in Hcy-thiolactonase activity in vitro and in vivo, produce more Hcy-thiolactone, and exhibit greater sensitivity to Hcy toxicity than wild type yeast cells. Our data suggest that BLH protects cells against Hcy toxicity by hydrolyzing intracellular Hcy-thiolactone. PMID:16769724

  16. Inhibition of soluble epoxide hydrolase attenuates hepatic fibrosis and endoplasmic reticulum stress induced by carbon tetrachloride in mice

    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 (CCl4)-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 CCl4-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 CCl4-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 CCl4-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 CCl4, 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

  17. POTENT UREA AND CARBAMATE INHIBITORS OF SOLUBLE EPOXIDE HYDROLASES. (R825433)

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  18. Development of the aza-crown ether metal complexes as artificial hydrolase.

    Yu, Lan; Li, Fang-zhen; Wu, Jiao-yi; Xie, Jia-qing; Li, Shuo

    2016-01-01

    Hydrolases play a crucial role in the biochemical process, which can catalyze the hydrolysis of various compounds like carboxylic esters, phosphoesters, amides, nucleic acids, peptides, and so on. The design of artificial hydrolases has attracted extensive attention due to their scientific significance and potential applications in the field of gene medicine and molecular biology. Numerous macrocyclic metal complexes have been used as artificial hydrolase in the catalytic hydrolysis of the organic substrate. Aza-crown ether for this comment is a special class of the macrocyclic ligand containing both the nitrogen atoms and oxygen atoms in the ring. The studies showed that the aza-crown complexes exhibited high activity of hydrolytic enzyme. However, the aza-crown ether metal complex as artificial hydrolase is still very limited because of its difficulty in synthesis. This review summarizes the development of the aza-crown ether metal complexes as the artificial hydrolase, including the synthesis and catalysis of the transition metal complexes and lanthanide metal complexes of aza-crown ethers. The purpose of this review is to highlight: (1) the relationship between the structure and hydrolytic activity of synthetic hydrolase; (2) the synergistic effect of metal sites and ligands in the course of organic compound hydrolysis; and (3) the design strategies of the aza-crown ethers as hydrolase. PMID:26460062

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

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

    2014-01-01

    The high resolution crystal structures of isatin hydrolase from Labrenzia aggregata in the apo and the product state, are described. These are the first structures of a functionally characterized metal-dependent hydrolase of this fold. Isatin hydrolase converts isatin to isatinate and belongs 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...

  20. Role of soluble epoxide hydrolase in the sex-specific vascular response to cerebral ischemia

    Zhang, Wenri; Iliff, Jeffrey J.; Campbell, Caitlyn J; Wang, Ruikang K.; Hurn, Patricia D.; Alkayed, Nabil J.

    2009-01-01

    Soluble epoxide hydrolase (sEH), a key enzyme in the metabolism of vasodilator eicosanoids called epoxyeicosatrienoic acids (EETs), is sexually dimorphic and suppressed by estrogen. We determined if the sex difference in blood flow during focal cerebral ischemia is linked to sEH. Soluble epoxide hydrolase expression in brain, hydrolase activity in cerebral vessels, and plasma 14,15-dihydroxyeicosatrienoic acid (14,15-DHET) were determined in male and female wild-type (WT) and sEH knockout (sE...

  1. Effect of phenobarbital on the level of translatable rat liver epoxide hydrolase mRNA.

    Pickett, C B; Lu, A Y

    1981-01-01

    Liver poly(A)+RNA isolated from untreated and phenobarbital-treated rats has been translated in the rabbit reticulocyte cell-fre system in order to determine the level of translationally active epoxide hydrolase (EC 3.3.2.3) mRNA. The in vitro translation systems were immunoprecipitated with rabbit IgG prepared against purified epoxide hydrolase, and the amount of epoxide hydrolase synthesized by the lysate programmed with control and phenobarbital poly(A)+RNA was quantitated. The level of tr...

  2. Primary structure and catalytic mechanism of the epoxide hydrolase from Agrobacterium radiobacter AD1

    Rink, R; Fennema, M.; Smids, M; Dehmel, U; Janssen, DB

    1997-01-01

    The epoxide hydrolase gene from Agrobacterium radiobacter AD1, a bacterium that is able to grow on epichlorohydrin as the sole carbon source, was cloned by means of the polymerase chain reaction with two degenerate primers based on the N-terminal and C-terminal sequences of the enzyme, The epoxide hydrolase gene coded for a protein of 294 amino acids with a molecular mass of 34 kDa, An identical epoxide hydrolase gene was cloned from chromosomal DNA of the closely related strain A, radiobacte...

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

    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. PMID:27139829

  4. Characterization of intracellular pteroylpolyglutamate hydrolase (PPH) from human intestinal mucosa

    Wang, T.T.Y.; Chandler, C.J.; Halsted, C.H.

    1986-03-01

    There are two forms of pteroylpolyglutamate hydrolase (PPH) in the human intestinal mucosa, one in the brush border membrane and the other intracellular; brush border PPH is an exopeptidase with optimal activity at pH 6.5 and a requirement for zinc. The presence study characterized human intracellular PPH and compared its properties to those of brush border PPH. Intracellular PPH was purified 30-fold. The enzyme had a MW of 75,000 by gel filtration, was optimally active at pH 4.5, and had an isoelectric point at pH 8.0. In contrast to brush border PPH, intracellular PPH was unstable at increasing temperatures, was unaffected by dialysis against chelating agents and showed no requirement for Zn/sup 2 +/. Using PteGlu/sub 2/(/sup 14/C)Glu as substrate, they demonstrated a K/sub m/ of 1.2 ..mu..M and increasing affinity for folates with longer glutamate chains. Intracellular PPH required the complete folic acid (PteGlu) moiety and a ..gamma..-glutamyl linkage for activity. Using ion exchange chromatography and an HPLC method to determine the hydrolytic products of the reaction, they found intracellular PPH could cleave both internal and terminal ..gamma..-glutamyl linkages, with PteGlu as an end product. After subcellular fractionation of the mucosa, PPH was found in the lysosomes. In summary, the distinct characteristics of brush border and intracellular PPH suggest that the two hydrolases serve different roles in folate metabolism.

  5. Role of haem oxygenase in the renoprotective effects of soluble epoxide hydrolase inhibition in diabetic spontaneously hypertensive rats.

    Elmarakby, Ahmed A; Faulkner, Jessica; Pye, Chelsey; Rouch, Katelyn; Alhashim, Abdulmohsin; Maddipati, Krishna Rao; Baban, Babak

    2013-10-01

    We have shown previously that inhibition of sEH (soluble epoxide hydrolase) increased EETs (epoxyeicosatrienoic acids) levels and reduced renal injury in diabetic mice and these changes were associated with induction of HO (haem oxygenase)-1. The present study determines whether the inhibition of HO negates the renoprotective effect of sEH inhibition in diabetic SHR (spontaneously hypertensive rats). After 6 weeks of induction of diabetes with streptozotocin, SHR were divided into the following groups: untreated, treated with the sEH inhibitor t-AUCB {trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid}, treated with the HO inhibitor SnMP (stannous mesoporphyrin), and treated with both inhibitors for 4 more weeks; non-diabetic SHR served as a control group. Induction of diabetes significantly increased renal sEH expression and decreased the renal EETs/DHETEs (dihydroxyeicosatrienoic acid) ratio without affecting HO-1 activity or expression in SHR. Inhibition of sEH with t-AUCB increased the renal EETs/DHETEs ratio and HO-1 activity in diabetic SHR; however, it did not significantly alter systolic blood pressure. Treatment of diabetic SHR with t-AUCB significantly reduced the elevation in urinary albumin and nephrin excretion, whereas co-administration of the HO inhibitor SnMP with t-AUCB prevented these changes. Immunohistochemical analysis revealed elevations in renal fibrosis as indicated by increased renal TGF-β (transforming growth factor β) levels and fibronectin expression in diabetic SHR and these changes were reduced with sEH inhibition. Co-administration of SnMP with t-AUCB prevented its ability to reduce renal fibrosis in diabetic SHR. In addition, SnMP treatment also prevented t-AUCB-induced decreases in renal macrophage infiltration, IL-17 expression and MCP-1 levels in diabetic SHR. These findings suggest that HO-1 induction is involved in the protective effect of sEH inhibition against diabetic renal injury. PMID:23611540

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

    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.

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

    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

  8. Erectogenic and Aphrodisiac Property of Moringa oleifera: Involvement of Soluble Epoxide Hydrolase Enzyme.

    Goswami, Sumanta Kumar; Inamdar, Mohammed Naseeruddin; Dethe, Shekhar M; Gururaj, Giligar M; Jamwal, Rohitash; Bhaskar, Anirban; Mundkinajeddu, Deepak; Agarwal, Amit

    2016-07-01

    Soluble epoxide hydrolase (sEH) inhibitors have been reported to improve penile erection; therefore, sEH could be useful for management of erectile dysfunction. Methanolic and aqueous extracts of 30 Indian medicinal plants were screened for their sEH inhibition potential. Fifteen extracts showed >50% inhibition when screened at 50 µg/mL in sEH inhibition assay. Methanolic extract of Moringa oleifera Lam. (Moringaceae) seeds (MEMO) was most potent with IC50 1.7 ± 0.1 µg/mL and was selected for in vitro studies on isolated rat corpus cavernosum smooth muscle and in vivo sexual behaviour studies on healthy and diabetic rats. Rats were divided into five groups, each containing six animals and treated orally with either water, vehicle (1% Tween-20), MEMO (45 and 90 mg/kg/day for 21 days), and standard drug, sildenafil (5 mg/kg/day for 7 days). An equal number of female rats were used, and the effect of MEMO and sildenafil was compared with that of vehicle. MEMO significantly relaxed isolated rat corpus cavernosum smooth muscle at 0.1-100 µg/mL in vitro and significantly increased (p < 0.05) sexual activity, intracavernous pressure/mean arterial pressure in normal and diabetic rats. The increase in erectile function of rats by MEMO could be because of its sEH inhibitory activity. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27020843

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

    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

  10. Structure-Guided Engineering of Molinate Hydrolase for the Degradation of Thiocarbamate Pesticides

    Leite, José P.; Duarte, Márcia; Paiva, Ana M.; Ferreira-da-Silva, Frederico; Matias, Pedro M.; Nunes, Olga C.; Gales, Luís

    2015-01-01

    Molinate is a recalcitrant thiocarbamate used to control grass weeds in rice fields. The recently described molinate hydrolase, from Gulosibacter molinativorax ON4T, plays a key role in the only known molinate degradation pathway ending in the formation of innocuous compounds. Here we report the crystal structure of recombinant molinate hydrolase at 2.27 Å. The structure reveals a homotetramer with a single mononuclear metal-dependent active site per monomer. The active site architecture show...

  11. Purification and characterization of a secreted recombinant phosphotriesterase (parathion hydrolase) from Streptomyces lividans.

    Rowland, S S; Speedie, M K; Pogell, B M

    1991-01-01

    A heterologous phosphotriesterase (parathion hydrolase), previously cloned from a Flavobacterium species into Streptomyces lividans, was secreted at high levels and purified to homogeneity. N-terminal analysis revealed that it had been processed in the same manner as the native membrane-bound Flavobacterium hydrolase. The enzyme consisted of a single polypeptide with an apparent molecular weight of 35,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Substrate sp...

  12. IN VITRO SOLUBLE EPOXIDE HYDROLASE ENZYME INHIBITORY ACTIVITY OF SOME NOVEL CHALCONE DERIVATIVES

    Kuppusamy Asokkumar; Lokeswari Prathyusha Tangella; Muthusamy Umamaheshwari; Thirumalaisamy Shivashanmugam; Varadharajan Subhadradevi; Puliyath Jagannath; Arumugam Madeswaran

    2012-01-01

    Objective Soluble epoxide hydrolase (sEH) belongs to the α/β -hydrolase superfamily, a subclass of α/β proteins. Chalcones are chemical compounds that show hopeful obliging efficacy in controlling numerous diseases. The main objective of the study is to evaluate the sEH inhibitory activity of some synthesized chalcone derivatives and identification of its mode of inhibition. Methods Four different chalcone derivatives (PC-1 to PC-4) were selected for synthesis by Claisen-Schmidt method. The i...

  13. Defects in fatty acid amide hydrolase 2 in a male with neurologic and psychiatric symptoms

    Sirrs, Sandra; van Karnebeek, Clara DM; Peng, Xiaoxue; Shyr, Casper; Tarailo-Graovac, Maja; Mandal, Rupasri; Testa, Daniel; Dubin, Devin; Carbonetti, Gregory; Glynn, Steven E.; Sayson, Bryan; Robinson, Wendy P.; Han, Beomsoo; Wishart, David; Ross, Colin J

    2015-01-01

    Background Fatty acid amide hydrolase 2 (FAAH2) is a hydrolase that mediates the degradation of endocannabinoids in man. Alterations in the endocannabinoid system are associated with a wide variety of neurologic and psychiatric conditions, but the phenotype and biochemical characterization of patients with genetic defects of FAAH2 activity have not previously been described. We report a male with autistic features with an onset before the age of 2 years who subsequently developed additional f...

  14. Corrosion inhibitors

    In this paper, we briefly describe the characteristics, cost and electrochemical nature of the corrosion phenomena as well as some of the technologies that are currently employed to minimize its effect. The main subject of the paper however, deals with the description, classification and mechanism of protection of the so-called corrosion inhibitors. Examples of the use of these substances in different aggressive environments are also presented as means to show that these compounds, or their combination, can in fact be used as excellent and relatively cheap technologies to control the corrosion of some metals. In the last part of the paper, the most commonly used techniques to evaluate the efficiency and performance of corrosion inhibitors are presented as well as some criteria to make a careful and proper selection of a corrosion inhibitor technology in a given situation. (Author) 151 refs

  15. Characterization and functional analysis of Trichinella spiralis Nudix hydrolase.

    Long, Shao Rong; Wang, Zhong Quan; Jiang, Peng; Liu, Ruo Dan; Qi, Xin; Liu, Pei; Ren, Hui Jun; Shi, Hai Ning; Cui, Jing

    2015-12-01

    Trichinella spiralis Nudix hydrolase (TsNd) was identified by screening a T7 phage display cDNA library from T. spiralis intestinal infective larvae (IIL), and vaccination of mice with recombinant TsNd protein (rTsNd) or TsNd DNA vaccine produced a partial protective immunity. The aim of this study was to identify the characteristics and biological functions of TsNd in the process of invasion and development of T. spiralis larvae. Transcription and expression of TsNd gene at all developmental stages of T. spiralis were observed by qPCR and immunofluorescent test (IFT). The rTsNd had the Nd enzymatic activity to dGTP, NAD, NADP and CoA. Its kinetic properties on the preferred substrate dGTP were calculated, and the Vmax, Km, and kcat/Km values at pH 8.0 were 3.19 μM min(-1) μg(-1), 370 μM, and 144 s(-1) M(-1), respectively, in reaction matrix containing 5 mM Zn(2+) and 2 mM DTT. The rTsNd was active from 25 °C to 50 °C, with optimal activity at 37 °C. rTsNd was able to bind specifically to mouse intestinal epithelial cells (IECs) and promoted the larval invasion of IECs, whereas anti-rTsNd antibodies inhibited the larval invasion of IECs in a dose-dependent manner. Anti-rTsNd antibodies could kill T. spiralis infective larvae by an ADCC-mediated mechanism. Our results showed that the rTsNd protein was able to interact with host IECs, had the Nudix hydrolasing activity and the enzymatic activity appeared to be essential indispensable for the T. spiralis larval invasion, development and survival in host. PMID:26545353

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

    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

  17. Actions of the FAAH inhibitor URB597 in neuropathic and inflammatory chronic pain models

    Jayamanne, Angelo; Greenwood, Ruth; Mitchell, Vanessa A; Aslan, Sevda; Piomelli, Daniele; Vaughan, Christopher W

    2005-01-01

    While cannabinoid receptor agonists have analgesic activity in chronic pain states, they produce a spectrum of central CB1 receptor-mediated motor and psychotropic side effects. The actions of endocannabinoids, such as anandamide are terminated by removal from the extracellular space, then subsequent enzymatic degradation by fatty-acid amide hydrolase (FAAH). In the present study, we compared the effect of a selective FAAH inhibitor, URB597, to that of a pan-cannabinoid receptor agonist HU210...

  18. Inhibitor and substrate binding by angiotensin-converting enzyme

    Wang, Xuemei; Wu, Shanshan; Xu, Dingguo;

    2011-01-01

    . In this work, we propose a model for an ACE Michaelis complex based on two known X-ray structures of inhibitor-enzyme complexes. Specifically, the human testis angiotensin-converting enzyme (tACE) complexed with two clinic drugs were first investigated using a combined quantum mechanical and molecular......Angiotensin-converting enzyme (ACE) is an important zinc-dependent hydrolase responsible for converting the inactive angiotensin I to the vasoconstrictor angiotensin II and for inactivating the vasodilator bradykinin. However, the substrate binding mode of ACE has not been completely understood...... computational protocol. Implications to ACE catalysis are discussed....

  19. Soluble epoxide hydrolase deficiency ameliorates acute pancreatitis in mice.

    Bettaieb, Ahmed; Morisseau, Christophe; Hammock, Bruce; Haj, Fawaz

    2014-10-01

    Acute pancreatitis (AP) is a frequent gastrointestinal disorder that causes significant morbidity and its incidence has been progressively increasing. AP starts as a local inflammation in the pancreas that often leads to systemic inflammatory response and complications. Soluble epoxide hydrolase (sEH) is a cytosolic enzyme whose inhibition in murine models has beneficial effects in inflammatory diseases, but its significance in AP remains unexplored. To investigate whether sEH may have a causal role in AP we utilized sEH knockout (KO) mice to determine the effects of sEH deficiency on ceruelin- and arginine-induced AP. sEH expression increased at the protein and messenger RNA levels, as well as sEH activity in the early phase of cerulein- and arginine-induced AP in mice. In addition, amylase and lipase levels were lower in cerulein-treated sEH KO mice compared with non-treated controls. Moreover, pancreatic mRNA and serum concentrations of the inflammatory cytokines IL-1ß and IL-6 were lower in sEH KO mice compared with controls. Further, sEH KO mice exhibited decreased cerulein- and arginine-induced NF-?B inflammatory response, MAPKs activation and decreased cell death. These findings demonstrate a novel role for sEH in the progression of cerulein- and arginine-induced AP. PMID:26461340

  20. Ubiquitin C-Terminal Hydrolase L1 in Tumorigenesis

    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.

  1. Investigation of the Bacillus cereus phosphonoacetaldehyde hydrolase catalytic mechanism

    The enzyme phosphonoacetaldehyde hydrolase (phosphonatase) from Bacillus cereus catalyzes the conversion of phosphonoacetaldehyde and phosphate. We have demonstrated that phosphonatase is inactivated when incubated with either acetaldehyde or phosphonoacetaldehyde for short time periods at low temperature in the presence of NaBH4. This result suggests that the Schiff base mechanism is operative since such treatment might be expected to inactivate the enzyme by reducing an iminium cation mechanistic intermediate. The inactivation process was shown to be highly specific for a single lysine residue. Incubation of phosphonatase with [3H]-NaBH4 and phosphonacetaldehyde [14C]-acetaldehyde and NaBH4 or [C2-3H]- phosphonoacetaldehyde and NaBH4 resulted in radiolabeled inactivated enzyme. Tryptic hydrolysis and reverse phase HPLC chromatography of the resulting digests demonstrated that the [C2 - 3H]- phosphonoacetaldehyde/NaBH4 methodology afforded the most specifically tritium labeled, inactivated phosphonatase. The radiolabeled, active site peptide was purified to homogeneity and its amino acid sequence was determined

  2. Epoxide hydrolase of Trichoderma reesei: Biochemical properties and conformational characterization.

    de Oliveira, Gabriel Stephani; Adriani, Patricia Pereira; Borges, Flavia Garcia; Lopes, Adriana Rios; Campana, Patricia T; Chambergo, Felipe S

    2016-08-01

    Epoxide hydrolases (EHs) are enzymes that are present in all living organisms and catalyze the hydrolysis of epoxides to the corresponding vicinal diols. EHs have biotechnological potential in chiral chemistry. We report the cloning, purification, enzymatic activity, and conformational analysis of the TrEH gene from Trichoderma reesei strain QM9414 using circular dichroism spectroscopy. The EH gene has an open reading frame encoding a protein of 343 amino acid residues, resulting in a molecular mass of 38.2kDa. The enzyme presents an optimum pH of 7.2, and it is highly active at temperatures ranging from 23 to 50°C and thermally inactivated at 70°C (t1/2=7.4min). The Michaelis constants (Km) were 4.6mM for racemic substrate, 21.7mM for (R)-(+)-styrene oxide and 3.0mM for (S)-(-)-styrene oxide. The kcat/Km analysis indicated that TrEH is enantioselective and preferentially hydrolyzes (S)-(-)-styrene oxide. The conformational stability studies suggested that, despite the extreme conditions (high temperatures and extremely acid and basic pHs), TrEH is able to maintain a considerable part of its regular structures, including the preservation of the native cores in some cases. The recombinant protein showed enantioselectivity that was distinct from other fungus EHs, making this protein a potential biotechnological tool. PMID:27177457

  3. Soluble epoxide hydrolase: A potential target for metabolic diseases.

    He, Jinlong; Wang, Chunjiong; Zhu, Yi; Ai, Ding

    2016-05-01

    Epoxyeicosatrienoic acids (EETs), important lipid mediators derived from arachidonic acid, have many beneficial effects in metabolic diseases, including atherosclerosis, hypertension, cardiac hypertrophy, diabetes, non-alcoholic fatty liver disease, and kidney disease. Epoxyeicosatrienoic acids can be further hydrolyzed to less active diols by the enzyme soluble epoxide hydrolase (sEH). Increasing evidence suggests that inhibition of sEH increases levels of EETs, which have anti-inflammatory effects and can prevent the development of hypertension, atherosclerosis, heart failure, fatty liver, and multiple organ fibrosis. Arachidonic acid is the most abundant omega-6 polyunsaturated fatty acid (PUFA) and shares the same set of enzymes with omega-3 PUFAs, such as docosahexaenoic acid and eicosapentaenoic acid. The omega-3 PUFAs and metabolites, such as regioisomeric epoxyeicosatetraenoic acids and epoxydocosapentaenoic acids, have been reported to have strong vasodilatory and anti-inflammatory effects. Therefore, sEH may be a potential therapeutic target for metabolic disorders. In this review, we focus on our and other recent studies of the functions of sEH, including the effects of its eicosanoid products from both omega-3 and omega-6 PUFAs, in various metabolic diseases. We also discuss the possible cellular and molecular mechanisms underlying the regulation of sEH. PMID:26621325

  4. Disrupting Dimerization Translocates Soluble Epoxide Hydrolase to Peroxisomes.

    Jonathan W Nelson

    Full Text Available The epoxyeicosatrienoic acid (EET neutralizing enzyme soluble epoxide hydrolase (sEH is a neuronal enzyme, which has been localized in both the cytosol and peroxisomes. The molecular basis for its dual localization remains unclear as sEH contains a functional peroxisomal targeting sequence (PTS. Recently, a missense polymorphism was identified in human sEH (R287Q that enhances its peroxisomal localization. This same polymorphism has also been shown to generate weaker sEH homo-dimers. Taken together, these observations suggest that dimerization may mask the sEH PTS and prevent peroxisome translocation. In the current study, we test the hypothesis that dimerization is a key regulator of sEH subcellular localization. Specifically, we altered the dimerization state of sEH by introducing substitutions in amino acids responsible for the dimer-stabilizing salt-bridge. Green Fluorescent Protein (GFP fusions of each of mutants were co-transfected into mouse primary cultured cortical neurons together with a PTS-linked red fluorescent protein to constitutively label peroxisomes. Labeled neurons were analyzed using confocal microscopy and co-localization of sEH with peroxisomes was quantified using Pearson's correlation coefficient. We find that dimer-competent sEH constructs preferentially localize to the cytosol, whereas constructs with weakened or disrupted dimerization were preferentially targeted to peroxisomes. We conclude that the sEH dimerization status is a key regulator of its peroxisomal localization.

  5. Recombinant production and characterisation of two related GH5 endo-β-1,4-mannanases from Aspergillus nidulans FGSC A4 showing distinctly different transglycosylation capacity

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

    2011-01-01

    The glycoside hydrolase family 5 (GH5) endo-β-1,4-mannanases ManA and ManC from Aspergillus nidulans FGSC A4 were produced in Pichia pastoris X33 and purified in high yields of 120 and 145mg/L, respectively, from the culture supernatants. Both enzymes showed increasing catalytic efficiency (kcat...

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

    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......, however, proposed for clan GH-H with GH31. A sequence alignment, based on the idea that residues equivalent in the primordial catalytic GH-H/GH31 (β/α)8-barrel may not be found in the present-day GH-H and GH31 structures at strictly equivalent positions, shows remote sequence homologies covering β3, β4, β......7 and β8 of the GH-H and GH31 (β/α)8-barrels. Structure comparison of GH13 α-amylase and GH31 α-xylosidase guided alignment of GH-H and GH31 members for construction of evolutionary trees. The closest sequence relationship displayed by GH31 is to GH77 of clan GH-H....

  7. 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

    Holt, S.; J. Fowler, C.; Rocksén, D.; Bucht, A.; Petersen, G.; Hansen, Harald S.; Valenti, M.; Di Marzo, V.

    2004-01-01

    lavage (BAL) fluid, which was not accompanied by epithelial cell injury. The treatment, however, did not change significantly the levels of anandamide and the related compound palmitoylethanolamide in the cell-free fraction of the BAL fluid. The activities of the anandamide synthetic enzymes N...... 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. © 2004 Elsevier Inc. All rights reserved....

  8. Inhibition of soluble epoxide hydrolase attenuates hepatic fibrosis and endoplasmic reticulum stress induced by carbon tetrachloride in mice

    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.

  9. Long-acting cocaine hydrolase for addiction therapy.

    Chen, Xiabin; Xue, Liu; Hou, Shurong; Jin, Zhenyu; Zhang, Ting; Zheng, Fang; Zhan, Chang-Guo

    2016-01-12

    Cocaine abuse is a world-wide public health and social problem without a US Food and Drug Administration-approved medication. An ideal anticocaine medication would accelerate cocaine metabolism, producing biologically inactive metabolites by administration of an efficient cocaine-specific exogenous enzyme. Our recent studies have led to the discovery of the desirable, highly efficient cocaine hydrolases (CocHs) that can efficiently detoxify and inactivate cocaine without affecting normal functions of the CNS. Preclinical and clinical data have demonstrated that these CocHs are safe for use in humans and are effective for accelerating cocaine metabolism. However, the actual therapeutic use of a CocH in cocaine addiction treatment is limited by its short biological half-life (e.g., 8 h or shorter in rats). Here we demonstrate a novel CocH form, a catalytic antibody analog, which is a fragment crystallizable (Fc)-fused CocH dimer (CocH-Fc) constructed by using CocH to replace the Fab region of human IgG1. The CocH-Fc not only has a high catalytic efficiency against cocaine but also, like an antibody, has a considerably longer biological half-life (e.g., ∼107 h in rats). A single dose of CocH-Fc was able to accelerate cocaine metabolism in rats even after 20 d and thus block cocaine-induced hyperactivity and toxicity for a long period. Given the general observation that the biological half-life of a protein drug is significantly longer in humans than in rodents, the CocH-Fc reported in this study could allow dosing once every 2-4 wk, or longer, for treatment of cocaine addiction in humans. PMID:26712009

  10. Conformational diversity and enantioconvergence in potato epoxide hydrolase 1.

    Bauer, P; Carlsson, Å Janfalk; Amrein, B A; Dobritzsch, D; Widersten, M; Kamerlin, S C L

    2016-06-28

    Potato epoxide hydrolase 1 (StEH1) is a biocatalytically important enzyme that exhibits rich enantio- and regioselectivity in the hydrolysis of chiral epoxide substrates. In particular, StEH1 has been demonstrated to enantioconvergently hydrolyze racemic mixes of styrene oxide (SO) to yield (R)-1-phenylethanediol. This work combines computational, crystallographic and biochemical analyses to understand both the origins of the enantioconvergent behavior of the wild-type enzyme, as well as shifts in activities and substrate binding preferences in an engineered StEH1 variant, R-C1B1, which contains four active site substitutions (W106L, L109Y, V141K and I155V). Our calculations are able to reproduce both the enantio- and regioselectivities of StEH1, and demonstrate a clear link between different substrate binding modes and the corresponding selectivity, with the preferred binding modes being shifted between the wild-type enzyme and the R-C1B1 variant. Additionally, we demonstrate that the observed changes in selectivity and the corresponding enantioconvergent behavior are due to a combination of steric and electrostatic effects that modulate both the accessibility of the different carbon atoms to the nucleophilic side chain of D105, as well as the interactions between the substrate and protein amino acid side chains and active site water molecules. Being able to computationally predict such subtle effects for different substrate enantiomers, as well as to understand their origin and how they are affected by mutations, is an important advance towards the computational design of improved biocatalysts for enantioselective synthesis. PMID:27049844

  11. Post-exposure administration of diazepam combined with soluble epoxide hydrolase inhibition stops seizures and modulates neuroinflammation in a murine model of acute TETS intoxication

    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 (GABAAR) function. We, therefore, investigated the effects of post-exposure administration of diazepam, a GABAAR 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 GABAAR 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 TETS

  12. Recombinant production, crystallization and X-ray crystallographic structure determination of the peptidyl-tRNA hydrolase of Pseudomonas aeruginosa

    Hughes, Ronny C.; McFeeters, Hana; Coates, Leighton; McFeeters, Robert L.

    2014-10-15

    The peptidyl-tRNA hydrolase enzyme from the pathogenic bacterium Pseudomonas aeruginosa (Pth; EC 3.1.1.29) has been cloned, expressed in Escherichia coli and crystallized for X-ray structural analysis. Suitable crystals were grown using the sitting-drop vapour-diffusion method after one week of incubation against a reservoir solution consisting of 20% polyethylene glycol 4000, 100 mM Tris pH 7.5, 10%(v/v) isopropyl alcohol. The crystals were used to obtain the three-dimensional structure of the native protein at 1.77 Å resolution. The structure was determined by molecular replacement of the crystallographic data processed in space group P6122 with unit-cell parameters a = b = 63.62,c = 155.20 Å, α = β = 90, γ = 120°. The asymmetric unit of the crystallographic lattice was composed of a single copy of the enzyme molecule with a 43% solvent fraction, corresponding to a Matthews coefficient of 2.43 Å3 Da-1. The crystallographic structure reported here will serve as the foundation for future structure-guided efforts towards the development of novel small-molecule inhibitors specific to bacterial Pths.

  13. Intracellular Self-Assembly of Cyclic d-Luciferin Nanoparticles for Persistent Bioluminescence Imaging of Fatty Acid Amide Hydrolase.

    Yuan, Yue; Wang, Fuqiang; Tang, Wei; Ding, Zhanling; Wang, Lin; Liang, Lili; Zheng, Zhen; Zhang, Huafeng; Liang, Gaolin

    2016-07-26

    Fatty acid amide hydrolase (FAAH) overexpression induces several disorder symptoms in nerve systems, and therefore long-term tracing of FAAH activity in vivo is of high importance but remains challenging. Current bioluminescence (BL) methods are limited in detecting FAAH activity within 5 h. Herein, by rational design of a latent BL probe (d-Cys-Lys-CBT)2 (1), we developed a "smart" method of intracellular reduction-controlled self-assembly and FAAH-directed disassembly of its cyclic d-luciferin-based nanoparticles (i.e., 1-NPs) for persistent BL imaging of FAAH activity in vitro, in cells, and in vivo. Using aminoluciferin methyl amide (AMA), Lys-amino-d-luciferin (Lys-Luc), and amino-d-luciferin (NH2-Luc) as control BL probes, we validated that the persistent BL of 1 from luciferase-expressing cells or tumors was controlled by the activity of intracellular FAAH. With the property of long-term tracing of FAAH activity in vivo of 1, we envision that our BL precursor 1 could probably be applied for in vivo screening of FAAH inhibitors and the diagnosis of their related diseases (or disorders) in the future. PMID:27348334

  14. Identification of the conserved spatial position of key active-site atoms in glycoside hydrolase 13 family members.

    Kumar, Vikash

    2010-07-19

    A computational study on the glycoside hydrolase 13 (GH13) family of the CAZy database has been carried out at the atomic level in order to identify the conserved positions that may be responsible for recognition of the substrate. Analysis with substrate analog-, inhibitor-, or product-bound 3D structures was carried out to find the atomic spatial arrangement of the amino acids that make -2, -1, +1, and +2 subsites and water oxygen atoms around the ligand. The identified conserved positions of subsites were independent from the nature of the amino acid. The -1 and +1 subsites have more conserved positions than the -2 and +2 subsites. Some of the clusters of the -1 and +1 subsites have atoms of the same chemical nature. A spatially conserved position for water, which is stabilized by a hydrogen bond with the carboxyl group of a proton donor (Glu) and Asp of the catalytic triad, was found in the -1 subsite of 75% of the enzymes subjected to analysis. This position could be the region of hydrolytic water. PMID:20557875

  15. Unraveling the substrate recognition mechanism and specificity of the unusual glycosyl hydrolase family 29 BT2192 from Bacteroides thetaiotaomicron.

    Guillotin, Laure; Lafite, Pierre; Daniellou, Richard

    2014-03-11

    Glycosyl hydrolase (GH) family 29 (CAZy database) consists of retaining α-l-fucosidases. We have identified BT2192, a protein from Bacteroides thetaiotaomicron, as the first GH29 representative exhibiting both weak α-l-fucosidase and β-d-galactosidase activities. Determination and analysis of X-ray structures of BT2192 in complex with β-d-galactoside competitive inhibitors showed a new binding mode different from that of known GH29 enzymes. Three point mutations, specific to BT2192, prevent the canonical GH29 substrate α-l-fucose from binding efficiently to the fucosidase-like active site relative to other GH29 enzymes. β-d-Galactoside analogues bind and interact in a second pocket, which is not visible in other reported GH29 structures. Molecular simulations helped in the assessment of the flexibility of both substrates in their respective pocket. Hydrolysis of the fucosyl moiety from the putative natural substrates like 3-fucosyllactose or Lewis(X) antigen would be mainly due to the efficient interactions with the galactosyl moiety, in the second binding site, located more than 6-7 Å apart. PMID:24527659

  16. Structure-guided engineering of molinate hydrolase for the degradation of thiocarbamate pesticides.

    José P Leite

    Full Text Available Molinate is a recalcitrant thiocarbamate used to control grass weeds in rice fields. The recently described molinate hydrolase, from Gulosibacter molinativorax ON4T, plays a key role in the only known molinate degradation pathway ending in the formation of innocuous compounds. Here we report the crystal structure of recombinant molinate hydrolase at 2.27 Å. The structure reveals a homotetramer with a single mononuclear metal-dependent active site per monomer. The active site architecture shows similarities with other amidohydrolases and enables us to propose a general acid-base catalysis mechanism for molinate hydrolysis. Molinate hydrolase is unable to degrade bulkier thiocarbamate pesticides such as thiobencarb which is used mostly in rice crops. Using a structural-based approach, we were able to generate a mutant (Arg187Ala that efficiently degrades thiobencarb. The engineered enzyme is suitable for the development of a broader thiocarbamate bioremediation system.

  17. Genetically reduced soluble epoxide hydrolase activity and risk of stroke and other cardiovascular disease

    Lee, Julie; Dahl, Morten; Grande, Peer; Tybjaerg-Hansen, Anne; Nordestgaard, Børge G

    2010-01-01

    BACKGROUND AND PURPOSE: The development of stroke has been linked to lowered levels of epoxyeicosatrienoic acids in the cerebral microvasculature. These substances are metabolized by the enzyme-soluble epoxide hydrolase encoded by the EPHX2 gene. We tested whether genetically reduced soluble...... epoxide hydrolase activity is associated with risk of ischemic stroke, myocardial infarction, and ischemic heart disease. METHODS: We genotyped participants from the Copenhagen City Heart Study (n=10 352), the Copenhagen General Population Study (n=26 042), the Copenhagen Carotid Stroke Study (n=398 cases......=0.08 to 1.00). Similar results were obtained for myocardial infarction and ischemic heart disease in the 3 studies. CONCLUSIONS: Our results show with significant power that genetically reduced soluble epoxide hydrolase activity is not a major risk factor for ischemic stroke, myocardial infarction...

  18. ISOLATION AND CHARACTERIZATION OF AN ACID, BILE SALT-INDEPENDENT, RETINYL ESTER HYDROLASE FROM RAT LIVER MICROSOMES

    Previous work in this laboratory has revealed the presence of both acidic and neutral bile-salt independent retinyl ester hydrolase activities in rat liver homogenates. Here we present the purification, identification and characterization of an acid retinyl ester hydrolase activity from solubilized ...

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

    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.

  20. A pro-nociceptive phenotype unmasked in mice lacking fatty-acid amide hydrolase.

    Carey, Lawrence M; Slivicki, Richard A; Leishman, Emma; Cornett, Ben; Mackie, Ken; Bradshaw, Heather; Hohmann, Andrea G

    2016-02-01

    Fatty-acid amide hydrolase (FAAH) is the major enzyme responsible for degradation of anandamide, an endocannabinoid. Pharmacological inhibition or genetic deletion of FAAH (FAAH KO) produces antinociception in preclinical pain models that is largely attributed to anandamide-induced activation of cannabinoid receptors. However, FAAH metabolizes a wide range of structurally related, biologically active lipid signaling molecules whose functions remain largely unknown. Some of these endogenous lipids, including anandamide itself, may exert pro-nociceptive effects under certain conditions. In our study, FAAH KO mice exhibited a characteristic analgesic phenotype in the tail flick test and in both formalin and carrageenan models of inflammatory nociception. Nonetheless, intradermal injection of the transient receptor potential channel V1 (TRPV1) agonist capsaicin increased nocifensive behavior as well as mechanical and heat hypersensitivity in FAAH KO relative to wild-type mice. This pro-nociceptive phenotype was accompanied by increases in capsaicin-evoked Fos-like immunoreactive (FLI) cells in spinal dorsal horn regions implicated in nociceptive processing and was attenuated by CB1 (AM251) and TRPV1 (AMG9810) antagonists. When central sensitization was established, FAAH KO mice displayed elevated levels of anandamide, other fatty-acid amides, and endogenous TRPV1 agonists in both paw skin and lumbar spinal cord relative to wild-type mice. Capsaicin decreased spinal cord 2-AG levels and increased arachidonic acid and prostaglandin E2 levels in both spinal cord and paw skin irrespective of genotype. Our studies identify a previously unrecognized pro-nociceptive phenotype in FAAH KO mice that was unmasked by capsaicin challenge. The heightened nociceptive response was mediated by CB1 and TRPV1 receptors and accompanied by enhanced spinal neuronal activation. Moreover, genetic deletion of FAAH has a profound impact on the peripheral and central lipidome. Thus, genetic

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

    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.

  2. Streptococcus pneumoniae Endohexosaminidase D, Structural and Mechanistic Insight into Substrate-Assisted Catalysis in Family 85 Glycoside Hydrolases

    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. Streptococcus pneumoniae Endohexosaminidase D, Structural and Mechanistic Insight into Substrate-Assisted Catalysis in Family 85 Glycoside Hydrolases

    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.

  4. Data set of optimal parameters for colorimetric red assay of epoxide hydrolase activity.

    de Oliveira, Gabriel Stephani; Adriani, Patricia Pereira; Borges, Flavia Garcia; Lopes, Adriana Rios; Campana, Patricia T; Chambergo, Felipe S

    2016-09-01

    The data presented in this article are related to the research article entitled "Epoxide hydrolase of Trichoderma reesei: Biochemical properties and conformational characterization" [1]. Epoxide hydrolases (EHs) are enzymes that catalyze the hydrolysis of epoxides to the corresponding vicinal diols. This article describes the optimal parameters for the colorimetric red assay to determine the enzymatic activity, with an emphasis on the characterization of the kinetic parameters, pH optimum and thermal stability of this enzyme. The effects of reagents that are not resistant to oxidation by sodium periodate on the reactions can generate false positives and interfere with the final results of the red assay. PMID:27366781

  5. Isolation, purification and characterization of a new organphosphorus hydrolase OPHC2

    WU Ningfeng; DENG Minjie; SHI Xiuyun; LIANG Guoyi; YAO Bin; FAN Yunliu

    2004-01-01

    A bacterium with the capability of degrading organphosphorus, identified as Pseudomonas pseudoalcaligenes, is isolated from OP-treated soil. The organphosphorus hydrolase OPHC2 from this bacterium has been purified and characterized. OPHC2 has optimum activity for the reaction at 65℃ and pH 9.0 with methyl parathion as a substrate, it also shows good thermal and pH stability. Most metal ions and chemicals have no effect on the activity of OPHC2. The analyses of nucleotide sequence encoding OPHC2 and amino acid sequence of OPHC2 show that there are lower homologies with those of organphosphorus hydrolase reported in GenBank.

  6. Phenotypic and genotypic characterization of peptidoglycan hydrolases of Lactobacillus sakei

    Afef Najjari

    2016-01-01

    Full Text Available Lactobacillus sakei, a lactic acid bacterium naturally found in fresh meat and sea products, is considered to be one of the most important bacterial species involved in meat fermentation and bio-preservation. Several enzymes of Lb. sakei species contributing to microbial safeguarding and organoleptic properties of fermented-meat were studied. However, the specific autolytic mechanisms and associated enzymes involved in Lb. sakei are not well understood. The autolytic phenotype of 22 Lb. sakei strains isolated from Tunisian meat and seafood products was evaluated under starvation conditions, at pH 6.5 and 8.5, and in the presence of different carbon sources. A higher autolytic rate was observed when cells were grown in the presence of glucose and incubated at pH 6.5. Almost all strains showed high resistance to mutanolysin, indicating a minor role of muramidases in Lb. sakei cell lysis. Using Micrococcus lysodeikticus cells as a substrate in activity gels zymogram, peptidoglycan hydrolase (PGH patterns for all strains was characterized by two lytic bands of ∼80 (B1 and ∼70 kDa (B2, except for strain BMG.167 which harbored two activity signals at a lower MW. Lytic activity was retained in high salt and in acid/basic conditions and was active toward cells of Lb. sakei, Listeria monocytogenes, Listeria ivanovii and Listeria innocua. Analysis of five putative PGH genes found in the Lb. sakei 23 K model strain genome, indicated that one gene, lsa1437, could encode a PGH (N-acetylmuramoyl-L-alanine amidase containing B1 and B2 as isoforms. According to this hypothesis, strain BMG.167 showed an allelic version of lsa1437 gene deleted of one of the five LysM domains, leading to a reduction in the MW of lytic bands and the high autolytic rate of this strain. Characterization of autolytic phenotype of Lb. sakei should expand the knowledge of their role in fermentation processes where they represent the dominant species.

  7. Phenotypic and genotypic characterization of peptidoglycan hydrolases of Lactobacillus sakei.

    Najjari, Afef; Amairi, Houda; Chaillou, Stéphane; Mora, Diego; Boudabous, Abdellatif; Zagorec, Monique; Ouzari, Hadda

    2016-01-01

    Lactobacillus sakei, a lactic acid bacterium naturally found in fresh meat and sea products, is considered to be one of the most important bacterial species involved in meat fermentation and bio-preservation. Several enzymes of Lb. sakei species contributing to microbial safeguarding and organoleptic properties of fermented-meat were studied. However, the specific autolytic mechanisms and associated enzymes involved in Lb. sakei are not well understood. The autolytic phenotype of 22 Lb. sakei strains isolated from Tunisian meat and seafood products was evaluated under starvation conditions, at pH 6.5 and 8.5, and in the presence of different carbon sources. A higher autolytic rate was observed when cells were grown in the presence of glucose and incubated at pH 6.5. Almost all strains showed high resistance to mutanolysin, indicating a minor role of muramidases in Lb. sakei cell lysis. Using Micrococcus lysodeikticus cells as a substrate in activity gels zymogram, peptidoglycan hydrolase (PGH) patterns for all strains was characterized by two lytic bands of ∼80 (B1) and ∼70 kDa (B2), except for strain BMG.167 which harbored two activity signals at a lower MW. Lytic activity was retained in high salt and in acid/basic conditions and was active toward cells of Lb. sakei, Listeria monocytogenes, Listeria ivanovii and Listeria innocua. Analysis of five putative PGH genes found in the Lb. sakei 23 K model strain genome, indicated that one gene, lsa1437, could encode a PGH (N-acetylmuramoyl-L-alanine amidase) containing B1 and B2 as isoforms. According to this hypothesis, strain BMG.167 showed an allelic version of lsa1437 gene deleted of one of the five LysM domains, leading to a reduction in the MW of lytic bands and the high autolytic rate of this strain. Characterization of autolytic phenotype of Lb. sakei should expand the knowledge of their role in fermentation processes where they represent the dominant species. PMID:26843981

  8. Conformational Variability of Organophosphorus Hydrolase upon Soman and Paraoxon Binding

    Gomes, Diego Eb; Lins, Roberto D.; Pascutti, Pedro G.; Lei, Chenghong; Soares, Thereza A.

    2011-12-31

    The bacterial enzyme organophosphorus hydrolase (OPH) exhibits both catalytic and substrate promiscuity. It hydrolyzes bonds in a variety of phosphotriester (P-O), phosphonothioate (P-S), phosphofluoridate (P-F) and phosphonocyanate (F-CN) compounds. However, its catalytic efficiency varies markedly for different substrates, limiting the broad-range application of OPH as catalyst in the bioremediation of pesticides and chemical war agents. In the present study, pK{sub a} calculations and multiple explicit-solvent molecular dynamics (MD) simulations were performed to characterize and contrast the structural dynamics of OPH bound to two substrates hydrolyzed with very distinct catalytic efficiencies: the nerve agent soman (O-pinacolyl-methyl-phosphonofluoridate) and the pesticide paraoxon (diethyl p-nitrophenyl phosphate). pK{sub a} calculations for the substrate-bound and unbound enzyme showed a significant pK{sub a} shift from standard values ({Delta}pK{sub a} = {+-} 3 units) for residues 254His and 275Arg. MD simulations of the doubly protonated 254His revealed a dynamic hydrogen bond network connecting the catalytic residue 301Asp via 254His to 232Asp, 233Asp, 275Arg and 235Asp, and is consistent with a previously postulated proton relay mechanism to ferry protons away from the active site with substrates that do not require activation of the leaving group. Hydrogen bonds between 301Asp and 254His were persistent in the OPH-paraoxon complex but not in the OPH-soman one, suggesting a potential role for such interaction in the more efficient hydrolysis of paraoxon over soman by OPH. These results are in line with previous mutational studies of residue 254His, which led to an increase of the catalytic efficiency of OPH over soman yet decreased its efficiency for paraoxon. In addition, comparative analysis of the molecular trajectories for OPH bound to soman and paraoxon suggests that binding of the latter facilitates the conformational transition of OPH from the

  9. Discovery of potent and reversible monoacylglycerol lipase inhibitors.

    King, Alvin R; Dotsey, Emmanuel Y; Lodola, Alessio; Jung, Kwang Mook; Ghomian, Azar; Qiu, Yan; Fu, Jin; Mor, Marco; Piomelli, Daniele

    2009-10-30

    Monoacylglycerol lipase (MGL) is a serine hydrolase involved in the biological deactivation of the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG). Previous efforts to design MGL inhibitors have focused on chemical scaffolds that irreversibly block the activity of this enzyme. Here, we describe two naturally occurring terpenoids, pristimerin and euphol, which inhibit MGL activity with high potency (median effective concentration, IC(50) = 93 nM and 315 nM, respectively) through a reversible mechanism. Mutational and modeling studies suggest that the two agents occupy a common hydrophobic pocket located within the putative lid domain of MGL, and each reversibly interacts with one of two adjacent cysteine residues (Cys(201) and Cys(208)) flanking such pocket. This previously unrecognized regulatory region might offer a molecular target for potent and reversible inhibitors of MGL. PMID:19875078

  10. Encoded Library Synthesis Using Chemical Ligation and the Discovery of sEH Inhibitors from a 334-Million Member Library

    Litovchick, Alexander; Dumelin, Christoph E.; Habeshian, Sevan; Gikunju, Diana; Guié, Marie-Aude; Centrella, Paolo; Zhang, Ying; Sigel, Eric A.; Cuozzo, John W.; Keefe, Anthony D.; Clark, Matthew A.

    2015-06-01

    A chemical ligation method for construction of DNA-encoded small-molecule libraries has been developed. Taking advantage of the ability of the Klenow fragment of DNA polymerase to accept templates with triazole linkages in place of phosphodiesters, we have designed a strategy for chemically ligating oligonucleotide tags using cycloaddition chemistry. We have utilized this strategy in the construction and selection of a small molecule library, and successfully identified inhibitors of the enzyme soluble epoxide hydrolase.

  11. Extracellular xylanolytic and pectinolytic hydrolase production by A. flavus isolates contributes to crop invasion

    Several atoxigenic Aspergillus flavus isolates, including some 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 ut...

  12. Genetically lowered microsomal epoxide hydrolase activity and tobacco-related cancer in 47,000 individuals

    Lee, Julie; Dahl, Morten; Nordestgaard, Børge G

    2011-01-01

    Two functional polymorphisms of the microsomal epoxide hydrolase (mEH) gene (EPHX1), Tyr113His (rs1051740) and His139Arg (rs2234922), have variably been found to influence susceptibility to various cancer forms. We tested whether genetically lowered mEH activity affects risk of developing cancer in...

  13. Biosynthesis of intestinal microvillar proteins. Intracellular processing of lactase-phlorizin hydrolase

    Danielsen, E M; Skovbjerg, H; Norén, Ove;

    1984-01-01

    The biosynthesis of pig small intestinal lactase-phlorizin hydrolase (EC 3.2.1.23-62) was studied by labelling of organ cultured mucosal explants with [35S]methionine. The earliest detactable form of the enzyme was an intracellular, membrane-bound polypeptide of Mr 225 000, sensitive to endo H as...

  14. Structure of the minimized α/β-hydrolase fold protein from Thermus thermophilus HB8

    The crystal structure of the minimized α/β-hydrolase fold protein encoded by the gene TTHA1544 from T. thermophilus HB8 has been determined at 2.0 Å resolution. The gene encoding TTHA1544 is a singleton found in the Thermus thermophilus HB8 genome and encodes a 131-amino-acid protein. The crystal structure of TTHA1544 has been determined at 2.0 Å resolution by the single-wavelength anomalous dispersion method in order to elucidate its function. There are two molecules in the asymmetric unit. Each molecule consists of four α-helices and six β-strands, with the β-strands composing a central β-sheet. A structural homology search revealed that the overall structure of TTHA1544 resembles the α/β-hydrolase fold, although TTHA1544 lacks the catalytic residues of a hydrolase. These results suggest that TTHA1544 represents the minimized α/β-hydrolase fold and that an additional component would be required for its activity

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

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

    2015-01-01

    The epoxide hydrolases (EHs) represent an attractive option for the synthesis of chiral epoxides and 1,2-diols which are valuable building blocks for the synthesis of several pharmaceutical compounds. A metagenomic approach has been used to identify two new members of the atypical EH limonene-1...

  16. Novel prokaryotic epoxide hydrolase and Baeyer-Villiger monooxygenase genes from metagenome

    Štěpánek, Václav; Kotík, Michael; Marešová, Helena; Valešová, Renata; Grulich, Michal

    Bratislava : Československá spoločnosť mikrobiologická, 2010. s. 57-57. ISBN 970-80-970477-8-8 Institutional research plan: CEZ:AV0Z50200510 Keywords : epoxide hydrolases * environmental DNA Subject RIV: EE - Microbiology, Virology

  17. Improved annotation of conjugated bile acid hydrolase superfamily members in Gram-positive bacteria

    Lambert, J.M.; Siezen, R.J.; Vos, de W.M.; Kleerebezem, M.

    2008-01-01

    Most Gram-positive bacteria inhabiting the gastrointestinal tract are capable of hydrolysing bile salts. Bile salt hydrolysis is thought to play an important role in various biological processes in the host. Therefore, correct annotation of bacterial bile salt hydrolases (Bsh) in public databases (E

  18. Oligogalacturonate hydrolase with unique substrate preference from the pulp of parsley roots

    Flodrová, Dana; Garajová, S.; Malovíková, A.; Mislovičová, D.; Omelková, J.; Stratilová, E.

    2009-01-01

    Roč. 64, č. 2 (2009), s. 228-234. ISSN 0006-3088 Institutional research plan: CEZ:AV0Z40310501 Keywords : exopolygalacturonase * oligogalacturonate hydrolase * Petroselium crispum Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.617, year: 2009

  19. Environmental DNA as a source of a novel epoxide hydrolase reacting with aliphatic terminal epoxides

    Kotík, Michael; Štěpánek, Václav; Marešová, Helena; Kyslík, Pavel; Archelas, A.

    2009-01-01

    Roč. 56, č. 4 (2009), s. 288-293. ISSN 1381-1177 R&D Projects: GA ČR GA204/06/0458 Institutional research plan: CEZ:AV0Z50200510 Keywords : environmental DNA * metagenome * epoxide hydrolase Subject RIV: EE - Microbiology, Virology Impact factor: 2.400, year: 2009

  20. EXPRESSION AND CHARACTERIZATION OF THE RECOMBINANT JUVENILE HORMONE EPOXIDE HYDROLASE (JHEH) FROM MANDUCA SEXTA. (R825433)

    The cDNA of the microsomal Juvenile Hormone Epoxide Hydrolase (JHEH) from Manduca sexta was expressed in vitro in the baculovirus system. In insect cell culture, the recombinant enzyme (Ms-JHEH) was produced at a high level (100 fold over background EH catalytic activit...

  1. Molecular modeling study for inhibition mechanism of human chymase and its application in inhibitor design.

    Mahreen Arooj

    Full Text Available Human chymase catalyzes the hydrolysis of peptide bonds. Three chymase inhibitors with very similar chemical structures but highly different inhibitory profiles towards the hydrolase function of chymase were selected with the aim of elucidating the origin of disparities in their biological activities. As a substrate (angiotensin-I bound crystal structure is not available, molecular docking was performed to dock the substrate into the active site. Molecular dynamics simulations of chymase complexes with inhibitors and substrate were performed to calculate the binding orientation of inhibitors and substrate as well as to characterize conformational changes in the active site. The results elucidate details of the 3D chymase structure as well as the importance of K40 in hydrolase function. Binding mode analysis showed that substitution of a heavier Cl atom at the phenyl ring of most active inhibitor produced a great deal of variation in its orientation causing the phosphinate group to interact strongly with residue K40. Dynamics simulations revealed the conformational variation in region of V36-F41 upon substrate and inhibitor binding induced a shift in the location of K40 thus changing its interactions with them. Chymase complexes with the most active compound and substrate were used for development of a hybrid pharmacophore model which was applied in databases screening. Finally, hits which bound well at the active site, exhibited key interactions and favorable electronic properties were identified as possible inhibitors for chymase. This study not only elucidates inhibitory mechanism of chymase inhibitors but also provides key structural insights which will aid in the rational design of novel potent inhibitors of the enzyme. In general, the strategy applied in the current study could be a promising computational approach and may be generally applicable to drug design for other enzymes.

  2. Characteristics, protein engineering and applications of microbial thermostable pullulanases and pullulan hydrolases.

    Nisha, M; Satyanarayana, T

    2016-07-01

    Pullulan hydrolyzing enzymes are endoacting, classified based on the substrate specificity and hydrolysis products as pullulanases (type I and II) and pullulan hydrolases (type I, II and III). Pullulanases and pullulan hydrolase type I are produced by bacteria and archaea. Among bacteria, many mesophilic, thermophilic and hyperthermophilic bacteria produce pullulanases and neopullulanases. While pullulan hydrolase type II and type III are produced by fungi and archaea, respectively. These are multi-domain proteins with three conserved catalytic acidic residues of the glycosyl hydrolases. The recent advances in molecular biology and protein engineering via mutagenesis and truncation led to improvement in thermostability, catalytic activity and substrate specificity. Pullulanases are debranching enzymes, which are widely employed in starch saccharification that minimizes the use of glucoamylase (approx. 50 %) and reduces the total reaction time of the industrial starch conversion process. The thermostable amylopullulanases are useful in one-step starch liquefaction and saccharification, which replaces amylolytic enzymes like α-amylase and glucoamylase, thus resulting in the reduction in the cost of sugar production. The enzymes also find application in making resistant starches and as an antistale in bread making. Panose and isopanose containing syrups are useful as prebiotics, while panose has also been reported to display anticarcinogenic activity. This review focuses on the distinguishing features of these enzymes based on the analysis of amino acid sequences and domain structure, besides highlighting recent advances in the molecular biology and protein engineering for enhancing their thermostability, catalytic activity and substrate specificity. This review also briefly summarizes the potential applications of pullulanases and pullulan hydrolases. PMID:27142298

  3. Characterization and purification of bile salt hydrolase from Lactobacillus sp. strain 100-100

    The authors have characterized and purified the bile salt hydrolase from Lactobacillus sp. strain 100-100. Bile salt hydrolase from cells of the strain was purified with column and high-performance liquid chromatography. The activity was assayed in whole cells and cell-free extracts with either a radiochemical assay involving [14C]taurocholic acid or a nonradioactive assay involving trinitrobenzene sulfonate. The activity was detectable only in stationary-phase cells. Within 20 min after conjugated bile acids were added to stationary-phase cultures of strain 100-100, the activity in whole cells increased to levels three- to fivefold higher than in cells from cultures grown in medium free of bile salts. In cell-free extracts, however, the activity was about equal whether or not the cells have been grown with bile salts present. When supernatant solutions from cultures grown in medium containing taurocholic acid were used to suspend cells grown in medium free of the bile salt, the bile salt hydrolase activity detected in whole cells increased two- to threefold. Two forms of the hydrolase were purified from the cells and designated hydrolases A and B. They eluted from anion-exchange high-performance liquid chromatography in two sets of fractions, A at 0.15 M NaCl and B at 0.18 M NaCl. Their apparent molecular weights in nondenaturing polyacrylamide gel electrophoresis were 115,000 and 105,000, respectively. However, discrepancies existed in the apparent molecular weights and number of peptides detected in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the two forms. Whether the enzyme exists in two forms in the cells remains to be determined

  4. Molecular Identification of β-Citrylglutamate Hydrolase as Glutamate Carboxypeptidase 3*

    Collard, François; Vertommen, Didier; Constantinescu, Stefan; Buts, Lieven; Van Schaftingen, Emile

    2011-01-01

    β-Citrylglutamate (BCG), a compound present in adult testis and in the CNS during the pre- and perinatal periods is synthesized by an intracellular enzyme encoded by the RIMKLB gene and hydrolyzed by an as yet unidentified ectoenzyme. To identify β-citrylglutamate hydrolase, this enzyme was partially purified from mouse testis and characterized. Interestingly, in the presence of Ca2+, the purified enzyme specifically hydrolyzed β-citrylglutamate and did not act on N-acetyl-aspartylglutamate (NAAG). However, both compounds were hydrolyzed in the presence of Mn2+. This behavior and the fact that the enzyme was glycosylated and membrane-bound suggested that β-citrylglutamate hydrolase belonged to the same family of protein as glutamate carboxypeptidase 2 (GCP2), the enzyme that catalyzes the hydrolysis of N-acetyl-aspartylglutamate. The mouse tissue distribution of β-citrylglutamate hydrolase was strikingly similar to that of the glutamate carboxypeptidase 3 (GCP3) mRNA, but not that of the GCP2 mRNA. Furthermore, similarly to β-citrylglutamate hydrolase purified from testis, recombinant GCP3 specifically hydrolyzed β-citrylglutamate in the presence of Ca2+, and acted on both N-acetyl-aspartylglutamate and β-citrylglutamate in the presence of Mn2+, whereas recombinant GCP2 only hydrolyzed N-acetyl-aspartylglutamate and this, in a metal-independent manner. A comparison of the structures of the catalytic sites of GCP2 and GCP3, as well as mutagenesis experiments revealed that a single amino acid substitution (Asn-519 in GCP2, Ser-509 in GCP3) is largely responsible for GCP3 being able to hydrolyze β-citrylglutamate. Based on the crystal structure of GCP3 and kinetic analysis, we propose that GCP3 forms a labile catalytic Zn-Ca cluster that is critical for its β-citrylglutamate hydrolase activity. PMID:21908619

  5. Molecular identification of β-citrylglutamate hydrolase as glutamate carboxypeptidase 3.

    Collard, François; Vertommen, Didier; Constantinescu, Stefan; Buts, Lieven; Van Schaftingen, Emile

    2011-11-01

    β-Citrylglutamate (BCG), a compound present in adult testis and in the CNS during the pre- and perinatal periods is synthesized by an intracellular enzyme encoded by the RIMKLB gene and hydrolyzed by an as yet unidentified ectoenzyme. To identify β-citrylglutamate hydrolase, this enzyme was partially purified from mouse testis and characterized. Interestingly, in the presence of Ca(2+), the purified enzyme specifically hydrolyzed β-citrylglutamate and did not act on N-acetyl-aspartylglutamate (NAAG). However, both compounds were hydrolyzed in the presence of Mn(2+). This behavior and the fact that the enzyme was glycosylated and membrane-bound suggested that β-citrylglutamate hydrolase belonged to the same family of protein as glutamate carboxypeptidase 2 (GCP2), the enzyme that catalyzes the hydrolysis of N-acetyl-aspartylglutamate. The mouse tissue distribution of β-citrylglutamate hydrolase was strikingly similar to that of the glutamate carboxypeptidase 3 (GCP3) mRNA, but not that of the GCP2 mRNA. Furthermore, similarly to β-citrylglutamate hydrolase purified from testis, recombinant GCP3 specifically hydrolyzed β-citrylglutamate in the presence of Ca(2+), and acted on both N-acetyl-aspartylglutamate and β-citrylglutamate in the presence of Mn(2+), whereas recombinant GCP2 only hydrolyzed N-acetyl-aspartylglutamate and this, in a metal-independent manner. A comparison of the structures of the catalytic sites of GCP2 and GCP3, as well as mutagenesis experiments revealed that a single amino acid substitution (Asn-519 in GCP2, Ser-509 in GCP3) is largely responsible for GCP3 being able to hydrolyze β-citrylglutamate. Based on the crystal structure of GCP3 and kinetic analysis, we propose that GCP3 forms a labile catalytic Zn-Ca cluster that is critical for its β-citrylglutamate hydrolase activity. PMID:21908619

  6. Cell- and ligand-specific dephosphorylation of acid hydrolases: Evidence that the mannose 6-phosphatase is controlled by compartmentalization

    Einstein, R.; Gabel, C.A. (Columbia Univ. College of Physicians and Surgeons, NY (USA))

    1991-01-01

    Mouse L cells that possess the cation-independent mannose 6-phosphate (Man 6-P)/insulin-like growth factor (IGF) II receptor change the extent to which they dephosphorylate endocytosed acid hydrolases in response to serum. To investigate the mechanism by which dephosphorylation competence is regulated, the dephosphorylation of individual acid hydrolases was studied in Man 6-P/IGF II receptor-positive and -deficient cell lines. 125I-labeled Man 6-P-containing acid hydrolases were proteolytically processed but remained phosphorylated when endocytosed by receptor-positive L cells maintained in the absence of serum; after the addition of serum, however, the cell-associated hydrolases were dephosphorylated. Individual hydrolases were dephosphorylated at distinct rates and to different extents. In contrast, the same hydrolases were dephosphorylated equally and completely after entry into Man 6-P/IGF II receptor-positive Chinese hamster ovary (CHO) cells. The dephosphorylation competence of Man 6-P/IGF II receptor-deficient mouse J774 cells was more limited. beta-Glucuronidase produced by these cells underwent a limited dephosphorylation in transit to lysosomes such that diphosphorylated oligosaccharides were converted to monophosphorylated species. The overall quantity of phosphorylated oligosaccharides associated with the enzyme, however, did not decrease within the lysosomal compartment. Likewise, beta-glucuronidase was not dephosphorylated when introduced into J774 cells via Fc receptor-mediated endocytosis. The CHO and J774 cell lysosomes, therefore, display opposite extremes with respect to their capacity to dephosphorylate acid hydrolases; within CHO cell lysosomes acid hydrolases are rapidly and efficiently dephosphorylated, but within J774 cell lysosomes the same acid hydrolases remain phosphorylated.

  7. Cell- and ligand-specific dephosphorylation of acid hydrolases: Evidence that the mannose 6-phosphatase is controlled by compartmentalization

    Mouse L cells that possess the cation-independent mannose 6-phosphate (Man 6-P)/insulin-like growth factor (IGF) II receptor change the extent to which they dephosphorylate endocytosed acid hydrolases in response to serum. To investigate the mechanism by which dephosphorylation competence is regulated, the dephosphorylation of individual acid hydrolases was studied in Man 6-P/IGF II receptor-positive and -deficient cell lines. 125I-labeled Man 6-P-containing acid hydrolases were proteolytically processed but remained phosphorylated when endocytosed by receptor-positive L cells maintained in the absence of serum; after the addition of serum, however, the cell-associated hydrolases were dephosphorylated. Individual hydrolases were dephosphorylated at distinct rates and to different extents. In contrast, the same hydrolases were dephosphorylated equally and completely after entry into Man 6-P/IGF II receptor-positive Chinese hamster ovary (CHO) cells. The dephosphorylation competence of Man 6-P/IGF II receptor-deficient mouse J774 cells was more limited. beta-Glucuronidase produced by these cells underwent a limited dephosphorylation in transit to lysosomes such that diphosphorylated oligosaccharides were converted to monophosphorylated species. The overall quantity of phosphorylated oligosaccharides associated with the enzyme, however, did not decrease within the lysosomal compartment. Likewise, beta-glucuronidase was not dephosphorylated when introduced into J774 cells via Fc receptor-mediated endocytosis. The CHO and J774 cell lysosomes, therefore, display opposite extremes with respect to their capacity to dephosphorylate acid hydrolases; within CHO cell lysosomes acid hydrolases are rapidly and efficiently dephosphorylated, but within J774 cell lysosomes the same acid hydrolases remain phosphorylated

  8. Synthesis and preclinical evaluation of [11C-carbonyl]PF-04457845 for neuroimaging of fatty acid amide hydrolase

    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 [11C-carbonyl]PF-04457845, an isotopologue of the potent irreversible FAAH inhibitor. Methods: PF-04457845 was radiolabeled at the carbonyl position via automated [11C]CO2-fixation. Ex vivo brain biodistribution of [11C-carbonyl]PF-04457845 was carried out in conscious rats. Specificity was determined by pre-administration of PF-04457845 or URB597 prior to [11C-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 [11C]CO2 (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 [11C-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 [11C-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: [11C-carbonyl]PF-04457845 was rapidly synthesized via an automated radiosynthesis. Ex vivo biodistribution studies in conscious rodents

  9. [{sup 11}C]CURB: Evaluation of a novel radiotracer for imaging fatty acid amide hydrolase by positron emission tomography

    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

  10. [11C]CURB: Evaluation of a novel radiotracer for imaging fatty acid amide hydrolase by positron emission tomography

    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 ([11C]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, [11C]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 [11C]CURB was irreversibly bound to FAAH. Conclusions: The

  11. Advances in the discovery of N-acylethanolamine acid amidase inhibitors.

    Bandiera, Tiziano; Ponzano, Stefano; Piomelli, Daniele

    2014-08-01

    N-Acylethanolamine acid amidase (NAAA) is a cysteine amidase that hydrolyzes saturated or monounsaturated fatty acid ethanolamides, such as palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). PEA has been shown to exert analgesic and anti-inflammatory effects by engaging peroxisome proliferator-activated receptor-α. Like other fatty acid ethanolamides, PEA is not stored in cells, but produced on demand from cell membrane precursors, and its actions are terminated by intracellular hydrolysis by either fatty acid amide hydrolase or NAAA. Endogenous levels of PEA and OEA have been shown to decrease during inflammation. Modulation of the tissue levels of PEA by inhibition of enzymes responsible for the breakdown of this lipid mediator may represent therefore a new therapeutic strategy for the treatment of pain and inflammation. While a large number of inhibitors of fatty acid amide hydrolase have been discovered, few compounds have been reported to inhibit NAAA activity. Here, we describe the most representative NAAA inhibitors and briefly highlight their pharmacological profile. A recent study has shown that a NAAA inhibitor attenuated heat hyperalgesia and mechanical allodynia caused by local inflammation or nerve damage in animal models of pain and inflammation. This finding encourages further exploration of the pharmacology of NAAA inhibitors. PMID:24798679

  12. Cathepsin D-mediated yolk protein degradation is blocked by acid phosphatase inhibitors.

    Fialho, Eliane; Nakamura, Angelica; Juliano, Luiz; Masuda, Hatisaburo; Silva-Neto, Mário A C

    2005-04-15

    Vitellin (VT) is a lipoglycophosphoprotein stored inside the eggs of every oviparous organism during oogenesis. In the blood-sucking bug Rhodnius prolixus, VT is deposited inside growing oocytes together with two acid hydrolases: acid phosphatase (AP) and cathepsin D (CD). Egg fertilization triggers AP activity and VT proteolysis in vivo [Insect Biochem. Mol. Biol. 2002 (32) 847]. Here, we show that CD is the main protease targeting VT proteolysis during egg development. CD activity in total egg homogenates is blocked by the classical aspartyl protease inhibitor, pepstatin A. Surprisingly, AP inhibitors such as NaF, Na+/K+ tartrate, and inorganic phosphate also block VT proteolysis, whereas this effect is not observed when tyrosine phosphatase inhibitors such as vanadate and phenylarsine oxide or an inhibitor of alkaline phosphatases such as levamisole are used in a VT proteolysis assay. NaF concentrations that block isolated AP activity do not affect the activity of partially purified CD. Therefore, a specific repressor of VT proteolysis must be dephosphorylated by AP in vivo. In conclusion, these results demonstrate for the first time that acid hydrolases act cooperatively to promote yolk degradation during egg development in arthropods. PMID:15797237

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

    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

  14. Soluble Epoxide Hydrolase Deficiency or Inhibition Attenuates Diet-induced Endoplasmic Reticulum Stress in Liver and Adipose Tissue*

    Bettaieb, Ahmed; Nagata, Naoto; AbouBechara, Daniel; Chahed, Samah; Morisseau, Christophe; Hammock, Bruce D; Haj, Fawaz G.

    2013-01-01

    Background: Soluble epoxide hydrolase (sEH) is a cytosolic enzyme whose pharmacological inhibition or targeted deletion in mice has beneficial effects, including improved insulin signaling in liver and adipose tissue.

  15. 3-D QSAR ANALYSIS OF INHIBITION OF MURINE SOLUBLE EPOXIDE HYDROLASE (MSEH) BY BENZOYLUREAS, ARYLUREAS, AND THEIR ANALOGUES. (R825433)

    Two hundred and seventy-one compounds including benzoylureas, arylureas and related compounds were assayed using recombinant murine soluble epoxide hydrolase (MsEH) produced from a baculovirus expression system. Among all the insect growth regulators assayed, 18 benzoylphenylu...

  16. Structure Determination and Characterization of the Vitamin B[superscript 6] Degradative Enzyme (E)-2-(Acetamidomethylene)succinate Hydrolase

    McCulloch, Kathryn M.; Mukherjee, Tathagata; Begley, Tadhg P.; Ealick, Steven E. (Cornell); (TAM)

    2010-06-22

    The gene identification and kinetic characterization of (E)-2-(acetamidomethylene)succinate (E-2AMS) hydrolase has recently been described. This enzyme catalyzes the final reaction in the degradation of vitamin B{sub 6} and produces succinic semialdehyde, acetate, ammonia, and carbon dioxide from E-2AMS. The structure of E-2AMS hydrolase was determined to 2.3 {angstrom} using SAD phasing. E-2AMS hydrolase is a member of the {alpha}/{beta} hydrolase superfamily and utilizes a serine/histidine/aspartic acid catalytic triad. Mutation of either the nucleophilic serine or the aspartate resulted in inactive enzyme. Mutation of an additional serine residue in the active site causes the enzyme to be unstable and is likely structurally important. The structure also provides insight into the mechanism of hydrolysis of E-2AMS and identifies several potential catalytically important residues.

  17. Epoxy fatty acids and inhibition of the soluble epoxide hydrolase selectively modulate GABA mediated neurotransmission to delay onset of seizures.

    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. Soluble epoxide hydrolase inhibition and gene deletion are protective against myocardial ischemia-reperfusion injury in vivo.

    Motoki, Atsuko; Merkel, Matthias J; Packwood, William H; Cao, Zhiping; Liu, Lijuan; Iliff, Jeffrey; Alkayed, Nabil J; Van Winkle, Donna M

    2008-11-01

    Soluble epoxide hydrolase (sEH) metabolizes epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids. EETs are formed from arachidonic acid during myocardial ischemia and play a protective role against ischemic cell death. Deletion of sEH has been shown to be protective against myocardial ischemia in the isolated heart preparation. We tested the hypothesis that sEH inactivation by targeted gene deletion or pharmacological inhibition reduces infarct size (I) after regional myocardial ischemia-reperfusion injury in vivo. Male C57BL\\6J wild-type or sEH knockout mice were subjected to 40 min of left coronary artery (LCA) occlusion and 2 h of reperfusion. Wild-type mice were injected intraperitoneally with 12-(3-adamantan-1-yl-ureido)-dodecanoic acid butyl ester (AUDA-BE), a sEH inhibitor, 30 min before LCA occlusion or during ischemia 10 min before reperfusion. 14,15-EET, the main substrate for sEH, was administered intravenously 15 min before LCA occlusion or during ischemia 5 min before reperfusion. The EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (EEZE) was given intravenously 15 min before reperfusion. Area at risk (AAR) and I were assessed using fluorescent microspheres and triphenyltetrazolium chloride, and I was expressed as I/AAR. I was significantly reduced in animals treated with AUDA-BE or 14,15-EET, independent of the time of administration. The cardioprotective effect of AUDA-BE was abolished by the EET antagonist 14,15-EEZE. Immunohistochemistry revealed abundant sEH protein expression in left ventricular tissue. Strategies to increase 14,15-EET, including sEH inactivation, may represent a novel therapeutic approach for cardioprotection against myocardial ischemia-reperfusion injury. PMID:18835921

  19. Juggling jobs: roles and mechanisms of multifunctional protease inhibitors in plants.

    Grosse-Holz, Friederike M; van der Hoorn, Renier A L

    2016-05-01

    Multifunctional protease inhibitors juggle jobs by targeting different enzymes and thereby often controlling more than one biological process. Here, we discuss the biological functions, mechanisms and evolution of three types of multifunctional protease inhibitors in plants. The first type is double-headed inhibitors, which feature two inhibitory sites targeting proteases with different specificities (e.g. Bowman-Birk inhibitors) or even different hydrolases (e.g. α-amylase/protease inhibitors preventing both early germination and seed predation). The second type consists of multidomain inhibitors which evolved by intragenic duplication and are released by processing (e.g. multicystatins and potato inhibitor II, implicated in tuber dormancy and defence, respectively). The third type consists of promiscuous inhibitory folds which resemble mouse traps that can inhibit different proteases cleaving the bait they offer (e.g. serpins, regulating cell death, and α-macroglobulins). Understanding how multifunctional inhibitors juggle biological jobs increases our knowledge of the connections between the networks they regulate. These examples show that multifunctionality evolved independently from a remarkable diversity of molecular mechanisms that can be exploited for crop improvement and provide concepts for protein design. PMID:26800491

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

    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°.

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

    The 31.3 kDa Ap4A hydrolase from Shigella flexneri 2a has been cloned, expressed and purified using an Escherichia coli expression system. Crystals of Ap4A hydrolase have been obtained by the hanging-drop technique at 291 K using PEG 550 MME as precipitant. Diadenosine tetraphosphate (Ap4A) hydrolase (EC 3.6.1.41) hydrolyzes Ap4A symmetrically in prokaryotes. It plays a potential role in organisms by regulating the concentration of Ap4A in vivo. To date, no three-dimensional structures of proteins with significant sequence homology to this protein have been determined. The 31.3 kDa Ap4A hydrolase from Shigella flexneri 2a has been cloned, expressed and purified using an Escherichia coli expression system. Crystals of Ap4A hydrolase have been obtained by the hanging-drop technique at 291 K using PEG 550 MME as precipitant. Ap4A hydrolase crystals diffract X-rays to 3.26 Å and belong to space group P21, with unit-cell parameters a = 118.9, b = 54.6, c = 128.5 Å, β = 95.7°

  2. New families in the classification of glycosyl hydrolases based on amino acid sequence similarities.

    Henrissat, B; Bairoch, A

    1993-01-01

    301 glycosyl hydrolases and related enzymes corresponding to 39 EC entries of the I.U.B. classification system have been classified into 35 families on the basis of amino-acid-sequence similarities [Henrissat (1991) Biochem. J. 280, 309-316]. Approximately half of the families were found to be monospecific (containing only one EC number), whereas the other half were found to be polyspecific (containing at least two EC numbers). A > 60% increase in sequence data for glycosyl hydrolases (181 additional enzymes or enzyme domains sequences have since become available) allowed us to update the classification not only by the addition of more members to already identified families, but also by the finding of ten new families. On the basis of a comparison of 482 sequences corresponding to 52 EC entries, 45 families, out of which 22 are polyspecific, can now be defined. This classification has been implemented in the SWISS-PROT protein sequence data bank. PMID:8352747

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

    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. PMID:26476165

  4. Hydrolases as Catalysts for Green Chemistry and Industrial Applications - Esterase, Lipase and Phytase

    Gaber, Yasser

    2012-01-01

    The use of enzymes in industrial applications has been recognised for providing clean processes with minimal impact on the environment. This thesis presents studies on engineering of enzymes and enzyme-based processes in the light of green chemistry and environmental sustainability, and focuses on three hydrolases: esterase, lipase and phytase. The use of esterase has been investigated to provide an alternative clean route for the synthesis of a chiral pharmaceutical compound, ...

  5. Dual roles of brain serine hydrolase KIAA1363 in ether lipid metabolism and organophosphate detoxification

    Serine hydrolase KIAA1363 is an acetyl monoalkylglycerol ether (AcMAGE) hydrolase involved in tumor cell invasiveness. It is also an organophosphate (OP) insecticide-detoxifying enzyme. The key to understanding these dual properties was the use of KIAA1363 +/+ (wildtype) and -/- (gene deficient) mice to define the role of this enzyme in brain and other tissues and its effectiveness in vivo in reducing OP toxicity. KIAA1363 was the primary AcMAGE hydrolase in brain, lung, heart and kidney and was highly sensitive to inactivation by chlorpyrifos oxon (CPO) (IC50 2 nM) [the bioactivated metabolite of the major insecticide chlorpyrifos (CPF)]. Although there was no difference in hydrolysis product monoalkylglycerol ether (MAGE) levels in +/+ and -/- mouse brains in vivo, isopropyl dodecylfluorophosphonate (30 mg/kg) and CPF (100 mg/kg) resulted in 23-51% decrease in brain MAGE levels consistent with inhibition of AcMAGE hydrolase activity. On incubating +/+ and -/- brain membranes with AcMAGE and cytidine-5'-diphosphocholine, the absence of KIAA1363 activity dramatically increased de novo formation of platelet-activating factor (PAF) and lyso-PAF, signifying that metabolically-stabilized AcMAGE can be converted to this bioactive lipid in brain. On considering detoxification, KIAA1363 -/- mice were significantly more sensitive than +/+ mice to ip-administered CPF (100 mg/kg) and parathion (10 mg/kg) with increased tremoring and mortality that correlated for CPF with greater brain acetylcholinesterase inhibition. Docking AcMAGE and CPO in a KIAA1363 active site model showed similar positioning of their acetyl and trichloropyridinyl moieties, respectively. This study establishes the relevance of KIAA1363 in ether lipid metabolism and OP detoxification

  6. GENETIC VARIATION IN SOLUBLE EPOXIDE HYDROLASE (EPHX2) IS ASSOCIATED WITH FOREARM VASODILATOR RESPONSES IN HUMANS

    Lee, Craig R.; Pretorius, Mias; Schuck, Robert N.; Burch, Lauranell H.; Bartlett, Jackie; Williams, Scott M.; ZELDIN, DARRYL C.; Brown, Nancy J.

    2010-01-01

    Cytochrome P450-derived epoxyeicosatrienoic acids are potent vasodilators in preclinical models and are hydrolyzed by soluble epoxide hydrolase (EPHX2). Associations between the EPHX2 Lys55Arg and Arg287Gln polymorphisms and cardiovascular disease risk have been reported; however, their impact on vascular function in humans has not been investigated. In 265 volunteers (198 white, 67 black American), forearm blood flow was measured by strain-gauge venous occlusion plethysmography at baseline a...

  7. Structural and Enzymatic Characterization of a Nucleoside Diphosphate Sugar Hydrolase from Bdellovibrio bacteriovorus

    de la Peña, Andres H.; Suarez, Allison; Duong-Ly, Krisna C.; Schoeffield, Andrew J.; Pizarro-Dupuy, Mario A.; Zarr, Melissa; Pineiro, Silvia A.; Amzel, L. Mario; Gabelli, Sandra B.

    2015-01-01

    Given the broad range of substrates hydrolyzed by Nudix (nucleoside diphosphate linked to X) enzymes, identification of sequence and structural elements that correctly predict a Nudix substrate or characterize a family is key to correctly annotate the myriad of Nudix enzymes. Here, we present the structure determination and characterization of Bd3179 –- a Nudix hydrolase from Bdellovibrio bacteriovorus–that we show localized in the periplasmic space of this obligate Gram-negative predator. We...

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

    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...

  9. Organophosphate Hydrolase in Conductometric Biosensor for the Detection of Organophosphate Pesticides

    Ani Mulyasuryani; Sasangka Prasetyawan

    2015-01-01

    The research has developed an enzyme biosensor for the detection organophosphate pesticide residues. The biosensor consists of a pair of screen-printed carbon electrode (SPCEs). One of electrodes contains immobilized organophosphate hydrolase (OPH) on a chitosan membrane by cross-linking it with glutaraldehyde. The area of the electrodes was optimized to 3, 5, and 7 mm2. The OPH was isolated from Pseudomonas putida, and was purified by the ammonium sulfate precipitation method, with 6444 ppm ...

  10. Development of an efficient 18O epoxide labeling method: Investigation of the cytosolic expoxide hydrolase mechanism

    Mechanistic studies of Cytosolic Epoxide Hydrolase (CEH) reveal an anti opening of the epoxide arising from base catalyzed activation of H2O, possibly in concert with acidic activation of the epoxide oxygen. Preliminary data on the hydrolysis of fatty acid oxides shows the CEH is able to discriminate between chemically equivalent epoxide carbons. The research is geared toward methodology development in which CEH could be used as a organic synthetic tool, with substrate and product regio-, stereo-, and enantioselectivity

  11. Inhibition of epoxide hydrolase by valproic acid in epileptic patients receiving carbamazepine.

    Robbins, D K; Wedlund, P J; Kuhn, R.; Baumann, R J; Levy, R.H.; Chang, S.L.

    1990-01-01

    The effect of valproic acid (VPA) on the disposition of carbamazepine-10,11-epoxide (epoxide) was studied in five epileptic patients on chronic carbamazepine (CBZ) therapy. The individual pharmacokinetic parameters influencing epoxide disposition were determined in the presence and absence of VPA. VPA significantly decreased the clearance of unbound epoxide (an in vivo index of epoxide hydrolase activity), but did not appear to affect epoxide formation. VPA also increased the free concentrati...

  12. Soluble Epoxide Hydrolase Inhibition: Targeting Multiple Mechanisms of Ischemic Brain Injury with a Single Agent

    Iliff, Jeffrey J.; Alkayed, Nabil J.

    2009-01-01

    Soluble epoxide hydrolase (sEH) is a key enzyme in the metabolic conversion and degradation of P450 eicosanoids called epoxyeicosatrienoic acids (EETs). Genetic variations in the sEH gene, designated EPHX2, are associated with ischemic stroke risk. In experimental studies, sEH inhibition and gene deletion reduce infarct size after focal cerebral ischemia in mice. Although the precise mechanism of protection afforded by sEH inhibition remains under investigation, EETs exhibit a wide array of p...

  13. The neutral cysteine protease bleomycin hydrolase is essential for epidermal integrity and bleomycin resistance

    Schwartz, Donald R.; Homanics, Gregg E.; Hoyt, Dale G.; Klein, Ed; Abernethy, John; Lazo, John S.

    1999-01-01

    The papain superfamily member bleomycin hydrolase (Blmh) is a neutral cysteine protease with structural similarity to a 20S proteasome. Bleomycin (BLM), a clinically used glycopeptide anticancer agent, is deaminated in vitro by Blmh. We used gene targeting to generate mice that lack Blmh and demonstrated that Blmh is the sole enzyme required for BLM deamination. Although some Blmh null mice were viable and reproduced, only about 65% of the expected number survived the neonatal period, reveali...

  14. Directed Evolution of an Enantioselective Epoxide Hydrolase : Uncovering the Source of Enantioselectivity at Each Evolutionary Stage

    Reetz, Manfred T.; Bocola, Marco; Wang, Li-Wen; Sanchis, Joaquin; Cronin, Annette; Arand, Michael; Zou, Jinyu; Archelas, Alain; Bottalla, Anne-Lise; Naworyta, Agata; Mowbray, Sherry L.

    2009-01-01

    Directed evolution of enzymes as enantioselective catalysts in organic chemistry is an alternative to traditional asymmetric catalysis using chiral transition-metal complexes or organocatalysts, the different approaches often being complementary. Moreover, directed evolution studies allow us to learn more about how enzymes perform mechanistically. The present study concerns a previously evolved highly enantioselective mutant of the epoxide hydrolase from Aspergillus niger in the hydrolytic ki...

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

    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.

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

    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 P43212, 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 P43212, 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

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

    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.

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

    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

  19. Post-exposure administration of diazepam combined with soluble epoxide hydrolase inhibition stops seizures and modulates neuroinflammation in a murine model of acute TETS intoxication

    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

  20. Characterization of a Nudix hydrolase from Deinococcus radiodurans with a marked specificity for (deoxyribonucleoside 5'-diphosphates

    Kamiya Hiroyuki

    2004-05-01

    Full Text Available Abstract Background Nudix hydrolases form a protein family whose function is to hydrolyse intracellular nucleotides and so regulate their levels and eliminate potentially toxic derivatives. The genome of the radioresistant bacterium Deinococcus radiodurans encodes 25 nudix hydrolases, an unexpectedly large number. These may contribute to radioresistance by removing mutagenic oxidised and otherwise damaged nucleotides. Characterisation of these hydrolases is necessary to understand the reason for their presence. Here, we report the cloning and characterisation of the DR0975 gene product, a nudix hydrolase that appears to be unique to this organism. Results The DR0975 gene was cloned and expressed as a 20 kDa histidine-tagged recombinant product in Escherichia coli. Substrate analysis of the purified enzyme showed it to act primarily as a phosphatase with a marked preference for (deoxynucleoside 5'-diphosphates (dGDP > ADP > dADP > GDP > dTDP > UDP > dCDP > CDP. Km for dGDP was 110 μM and kcat was 0.18 s-1 under optimal assay conditions (pH 9.4, 7.5 mM Mg2+. 8-Hydroxy-2'-deoxyguanosine 5'-diphosphate (8-OH-dGDP was also a substrate with a Km of 170 μM and kcat of 0.13 s-1. Thus, DR0975 showed no preference for 8-OH-dGDP over dGDP. Limited pyrophosphatase activity was also observed with NADH and some (diadenosine polyphosphates but no other substrates. Expression of the DR0975 gene was undetectable in logarithmic phase cells but was induced at least 30-fold in stationary phase. Superoxide, but not peroxide, stress and slow, but not rapid, dehydration both caused a slight induction of the DR0975 gene. Conclusion Nucleotide substrates for nudix hydrolases conform to the structure NDP-X, where X can be one of several moieties. Thus, a preference for (dNDPs themselves is most unusual. The lack of preference for 8-OH-dGDP over dGDP as a substrate combined with the induction in stationary phase, but not by peroxide or superoxide, suggests that the

  1. Proton pump inhibitors

    Proton pump inhibitors (PPIs) are medicines that work by reducing the amount of stomach acid made by glands in ... Proton pump inhibitors are used to: Relieve symptoms of acid reflux, or gastroesophageal reflux disease (GERD). This is a ...

  2. Proton pump inhibitors

    Proton pump inhibitors (PPIs) are medicines that work by reducing the amount of stomach acid made by ... Proton pump inhibitors are used to: Relieve symptoms of acid reflux, or gastroesophageal reflux disease (GERD). This ...

  3. Preclinical Characterization of the FAAH Inhibitor JNJ-42165279.

    Keith, John M; Jones, William M; Tichenor, Mark; Liu, Jing; Seierstad, Mark; Palmer, James A; Webb, Michael; Karbarz, Mark; Scott, Brian P; Wilson, Sandy J; Luo, Lin; Wennerholm, Michelle L; Chang, Leon; Rizzolio, Michele; Rynberg, Raymond; Chaplan, Sandra R; Breitenbucher, J Guy

    2015-12-10

    The pre-clinical characterization of the aryl piperazinyl urea inhibitor of fatty acid amide hydrolase (FAAH) JNJ-42165279 is described. JNJ-42165279 covalently inactivates the FAAH enzyme, but is highly selective with regard to other enzymes, ion channels, transporters, and receptors. JNJ-42165279 exhibited excellent ADME and pharmacodynamic properties as evidenced by its ability to block FAAH in the brain and periphery of rats and thereby cause an elevation of the concentrations of anandamide (AEA), oleoyl ethanolamide (OEA), and palmitoyl ethanolamide (PEA). The compound was also efficacious in the spinal nerve ligation (SNL) model of neuropathic pain. The combination of good physical, ADME, and PD properties of JNJ-42165279 supported it entering the clinical portfolio. PMID:26713105

  4. Systemic and spinal administration of FAAH, MAGL inhibitors and dual FAAH/MAGL inhibitors produce antipruritic effect in mice.

    Yesilyurt, Ozgur; Cayirli, Mutlu; Sakin, Yusuf Serdar; Seyrek, Melik; Akar, Ahmet; Dogrul, Ahmet

    2016-07-01

    The increase of endocannabinoid tonus by inhibiting fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL) represents a promising therapeutic approach in a variety of disease to overcome serious central side effects of exocannabinoids. Recent studies reported that systemic administration of FAAH and MAGL inhibitors produce antipruritic action. Dual FAAH/MAGL inhibitors have also been described to get enhanced endocannabinoid therapeutic effect. In this study, we examined and compared dose-related antipruritic effects of systemic (intraperitoneal; ip) or intrathecal (it) administration of selective FAAH inhibitor PF-3845 (5, 10, and 20 mg/kg, i.p.; 1, 5, and 10 µg, i.t.), MAGL inhibitor JZL184 (4, 20, and 40 mg/kg, i.p.; 1, 5, and 10 µg, i.t.) and dual FAAH/MAGL inhibitor JZL195 (2, 5, and 20 mg/kg, i.p.; 1, 5, and 10 µg, i.t.) on serotonin (5-HT)-induced scratching model. Serotonin (25 μg) was injected intradermally in a volume of 50 μl into the rostral part of skin on the back of male Balb-C mice. Both systemic or intrathecal administration of PF-3845, JZL184 or JZL195 produced similar dose-dependent antipruritic effects. Our results suggest that endocannabinoid-degrading enzymes FAAH and MAGL are involved in pruritic process at spinal level. FAAH, MAGL or dual FAAH/MAGL inhibitors have promising antipruritic effects, at least, in part through spinal site of action. PMID:27126057

  5. Combretastatin A-4 and Derivatives: Potential Fungicides Targeting Fungal Tubulin.

    Ma, Zhong-lin; Yan, Xiao-jing; Zhao, Lei; Zhou, Jiu-jiu; Pang, Wan; Kai, Zhen-peng; Wu, Fan-hong

    2016-02-01

    Combretastatin A-4, first isolated from the African willow tree Combretum caffrum, is a tubulin polymerization inhibitor in medicine. It was first postulated as a potential fungicide targeting fungal tubulin for plant disease control in this study. Combretastatin A-4 and its derivatives were synthesized and tested against Rhizoctonia solani and Pyricularia oryzae. Several compounds have EC50 values similar to or better than that of isoprothiolane, which is widely used for rice disease control. Structure-activity relationship study indicated the the cis configuration and hydroxyl group in combretastatin A-4 are crucial to the antifungal effect. Molecular modeling indicated the binding sites of combretastatin A-4 and carbendazim on fungal tubulin are totally different. The bioactivity of combretastatin A-4 and its derivatives against carbendazim-resistant strains was demonstrated in this study. The results provide a new approach for fungicide discovery and fungicide resistance management. PMID:26711170

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

    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.

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

    Patricia Marie Legler

    2014-07-01

    Full Text Available 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.

  8. Lysosomal degradation of receptor-bound urokinase-type plasminogen activator is enhanced by its inhibitors in human trophoblastic choriocarcinoma cells

    Jensen, Poul Henning; Christensen, Erik Ilsø; Ebbesen, P.;

    1990-01-01

    apparently intact form in the medium or was still cell associated. The degradation could be inhibited by inhibitors of vesicle transport and lysosomal hydrolases. By electron microscopic autoradiography, both 125I-u-PA and 125I-u-PA-inhibitor complexes were located over the cell membrane at 4 degrees C, with...... the highest density of grains over the membrane at cell-cell interphases, but, after incubation at 37 degrees C, 17 and 27% of the grains for u-PA and u-PA-PAI-1 complexes, respectively, appeared over lysosomal-like bodies. These findings suggest that the u-PA receptor possesses a clearance function...

  9. Discovery of MK-3168: A PET Tracer for Imaging Brain Fatty Acid Amide Hydrolase.

    Liu, Ping; Hamill, Terence G; Chioda, Marc; Chobanian, Harry; Fung, Selena; Guo, Yan; Chang, Linda; Bakshi, Raman; Hong, Qingmei; Dellureficio, James; Lin, Linus S; Abbadie, Catherine; Alexander, Jessica; Jin, Hong; Mandala, Suzanne; Shiao, Lin-Lin; Li, Wenping; Sanabria, Sandra; Williams, David; Zeng, Zhizhen; Hajdu, Richard; Jochnowitz, Nina; Rosenbach, Mark; Karanam, Bindhu; Madeira, Maria; Salituro, Gino; Powell, Joyce; Xu, Ling; Terebetski, Jenna L; Leone, Joseph F; Miller, Patricia; Cook, Jacquelynn; Holahan, Marie; Joshi, Aniket; O'Malley, Stacey; Purcell, Mona; Posavec, Diane; Chen, Tsing-Bau; Riffel, Kerry; Williams, Mangay; Hargreaves, Richard; Sullivan, Kathleen A; Nargund, Ravi P; DeVita, Robert J

    2013-06-13

    We report herein the discovery of a fatty acid amide hydrolase (FAAH) positron emission tomography (PET) tracer. Starting from a pyrazole lead, medicinal chemistry efforts directed toward reducing lipophilicity led to the synthesis of a series of imidazole analogues. Compound 6 was chosen for further profiling due to its appropriate physical chemical properties and excellent FAAH inhibition potency across species. [(11)C]-6 (MK-3168) exhibited good brain uptake and FAAH-specific signal in rhesus monkeys and is a suitable PET tracer for imaging FAAH in the brain. PMID:24900701

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

    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 and...... lysosomes of glycans originating from e.g. terminally misfolded proteins. A divalent metal ion typically resides in the active site, and is essential for enzyme activity. Recently, the α-mannosidase II from Sulfolobus solfataricus (ManA) was purified and characterized as a Zn2+-containing enzyme, but also...

  11. Characterization of Two New Glycosyl Hydrolases from the Lactic Acid Bacterium Carnobacterium piscicola Strain BA

    Coombs, Jonna; Brenchley, Jean E.

    2001-01-01

    Three genes with homology to glycosyl hydrolases were detected on a DNA fragment cloned from a psychrophilic lactic acid bacterium isolate, Carnobacterium piscicola strain BA. A 2.2-kb region corresponding to an α-galactosidase gene, agaA, was followed by two genes in the same orientation, bgaB, encoding a 2-kb β-galactosidase, and bgaC, encoding a structurally distinct 1.76-kb β-galactosidase. This gene arrangement had not been observed in other lactic acid bacteria, including Lactococcus la...

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

    Bicalho, Beatriz; Chen, Lu S.; Marsaioli, Anita J. [Universidade Estadual de Campinas, SP (Brazil). Inst. de Quimica]. E-mail: anita@iqm.unicamp.br; Grognux, Johann; Reymond, Jean-Louis [University of Berne (Switzerland). Dept. of Chemistry and Biochemistry

    2004-12-01

    Biocatalysis reactions were performed on microtiter plates (200 {mu}L) aiming at the utilization of fluorogenic substrates (100 {mu}mol L{sup -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)

  13. Detoxication strategy of epoxide hydrolase-the basis for a novel threshold for definable genotoxic carcinogens

    Oesch, Franz; Hengstler, Jan Georg; Arand, Michael

    2004-01-01

    From our recent work on the three-dimensional structure of epoxide hydrolases we theoretically deduced the likelihood of a two-step catalytic mechanism that we and others have subsequently experimentally confirmed. Analysis of the rate of the two steps by us and by others show that the first step—responsible for removal of the reactive epoxide from the system—works extraordinarily fast (typically three orders of magnitude faster than the second step), sucking up the epoxide like a sponge. Reg...

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

    Patricia Marie Legler; Susanne eBoisvert; Compton, Jaimee R.; Millard, Charles B.

    2014-01-01

    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 nucl...

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

    Legler, Patricia M.; Boisvert, Susanne M.; Compton, Jaimee R.; Millard, Charles B.

    2014-01-01

    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 nucleo...

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

    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)

  17. Genetic variants in microsomal epoxide hydrolase and N-acetyltransferase 2 in susceptibility of IBD in the Danish population

    Ernst, Anja; Andersen, Vibeke; Østergaard, Mette;

    induce or sustain an immune response. Changes in detoxification of substances that causes epithelial damage may confer susceptibility to IBD. Hence, polymorphic enzymes involved in the detoxification processes may be risk factors of IBD. Methods. The two biotransformation enzymes microsomal epoxide...... hydrolase and N-acetyltransferase 2 were genotyped using TaqMan based Real-Time PCR in 388 patients with Crohn's disease (CD), 565 patients with ulcerative colitis (UC) and 796 healthy Danish controls. Results. No association was found between low microsomal epoxide hydrolase activity or slow N......-acetyltransferase 2 acetylator status and IBD. An association between high activity of microsomal epoxide hydrolase and disease diagnosis before age 40 in CD with an OR of 2.2(1.1- 4.2) P=0.02) was found. No other phenotypic associations were found for the two enzymes and IBD, regarding age at onset, disease location...

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

    Hansen, Michael Riis; Dandanell, Gert

    2005-01-01

    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......C most likely prefers the neutral form of xanthosine....

  19. Partial purification and characterization of an inducible indole-3-acetyl-L-aspartic acid hydrolase from Enterobacter agglomerans

    Chou, Jyh-Ching [Department of Agriculture, Beltsville, MD (United States)]|[Univ. of Maryland, College Park, MD (United States); Cohen, J.D.; Mulbry, W.W. [Department of Agriculture, Beltsville, MD (United States)] [and others

    1996-11-01

    Indole-3-acetyl-amino acid conjugate hydrolases are believed to be important in the regulation of indole-3-acetic acid (IAA) metabolism in plants and therefore have potential uses for the alteration of plant IAA metabolism. To isolate bacterial strains exhibiting significant indole-3-acetyl-aspartate (IAA-Asp) hydrolase activity, a sewage sludge inoculation was cultured under conditions in which IAA-Asp served as the sole source of carbon and nitrogen. One isolate, Enterobacter agglomerans, showed hydrolase activity inducible by IAA-L-Asp or N-acetyl-L-Asp but not by IAA, (NH{sub 4}){sub 2}SO{sub 4}, urea, or indoleacetamide. Among a total of 17 IAA conjugates tested as potential substrates, the enzyme had an exclusively high substrate specificity for IAA-L-Asp of 13.5 mM. The optimal pH for this enzyme was between 8.0 and 8.5. In extraction buffer containing 0.8 mM Mg{sup 2+} the hydrolase activity was inhibited to 80% by 1 mM dithiothreitol and to 60% by 1 mm CuSO{sub 4}; the activity was increased by 40% with 1mM MnSO{sub 4}. However, in extraction buffer with no trace elements, the hydrolase activity was inhibited to 50% by either 1 mM dithiothreitol or 1% Triton X-100 (Sigma). These results suggest that disulfide bonding might be essential for enzyme activity. Purification of the hydrolase by hydroxyapatite and TSK-phenyl (HP-Genenchem, South San Francisco, CA) preparative high-performance liquid chromatography yielded a major 45-kD polypeptide as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 45 refs., 5 figs., 3 tabs.

  20. Pharmacological blockade of the fatty acid amide hydrolase (FAAH) alters neural proliferation, apoptosis and gliosis in the rat hippocampus, hypothalamus and striatum in a negative energy context.

    Rivera, Patricia; Bindila, Laura; Pastor, Antoni; Pérez-Martín, Margarita; Pavón, Francisco J; Serrano, Antonia; de la Torre, Rafael; Lutz, Beat; Rodríguez de Fonseca, Fernando; Suárez, Juan

    2015-01-01

    Endocannabinoids participate in the control of neurogenesis, neural cell death and gliosis. The pharmacological effect of the fatty acid amide hydrolase (FAAH) inhibitor URB597, which limits the endocannabinoid degradation, was investigated in the present study. Cell proliferation (phospho-H3(+) or BrdU(+) cells) of the main adult neurogenic zones as well as apoptosis (cleaved caspase-3(+)), astroglia (GFAP(+)), and microglia (Iba1(+) cells) were analyzed in the hippocampus, hypothalamus and striatum of rats intraperitoneally treated with URB597 (0.3 mg/kg/day) at one dose/4-days resting or 5 doses (1 dose/day). Repeated URB597 treatment increased the plasma levels of the N-acylethanolamines oleoylethanolamide, palmitoylethanolamide and arachidonoylethanolamine, reduced the plasma levels of glucose, triglycerides and cholesterol, and induced a transitory body weight decrease. The hippocampi of repeated URB597-treated rats showed a reduced number of phospho-H3(+) and BrdU(+) subgranular cells as well as GFAP(+), Iba1(+) and cleaved caspase-3(+) cells, which was accompanied with decreased hippocampal expression of the cannabinoid CB1 receptor gene Cnr1 and Faah. In the hypothalami of these rats, the number of phospho-H3(+), GFAP(+) and 3-weeks-old BrdU(+) cells was specifically decreased. The reduced striatal expression of CB1 receptor in repeated URB597-treated rats was only associated with a reduced apoptosis. In contrast, the striatum of acute URB597-treated rats showed an increased number of subventricular proliferative, astroglial and apoptotic cells, which was accompanied with increased Faah expression. Main results indicated that FAAH inhibitor URB597 decreased neural proliferation, glia and apoptosis in a brain region-dependent manner, which were coupled to local changes in Faah and/or Cnr1 expression and a negative energy context. PMID:25870539

  1. Pharmacological blockade of the fatty acid amide hydrolase (FAAH alters neural proliferation, apoptosis and gliosis in the rat hippocampus, hypothalamus and striatum in a negative energy context

    Patricia eRivera

    2015-03-01

    Full Text Available Endocannabinoids participate in the control of neurogenesis, neural cell death and gliosis. The pharmacological effect of the fatty acid amide hydrolase (FAAH inhibitor URB597, which limits the endocannabinoid degradation, was investigated in the present study. Cell proliferation (phospho-H3+ or BrdU+ cells of the main adult neurogenic zones as well as apoptosis (cleaved caspase-3+, astroglia (GFAP+, and microglia (Iba1+ cells were analyzed in the hippocampus, hypothalamus and striatum of rats intraperitoneally treated with URB597 (0.3 mg/kg/day at one dose/4-days resting or 5 doses (1 dose/day. Repeated URB597 treatment increased the plasma levels of the endocannabinoids oleoylethanolamide, palmitoylethanolamide and arachidonoylethanolamine, reduced the plasma levels of glucose, triglycerides and cholesterol, and induced a transitory body weight decrease. The hippocampi of repeated URB597-treated rats showed a reduced number of phospho-H3+ and BrdU+ subgranular cells as well as GFAP+, Iba1+ and cleaved caspase-3+ cells, which was accompanied with decreased hippocampal expressions of cannabinoid CB1 receptor and FAAH. In the hypothalami of these rats, the number of phospho-H3+, GFAP+ and 3-weeks-old BrdU+ cells was specifically decreased. The reduced striatal expression of CB1 receptor in repeated URB597-treated rats was only associated with a reduced apoptosis. In contrast, the striatum of acute URB597-treated rats showed an increased number of subventricular proliferative, astroglial and apoptotic cells, which was accompanied with increased Faah expression. Main results indicated that FAAH inhibitor URB597 decreased neural proliferation, glia and apoptosis in a brain region-dependent manner, which were coupled to local changes in FAAH and/or CB1 receptor expressions and a negative energy context.

  2. The synthesis and in vivo evaluation of [18F]PF-9811: a novel PET ligand for imaging brain fatty acid amide hydrolase (FAAH)

    Introduction: Fatty acid amide hydrolase (FAAH) is responsible for the enzymatic degradation of the fatty acid amide family of signaling lipids, including the endogenous cannabinoid (endocannabinoid) anandamide. The involvement of the endocannabinoid system in pain and other nervous system disorders has made FAAH an attractive target for drug development. Companion molecular imaging probes are needed, however, to assess FAAH inhibition in the nervous system in vivo. We report here the synthesis and in vivo evaluation of [18F]PF-9811, a novel PET ligand for non-invasive imaging of FAAH in the brain. Methods: The potency and selectivity of unlabeled PF-9811 were determined by activity-based protein profiling (ABPP) both in vitro and in vivo. [18F]PF-9811 was synthesized in a 3-step, one-pot reaction sequence, followed by HPLC purification. Biological evaluation was performed by biodistribution and dynamic PET imaging studies in male rats. The specificity of [18F]PF-9811 uptake was evaluated by pre-administration of PF-04457845, a potent and selective FAAH inhibitor, 1 h prior to radiotracer injection. Results: Biodistribution studies show good uptake (SUV ∼ 0.8 at 90 min) of [18F]PF-9811 in rat brain, with significant reduction of the radiotracer in all brain regions (37%–73% at 90 min) in blocking experiments. Dynamic PET imaging experiments in rat confirmed the heterogeneous uptake of [18F]PF-9811 in brain regions with high FAAH enzymatic activity, as well as statistically significant reductions in signal following pre-administration of the blocking compound PF-04457845. Conclusions: [18F]PF-9811 is a promising PET imaging agent for FAAH. Biodistribution and PET imaging experiments show that the tracer has good uptake in brain, regional heterogeneity, and specific binding as determined by blocking experiments with the highly potent and selective FAAH inhibitor, PF-04457845.

  3. Crystallization and preliminary X-ray diffraction analysis of the amidase domain of allophanate hydrolase from Pseudomonas sp. strain ADP

    The amidase domain of the allophanate hydrolase AtzF from Pseudomonas sp. strain ADP has been crystallized and preliminary X-ray diffraction data have been collected. The allophanate hydrolase from Pseudomonas sp. strain ADP was expressed and purified, and a tryptic digest fragment was subsequently identified, expressed and purified. This 50 kDa construct retained amidase activity and was crystallized. The crystals diffracted to 2.5 Å resolution and adopted space group P21, with unit-cell parameters a = 82.4, b = 179.2, c = 112.6 Å, β = 106.6°

  4. X-Ray Diffraction Structure of a plant Glycosyl Hydrolase family 32 protein: Fructan 1-Exohydrolase IIa of Cichorium intybus

    Verhaest, Maureen; Van den Ende, Wim; Le Roy, Katrien; De Ranter, Camiel; Van Laere, André; Rabijns, Anja

    2005-01-01

    Fructan 1-exohydrolase, an enzyme involved in fructan degradation, belongs to the glycosyl hydrolase family 32. The structure of isoenzyme 1-FEH IIa from Cichorium intybus is described at a resolution of 2.35 Å. The structure consists of an N-terminal fivefold β-propeller domain connected to two C-terminal β-sheets. The putative active site is located entirely in the β-propeller domain and is formed by amino acids which are highly conserved within glycosyl hydrolase family 32. The fructan-bin...

  5. Sequence and Expression Analyses of Cytophaga-Like Hydrolases in a Western Arctic Metagenomic Library and the Sargasso Sea†

    Cottrell, Matthew T.; Yu, Liying; Kirchman, David L.

    2005-01-01

    Sequence analysis of environmental DNA promises to provide new insights into the ecology and biogeochemistry of uncultured marine microbes. In this study we used the Sargasso Sea Whole Genome Sequence (WGS) data set to search for hydrolases used by Cytophaga-like bacteria to degrade biopolymers such as polysaccharides and proteins. Analysis of the Sargasso WGS data for contigs bearing both the 16S rRNA genes of Cytophaga-like bacteria and hydrolase genes revealed a cellulase gene (celM) most ...

  6. Effect of limited proteolysis on the stability and enzymatic activity of human placental S-adenosylhomocysteine hydrolase.

    Huang, H; Yuan, C. S.; Borchardt, R. T.

    1997-01-01

    Human placental S-adenosylhomocysteine (AdoHcy) hydrolase was subjected to limited papain digestion. The multiple cleavage sites in the enzyme were identified to be Lys94-Ala95, Tyr100-Ala101, Glu243-Ile244, Met367-Ala368, Gln369-Ile370, and Gly382-Val383. Despite multiple cleavage sites in the backbone of the protein, the digested enzyme was able to maintain its quaternary structure and retain its full catalytic activity. The enzyme activity of the partially digested AdoHcy hydrolase was ess...

  7. Differential Recognition and Hydrolysis of Host Carbohydrate Antigens by Streptococcus pneumoniae Family 98 Glycoside Hydrolases

    Higgins, M.; Whitworth, G; El Warry, N; Randriantsoa, M; Samain, E; Burke, R; Vocadlo, D; Boraston, A

    2009-01-01

    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.

  8. Structural and kinetic insights into the mechanism of 5-hydroxyisourate hydrolase from Klebsiella pneumoniae

    French, Jarrod B.; Ealick, Steven E., E-mail: see3@cornell.edu [Cornell University, Ithaca, NY 14853-1301 (United States)

    2011-08-01

    The crystal structure of 5-hydroxyisourate hydrolase from K. pneumoniae and the steady-state kinetic parameters of the native enzyme as well as several mutants provide insights into the catalytic mechanism of this enzyme and the possible roles of the active-site residues. The stereospecific oxidative degradation of uric acid to (S)-allantoin has recently been demonstrated to proceed via two unstable intermediates and requires three separate enzymatic reactions. The second step of this reaction, the conversion of 5-hydroxyisourate (HIU) to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline, is catalyzed by HIU hydrolase (HIUH). The high-resolution crystal structure of HIUH from the opportunistic pathogen Klebsiella pneumoniae (KpHIUH) has been determined. KpHIUH is a homotetrameric protein that, based on sequence and structural similarity, belongs to the transthyretin-related protein family. In addition, the steady-state kinetic parameters for this enzyme and four active-site mutants have been measured. These data provide valuable insight into the functional roles of the active-site residues. Based upon the structural and kinetic data, a mechanism is proposed for the KpHIUH-catalyzed reaction.

  9. Molecular cloning, expression and characterization of acylpeptide hydrolase in the silkworm, Bombyx mori.

    Fu, Ping; Sun, Wei; Zhang, Ze

    2016-04-10

    Acylpeptide hydrolase (APH) can catalyze the release of the N-terminal amino acid from acetylated peptides. There were many documented examples of this enzyme in various prokaryotic and eukaryotic organisms. However, knowledge about APH in insects still remains unknown. In this study, we cloned and sequenced a putative silkworm Bombyx mori APH (BmAPH) gene. The BmAPH gene encodes a protein of 710 amino acids with a predicted molecular mass of 78.5kDa. The putative BmAPH and mammal APHs share about 36% amino acid sequence identity, yet key catalytic residues are conserved (Ser566, Asp654, and His686). Expression and purification of the recombinant BmAPH in Escherichia coli showed that it has acylpeptide hydrolase activity toward the traditional substrate, Ac-Ala-pNA. Furthermore, organophosphorus (OP) insecticides, chlorpyrifos, phoxim, and malathion, significantly inhibited the activity of the APH both in vitro and in vivo. In addition, BmAPH was expressed in all tested tissues and developmental stages of the silkworm. Finally, immunohistochemistry analysis showed that BmAPH protein was localized in the basement membranes. These results suggested that BmAPH may be involved in enhancing silkworm tolerance to the OP insecticides. In a word, our results provide evidence for understanding of the biological function of APH in insects. PMID:26778207

  10. Direct detection, cloning and characterization of a glucoside hydrolase from forest soil.

    Hua, Mei; Zhao, Shubo; Zhang, Lili; Liu, Dongbo; Xia, Hongmei; Li, Fan; Chen, Shan

    2015-06-01

    A glucoside hydrolase gene, egl01, was cloned from the soil DNA of Changbai Mountain forest by homologous PCR amplification. The deduced sequence of 517 amino acids included a catalytic domain of glycoside hydrolase family 5 and was homologous to a putative cellulase from Bacillus licheniformis. The recombinant enzyme, Egl01, was maximally active at pH 5 and 50 °C and it was stable at pH 3-9, 4-50 °C, and also stable in the presence of metal ions, organic solvents, surfactants and salt. Its activity was above 120 % in 2-3 M NaCl/KCl and over 70 % was retained in 1-4 M NaCl/KCl for 6d. Egl01 hydrolyzed carboxymethyl cellulose, beechwood xylan, crop stalk, laminarin, filter paper, and avicel but not pNPG, indicating its broad substrate specificity. These properties make this recombinant enzyme a promising candidate for industrial applications. PMID:25700816