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Sample records for amidohydrolases

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-10-25

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

  2. Synthesis and characterization of diazomethylarachidonyl ketone: an irreversible inhibitor of N-arachidonylethanolamine amidohydrolase.

    Science.gov (United States)

    Edgemond, W S; Greenberg, M J; McGinley, P J; Muthian, S; Campbell, W B; Hillard, C J

    1998-07-01

    N-Arachidonylethanolamine (AEA), a putative endogenous agonist of neuronal (CB1) cannabinoid receptors, is a substrate for N-arachidonylethanolamine amidohydrolase (AEA amidohydrolase), a serine amidase present in cell membranes. Following a strategy that has been used to develop inhibitors that covalently bind to the active site of serine peptidases, diazomethyl arachidonyl ketone (DAK) was synthesized and its effects on AEA amidohydrolase were determined. DAK inhibits the hydrolysis of AEA by rat brain membranes with an IC50 value of 0.5 microM. At low concentrations, DAK reduces the Vmax and increases the K(m) of the enzyme for its substrate AEA, which suggests that it is both a competitive and noncompetitive inhibitor. At higher concentrations, DAK inhibition is completely noncompetitive. DAK inhibition of membrane-associated AEA amidohydrolase is irreversible because hydrolytic activity is not restored with extensive washing or dialysis of the membranes. Furthermore, DAK inhibition is not reversible by anion exchange chromatography of the subsequently solubilized enzyme. In contrast, DAK inhibition of detergent-solubilized enzyme exhibits competitive kinetics and is reversible upon ion exchange chromatography. Exposure of C6 glioma cells to DAK results in concentration-related inhibition of AEA amidohydrolase activity in cellular membranes with an IC50 value of 0.3 microM. In summary, these studies demonstrate that DAK is an irreversible inhibitor of AEA amidohydrolase in its native membrane and provides a useful tool with which to study the role of AEA amidohydrolase in the termination of action of AEA.

  3. Thermodynamics of ligand binding to histone deacetylase like amidohydrolase from Bordetella/Alcaligenes.

    Science.gov (United States)

    Meyners, Christian; Baud, Matthias G J; Fuchter, Matthew J; Meyer-Almes, Franz-Josef

    2014-03-01

    Thermodynamic studies on ligand-protein binding have become increasingly important in the process of drug design. In combination with structural data and molecular dynamics simulations, thermodynamic studies provide relevant information about the mode of interaction between compounds and their target proteins and therefore build a sound basis for further drug optimization. Using the example of histone deacetylases (HDACs), particularly the histone deacetylase like amidohydrolase (HDAH) from Bordetella/Alcaligenes, a novel sensitive competitive fluorescence resonance energy transfer-based binding assay was developed and the thermodynamics of interaction of both fluorescent ligands and inhibitors to histone deacetylase like amidohydrolase were investigated. The assay consumes only small amounts of valuable target proteins and is suitable for fast kinetic and mechanistic studies as well as high throughput screening applications. Binding affinity increased with increasing length of aliphatic spacers (n = 4-7) between the hydroxamate moiety and the dansyl head group of ligand probes. Van't Hoff plots revealed an optimum in enthalpy contribution to the free energy of binding for the dansyl-ligand with hexyl spacer. The selectivity in the series of dansyl-ligands against human class I HDAC1 but not class II HDACs 4 and 6 increased with the ratio of ΔH(0)/ΔG(0). The data clearly emphasize the importance of thermodynamic signatures as useful general guidance for the optimization of ligands or rational drug design.

  4. Inhibition of a Putative Dihydropyrimidinase from Pseudomonas aeruginosa PAO1 by Flavonoids and Substrates of Cyclic Amidohydrolases.

    Directory of Open Access Journals (Sweden)

    Cheng-Yang Huang

    Full Text Available Dihydropyrimidinase is a member of the cyclic amidohydrolase family, which also includes allantoinase, dihydroorotase, hydantoinase, and imidase. These metalloenzymes possess very similar active sites and may use a similar mechanism for catalysis. However, whether the substrates and inhibitors of other cyclic amidohydrolases can inhibit dihydropyrimidinase remains unclear. This study investigated the inhibition of dihydropyrimidinase by flavonoids and substrates of other cyclic amidohydrolases. Allantoin, dihydroorotate, 5-hydantoin acetic acid, acetohydroxamate, orotic acid, and 3-amino-1,2,4-triazole could slightly inhibit dihydropyrimidinase, and the IC50 values of these compounds were within the millimolar range. The inhibition of dihydropyrimidinase by flavonoids, such as myricetin, quercetin, kaempferol, galangin, dihydromyricetin, and myricitrin, was also investigated. Some of these compounds are known as inhibitors of allantoinase and dihydroorotase. Although the inhibitory effects of these flavonoids on dihydropyrimidinase were substrate-dependent, dihydromyricetin significantly inhibited dihydropyrimidinase with IC50 values of 48 and 40 μM for the substrates dihydrouracil and 5-propyl-hydantoin, respectively. The results from the Lineweaver-Burk plot indicated that dihydromyricetin was a competitive inhibitor. Results from fluorescence quenching analysis indicated that dihydromyricetin could form a stable complex with dihydropyrimidinase with the K(d value of 22.6 μM. A structural study using PatchDock showed that dihydromyricetin was docked in the active site pocket of dihydropyrimidinase, which was consistent with the findings from kinetic and fluorescence studies. This study was the first to demonstrate that naturally occurring product dihydromyricetin inhibited dihydropyrimidinase, even more than the substrate analogs (>3 orders of magnitude. These flavonols, particularly myricetin, may serve as drug leads and dirty drugs (for

  5. Functional Identification of Incorrectly Annotated Prolidases from the Amidohydrolase Superfamily of Enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Xiang, D.; Patskovsky, Y; Xu, C; Meyer, A; Sauder, J; Burley, S; Almo, S; Raushel, F

    2009-01-01

    The substrate profiles for two proteins from Caulobacter crescentus CB15 (Cc2672 and Cc3125) and one protein (Sgx9359b) derived from a DNA sequence (gi|44368820) isolated from the Sargasso Sea were determined using combinatorial libraries of dipeptides and N-acyl derivatives of amino acids. These proteins are members of the amidohydrolase superfamily and are currently misannotated in NCBI as catalyzing the hydrolysis of l-Xaa-l-Pro dipeptides. Cc2672 was shown to catalyze the hydrolysis of l-Xaa-l-Arg/Lys dipeptides and the N-acetyl and N-formyl derivatives of lysine and arginine. This enzyme will also hydrolyze longer peptides that terminate in either lysine or arginine. The N-methyl phosphonate derivative of l-lysine was a potent competitive inhibitor of Cc2672 with a Ki value of 120 nM. Cc3125 was shown to catalyze the hydrolysis of l-Xaa-l-Arg/Lys dipeptides but will not hydrolyze tripeptides or the N-formyl and N-acetyl derivatives of lysine or arginine. The substrate profile for Sgx9359b is similar to that of Cc2672 except that compounds with a C-terminal lysine are not recognized as substrates. The X-ray structure of Sgx9359b was determined to a resolution of 2.3 Angstroms. The protein folds as a (e/a)8-barrel and self-associates to form a homooctamer. The active site is composed of a binuclear metal center similar to that found in phosphotriesterase and dihydroorotase. In one crystal form, arginine was bound adventitiously to the eight active sites within the octamer. The orientation of the arginine in the active site identified the structural determinants for recognition of the a-carboxylate and the positively charged side chains of arginine-containing substrates. This information was used to identify 18 other bacterial sequences that possess identical or similar substrate profiles.

  6. X-RAY STRUCTURE OF ILL2, AN AUXIN-CONJUGATE AMIDOHYDROLASE FROM ARABIDOPSIS THALIANA

    Science.gov (United States)

    Bitto, Eduard; Bingman, Craig A.; Bittova, Lenka; Houston, Norma L.; Boston, Rebecca S.; Fox, Brian G.; Phillips, George N.

    2008-01-01

    The plant hormone indole-3-acetic acid (IAA) is the most abundant natural auxin involved in many aspects of plant development and growth. The IAA levels in plants are modulated by a specific group of amidohydrolases from the peptidase M20D family that release the active hormone from its conjugated storage forms. Here we describe the X-ray crystal structure of IAA-amino acid hydrolase IAA-leucine resistant-like gene 2 (ILL2) from Arabidopsis thaliana at 2.0 Å resolution. ILL2 preferentially hydrolyses the auxin-amino acid conjugate N-(indol-3-acetyl)-alanine. The overall structure of ILL2 is reminiscent of dinuclear metallopeptidases from the M20 peptidase family. The structure consists of two domains, a larger catalytic domain with 3-layer αβα sandwich architecture and aminopeptidase topology and a smaller satellite domain with 2-layer αβ sandwich architecture and alpha-beta plaits topology. The metal coordinating residues in the active site of ILL2 include a conserved cysteine that clearly distinguishes this protein from previously structurally characterized members of the M20 peptidase family. Modeling of N-(indol-3-acetyl)-alanine into the active site of ILL2 suggests that Leu175 serves as a key determinant for the amino acid side chain specificity of this enzyme. Furthermore, a hydrophobic pocket nearby the catalytic dimetal center likely recognizes the indolyl moiety of the substrate. Finally, the active site of ILL2 harbors an absolutely conserved glutamate (Glu172), which is well positioned to act as a general acid-base residue. Overall, the structure of ILL2 suggests that this enzyme likely uses a catalytic mechanism that follows the paradigm established for the other enzymes of the M20 peptidase family. PMID:18543330

  7. Functional Annotation of Two New Carboxypeptidases from the Amidohydrolase Superfamily of Enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Xiang, D.; Xu, C; Kumaran, D; Brown, A; Sauder, M; Burley, S; Swaminathan, S; Raushel, F

    2009-01-01

    Two proteins from the amidohydrolase superfamily of enzymes were cloned, expressed, and purified to homogeneity. The first protein, Cc0300, was from Caulobacter crescentus CB-15 (Cc0300), while the second one (Sgx9355e) was derived from an environmental DNA sequence originally isolated from the Sargasso Sea (gi|44371129). The catalytic functions and the substrate profiles for the two enzymes were determined with the aid of combinatorial dipeptide libraries. Both enzymes were shown to catalyze the hydrolysis of l-Xaa-l-Xaa dipeptides in which the amino acid at the N-terminus was relatively unimportant. These enzymes were specific for hydrophobic amino acids at the C-terminus. With Cc0300, substrates terminating in isoleucine, leucine, phenylalanine, tyrosine, valine, methionine, and tryptophan were hydrolyzed. The same specificity was observed with Sgx9355e, but this protein was also able to hydrolyze peptides terminating in threonine. Both enzymes were able to hydrolyze N-acetyl and N-formyl derivatives of the hydrophobic amino acids and tripeptides. The best substrates identified for Cc0300 were l-Ala-l-Leu with kcat and kcat/Km values of 37 s-1 and 1.1 x 105 M-1 s-1, respectively, and N-formyl-l-Tyr with kcat and kcat/Km values of 33 s-1 and 3.9 x 105 M-1 s-1, respectively. The best substrate identified for Sgx9355e was l-Ala-l-Phe with kcat and kcat/Km values of 0.41 s-1 and 5.8 x 103 M-1 s-1. The three-dimensional structure of Sgx9355e was determined to a resolution of 2.33 Angstroms with l-methionine bound in the active site. The a-carboxylate of the methionine is ion-paired to His-237 and also hydrogen bonded to the backbone amide groups of Val-201 and Leu-202. The a-amino group of the bound methionine interacts with Asp-328. The structural determinants for substrate recognition were identified and compared with other enzymes in this superfamily that hydrolyze dipeptides with different specificities.

  8. Biochemical and structural characterization of Klebsiella pneumoniae oxamate amidohydrolase in the uric acid degradation pathway

    Energy Technology Data Exchange (ETDEWEB)

    Hicks, Katherine A.; Ealick, Steven E.

    2016-05-25

    HpxW from the ubiquitous pathogenKlebsiella pneumoniaeis involved in a novel uric acid degradation pathway downstream from the formation of oxalurate. Specifically, HpxW is an oxamate amidohydrolase which catalyzes the conversion of oxamate to oxalate and is a member of the Ntn-hydrolase superfamily. HpxW is autoprocessed from an inactive precursor to form a heterodimer, resulting in a 35.5 kDa α subunit and a 20 kDa β subunit. Here, the structure of HpxW is presented and the substrate complex is modeled. In addition, the steady-state kinetics of this enzyme and two active-site variants were characterized. These structural and biochemical studies provide further insight into this class of enzymes and allow a mechanism for catalysis consistent with other members of the Ntn-hydrolase superfamily to be proposed.

  9. Identification of nicotinamide mononucleotide deamidase of the bacterial pyridine nucleotide cycle reveals a novel broadly conserved amidohydrolase family.

    Science.gov (United States)

    Galeazzi, Luca; Bocci, Paola; Amici, Adolfo; Brunetti, Lucia; Ruggieri, Silverio; Romine, Margaret; Reed, Samantha; Osterman, Andrei L; Rodionov, Dmitry A; Sorci, Leonardo; Raffaelli, Nadia

    2011-11-18

    The pyridine nucleotide cycle is a network of salvage and recycling routes maintaining homeostasis of NAD(P) cofactor pool in the cell. Nicotinamide mononucleotide (NMN) deamidase (EC 3.5.1.42), one of the key enzymes of the bacterial pyridine nucleotide cycle, was originally described in Enterobacteria, but the corresponding gene eluded identification for over 30 years. A genomics-based reconstruction of NAD metabolism across hundreds of bacterial species suggested that NMN deamidase reaction is the only possible way of nicotinamide salvage in the marine bacterium Shewanella oneidensis. This prediction was verified via purification of native NMN deamidase from S. oneidensis followed by the identification of the respective gene, termed pncC. Enzymatic characterization of the PncC protein, as well as phenotype analysis of deletion mutants, confirmed its proposed biochemical and physiological function in S. oneidensis. Of the three PncC homologs present in Escherichia coli, NMN deamidase activity was confirmed only for the recombinant purified product of the ygaD gene. A comparative analysis at the level of sequence and three-dimensional structure, which is available for one of the PncC family member, shows no homology with any previously described amidohydrolases. Multiple alignment analysis of functional and nonfunctional PncC homologs, together with NMN docking experiments, allowed us to tentatively identify the active site area and conserved residues therein. An observed broad phylogenomic distribution of predicted functional PncCs in the bacterial kingdom is consistent with a possible role in detoxification of NMN, resulting from NAD utilization by DNA ligase.

  10. IDENTIFICATION OF NICOTINAMIDE MONONUCLEOTIDE DEAMIDASE OF THE BACTERIAL PYRIDINE NUCLEOTIDE CYCLE REVEALS A NOVEL BROADLY CONSERVED AMIDOHYDROLASE FAMILY

    Energy Technology Data Exchange (ETDEWEB)

    Galeazzi, Luca; Bocci, Paolo; Amici, Adolfo; Brunetti, Lucia; Ruggieri, Silverio; Romine, Margaret F.; Reed, Samantha B.; Osterman, Andrei; Rodionov, Dmitry A.; Sorci, Leonardo; Raffaelli, Nadia

    2011-09-27

    The pyridine nucleotide cycle (PNC) is a network of salvage and recycling routes maintaining homeostasis of NAD(P) cofactor pool in the cell. Nicotinamide mononucleotide (NMN) deamidase (EC 3.5.1.42), one of the key enzymes of the bacterial PNC was originally described in Enterobacteria, but the corresponding gene eluded identification for over 30 years. A genomics-based reconstruction of NAD metabolism across hundreds bacterial species suggested that NMN deamidase reaction is the only possible way of nicotinamide salvage in the marine bacterium Shewanella oneidensis. This prediction was verified via purification of native NMN deamidase from S. oneidensis followed by the identification of the respective gene, termed pncC. Enzymatic characterization of the PncC protein, as well as phenotype analysis of deletion mutants, confirmed its proposed biochemical and physiological function in S. oneidensis. Of the three PncC homologs present in E. coli, NMN deamidase activity was confirmed only for the recombinant purified product of the ygaD gene. A comparative analysis at the level of sequence and three dimensional structure, which is available for one of the PncC family member, shows no homology with any previously described amidohydrolases. Multiple alignment analysis of functional and non functional PncC homologs, together with NMN docking experiments, allowed us to tentatively identify the active site area and conserved residues therein. An observed broad phylogenomic distribution of predicted functional PncCs in bacterial kingdom is consistent with a possible role in detoxification of NMN, resulting from NAD utilization by DNA ligase.

  11. Annotating Enzymes of Uncertain Function: The Deacylation of d-Amino Acids by Members of the Amidohydrolase Superfamily

    Energy Technology Data Exchange (ETDEWEB)

    Cummings, J.; Fedorov, A; Xu, C; Brown, S; Fedorov, E; Babbitt, P; Almo, S; Raushel, F

    2009-01-01

    The catalytic activities of three members of the amidohydrolase superfamily were discovered using amino acid substrate libraries. Bb3285 from Bordetella bronchiseptica, Gox1177 from Gluconobacter oxidans, and Sco4986 from Streptomyces coelicolor are currently annotated as d-aminoacylases or N-acetyl-d-glutamate deacetylases. These three enzymes are 22-34% identical to one another in amino acid sequence. Substrate libraries containing nearly all combinations of N-formyl-d-Xaa, N-acetyl-d-Xaa, N-succinyl-d-Xaa, and l-Xaa-d-Xaa were used to establish the substrate profiles for these enzymes. It was demonstrated that Bb3285 is restricted to the hydrolysis of N-acyl-substituted derivatives of d-glutamate. The best substrates for this enzyme are N-formyl-d-glutamate (k{sub cat}/K{sub m} = 5.8 x 10{sup 6} M{sup -1} s{sup -1}), N-acetyl-d-glutamate (k{sub cat}/K{sub m} = 5.2 x 10{sup 6} M{sup -1} s{sup -1}), and l-methionine-d-glutamate (k{sub cat}/K{sub m} = 3.4 x 10{sup 5} M{sup -1} s{sup -1}). Gox1177 and Sco4986 preferentially hydrolyze N-acyl-substituted derivatives of hydrophobic d-amino acids. The best substrates for Gox1177 are N-acetyl-d-leucine (k{sub cat}/K{sub m} = 3.2 x 104 M{sup -1} s-1), N-acetyl-d-tryptophan (kcat/Km = 4.1 x 104 M-1 s-1), and l-tyrosine-d-leucine (kcat/Km = 1.5 x 104 M-1 s-1). A fourth protein, Bb2785 from B. bronchiseptica, did not have d-aminoacylase activity. The best substrates for Sco4986 are N-acetyl-d-phenylalanine and N-acetyl-d-tryptophan. The three-dimensional structures of Bb3285 in the presence of the product acetate or a potent mimic of the tetrahedral intermediate were determined by X-ray diffraction methods. The side chain of the d-glutamate moiety of the inhibitor is ion-paired to Arg-295, while the {alpha}-carboxylate is ion-paired with Lys-250 and Arg-376. These results have revealed the chemical and structural determinants for substrate specificity in this protein. Bioinformatic analyses of an additional {approx}250

  12. At the Perphery of the Amidohydrolase Superfamily: Bh0493 from Bacillus halodurans Catalyzes the Isomerization of D-Galacturonate to D-Tagaturonate

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    Nguyen,T.; Brown, S.; Fedorov, A.; Fedorov, E.; Babbitt, P.; Almo, S.; Raushel, F.

    2008-01-01

    The amidohydrolase superfamily is a functionally diverse set of enzymes that catalyzes predominantly hydrolysis reactions involving sugars, nucleic acids, amino acids, and organophosphate esters. One of the most divergent members of this superfamily, uronate isomerase from Escherichia coli, catalyzes the isomerization of d-glucuronate to d-fructuronate and d-galacturonate to d-tagaturonate and is the only uronate isomerase in this organism. A gene encoding a putative uronate isomerase in Bacillus halodurans (Bh0705) was identified based on sequence similarity to uronate isomerases from other organisms. Kinetic evidence indicates that Bh0705 is relatively specific for the isomerization of d-glucuronate to d-fructuronate, confirming this functional assignment. Despite a low sequence identity to all other characterized uronate isomerases, phylogenetic and network-based analysis suggests that a second gene in this organism, Bh0493, is also a uronate isomerase, although it is an outlier in the group, with <20% sequence identity to any other characterized uronate isomerase from another species. The elucidation of the X-ray structure at a resolution of 2.0 Angstroms confirms that Bh0493 is a member of the amidohydrolase superfamily with conserved residues common to other members of the uronate isomerase family. Functional characterization of this protein shows that unlike Bh0705, Bh0493 can utilize both d-glucuronate and d-galacturonate as substrates. In B. halodurans, Bh0705 is found in an operon for the metabolism of d-glucuronate, whereas Bh0493 is in an operon for the metabolism of d-galacturonate. These results provide the first identification of a uronate isomerase that operates in a pathway distinct from that for d-glucuronate. While most organisms that contain this pathway have only one gene for a uronate isomerase, sequence analysis and operon context show that five other organisms also appear to have two genes and one organism appears to have three genes for

  13. Low-temperature-induced expression of rice ureidoglycolate amidohydrolase is mediated by a C-repeat/dehydration-responsive element that specifically interacts with rice C-repeat-binding factor 3

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    Juan eLi

    2015-11-01

    Full Text Available Nitrogen recycling and redistribution are important for the environmental stress response of plants. In non nitrogen-fixing plants, ureide metabolism is crucial to nitrogen recycling from organic sources. Various studies have suggested that the rate-limiting components of ureide metabolism respond to environmental stresses. However, the underlying regulation mechanism is not well understood. In this report, rice ureidoglycolate amidohydrolase (OsUAH, which is a recently identified enzyme catalyzing the final step of ureide degradation, was identified as low-temperature- (LT but not abscisic acid- (ABA regulated. To elucidate the LT regulatory mechanism at the transcriptional level, we isolated and characterized the promoter region of OsUAH (POsUAH. Series deletions revealed that a minimal region between -522 and -420 relative to the transcriptional start site was sufficient for the cold induction of POsUAH. Detailed analyses of this 103-bp fragment indicated that a C-repeat/dehydration-responsive (CRT/DRE element localized at position -434 was essential for LT-responsive expression. A rice C-repeat-binding factors/DRE-binding proteins 1 (CBFs/DREB1s subfamily member, OsCBF3, was screened to specifically bind to the CRT/DRE element in the minimal region both in yeast one-hybrid assays and in in vitro gel-shift analysis. Moreover, the promoter could be exclusively trans-activated by the interaction between the CRT/DRE element and OsCBF3 in vivo. These findings may help to elucidate the regulation mechanism of stress-responsive ureide metabolism genes and provide an example of the member-specific manipulation of the CBF/DREB1 subfamily.

  14. Association analysis between N-acylsphingosine amidohydrolase gene and symptom quantitative trait of schizophrenia in Chinese Han population%酸性神经酰胺酶基因与精神分裂症症状数量性状的关联研究

    Institute of Scientific and Technical Information of China (English)

    张怀惠; 崔东红; 朱紫青; 江开达; 江三多

    2008-01-01

    目的 探讨酸性神经酰胺酶基因(ASAH1)与精神分裂症症状数量性状的关联.方法 应用聚合酶链反应及限制性片段长度多态性技术,对254例精神分裂症患者ASAH1基因的3个单核苷酸多态性(rs3753118、rs3753116及rs7830490)进行检测;使用阳性和阴性症状量表(PANSS)评定患者疾病表型的数量性状.结果 (1)患者rs3753118位点3种基因型间的阳性症状分的差异有统计学意义(F=3.506,P<0.05);rs3753116位点3种基因型间的PANSS总分及阴性症状分的差异均有统计学意义(F=3.548,P<0.05;F=3.358,P<0.05).经两两比较,rs3753118位点C/C基因型组患者的阳性症状分及一般精神病理分明显高于C/T基因型组患者;rs3753116位点G/G基因型组患者的PANSS总分及阴性症状分明显高于A/G基因型组患者,且G/G基因型组患者的阴性症状分明显高于A/A基因型组患者,差异均有统计学意义(P均<0.05).(2)患者3个位点各等位基因间的PANSS量表评分的差异无统计学意义(P>0.05).(3)患者rs3753118C-rs3753116G单体型者及rs3753118C-rs3753116G-rs7830490A单体型者,其PANSS总分及阴性症状分均明显高于参照单体型者,差异均有统计学意义(P均<0.05).结论 ASAH1基因区域可能存在精神分裂症症状数量性状位点.%Objective To explore the association between N-acylsphingosine amidohydrolase(ASAHl)gene and symptom quantitative trait of schizophrenia in Chinese Han Population.Methods Three polymorphic markers(rs3753118,rs3753116 and rs7830490)in ASAH1 gene were genotyped with polymerase chain reaction and restriction fragment length polymorphism in 254 schizophrenic patients.The Positive and Negative Syndrome Scale (PANSS) was used to quantify the phenotypes of schizophrenia.Results (1)There were significant difierence in the positive subseale score among patients with different genotypes of rs3753118(F=3.506,P0.05).(3)The PANSS total score and the negative score in patients with rs

  15. Features of phospho- and amidohydrolases functioning in edaphotopes polluted by ore mill effluents

    Directory of Open Access Journals (Sweden)

    O. M. Artyushenko

    2006-02-01

    Full Text Available Influence of aerotechnogenic contamination of soils on activity of some hydrolytic enzymes of nitrogen and phosphorus cycles is examined. Biochemical mobilization of organophosphorous and nitrogen-bearing compounds in soils polluted by heavy metals is depressed to a variable extent. In descending order of sensitivity to the pollution, the studied enzymes ranked as follows: urease > alkaline phosphatase > arginase > АТPase > acid phosphatase > amidase.

  16. Dihydropyrimidine amidohydrolases and dihydroorotases share the same origin and several enzymatic properties

    DEFF Research Database (Denmark)

    Gojkovic, Zoran; Rislund, L.; Andersen, B.

    2003-01-01

    was catalyzed most efficiently at pH 8.0 and competitively inhibited by the reaction product, N-carbamyl-beta-alanine. At lower pH values DHPases catalyzed the reverse reaction, the closing of the ring. Apparently, eukaryote DHPases are enzymatically as well as phylogenetically related to the de novo...

  17. A fluorescence lifetime-based binding assay for acetylpolyamine amidohydrolases from Pseudomonas aeruginosa using a [1,3]dioxolo[4,5-f][1,3]benzodioxole (DBD) ligand probe.

    Science.gov (United States)

    Meyners, Christian; Wawrzinek, Robert; Krämer, Andreas; Hinz, Steffen; Wessig, Pablo; Meyer-Almes, Franz-Josef

    2014-08-01

    High-throughput assays for drug screening applications have to fulfill particular specifications. Besides the capability to identify even compounds with low potency, one of the major issues is to minimize the number of false-positive hits in a screening campaign in order to reduce the logistic effort for the subsequent cherry picking and confirmation procedure. In this respect, fluorescence lifetime (FLT) appears as an ideal readout parameter that is supposed to be robust against autofluorescent and light-absorbing compounds, the most common source of systematic false positives. The extraordinary fluorescence features of the recently discovered [1,3]dioxolo[4,5-f][1,3] benzodioxole dyes were exploited to develop an FLT-based binding assay with exceptionally robust readout. The assay setup was comprehensively validated and shown to comply not only with all requirements for a powerful high-throughput screening assay but also to be suitable to determine accurate binding constants for inhibitors against enzymes of the histone deacetylase family. Using the described binding assay, the first inhibitors against three members of this enzyme family from Pseudomonas aeruginosa were identified. The compounds were characterized in terms of potency and selectivity profile. The novel ligand probe should also be applicable to other homologues of the histone deacetylase family that are inhibited by N-hydroxy-N'-phenyloctandiamide.

  18. NCBI nr-aa BLAST: CBRC-DNOV-01-1277 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DNOV-01-1277 ref|YP_319048.1| N-formylglutamate amidohydrolase [Nitrobacter winogradsky...i Nb-255] gb|ABA05696.1| N-formylglutamate amidohydrolase [Nitrobacter winogradskyi Nb-255] YP_319048.1 2.3 30% ...

  19. NCBI nr-aa BLAST: CBRC-DNOV-01-0806 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DNOV-01-0806 ref|ZP_01156682.1| N-formylglutamate amidohydrolase [Oceanicola g...ranulosus HTCC2516] gb|EAR51161.1| N-formylglutamate amidohydrolase [Oceanicola granulosus HTCC2516] ZP_01156682.1 0.36 32% ...

  20. NCBI nr-aa BLAST: CBRC-GACU-09-0002 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-GACU-09-0002 ref|YP_594114.1| amidohydrolase [Deinococcus geothermalis DSM 113...00] gb|ABF44040.1| amidohydrolase [Deinococcus geothermalis DSM 11300] YP_594114.1 5.5 28% ...

  1. Enzymatic synthesis oF L-tryptophan from D,L-2-amino-delta2-thiazoline-4-carboxylic acid and indole by Pseudomonas sp. TS1138 L-2-amino-delta2-thiazoline-4-carboxylic acid hydrolase, S-carbamyl-L-cysteine amidohydrolase, and Escherichia coli L-tryptophanase.

    Science.gov (United States)

    Du, J; Duan, J J; Zhang, Q; Hou, J; Bai, F; Chen, N; Bai, G

    2012-01-01

    L-Tryptophan (L-Trp) is an essential amino acid. It is widely used in medical, health and food products, so a low-cost supply is needed. There are 4 methods for L-Trp production: chemical synthesis, extraction, enzymatic synthesis, and fermentation. In this study, we produced a recombinant bacterial strain pET-tnaA of Escherichia coli which has the L-tryptophanase gene. Using the pET-tnaA E. coli and the strain TS1138 of Pseudomonas sp., a one-pot enzymatic synthesis of L-Trp was developed. Pseudomonas sp. TS1138 was added to a solution of D,L-2-amino-delta2-thiazoline-4-carboxylic acid (DL-ATC) to convert it to L-cysteine (L-Cys). After concentration, E. coli BL21 (DE 3) cells including plasmid pET-tnaA, indole, and pyridoxal 5'-phosphate were added. At the optimum conditions, the conversion rates of DL-ATC and L-Cys were 95.4% and 92.1%, respectively. After purifying using macroporous resin S8 and NKA-II, 10.32 g of L-Trp of 98.3% purity was obtained. This study established methods for one-pot enzymatic synthesis and separation of L-Trp. This method of producing L-Trp is more environmentally sound than methods using chemical synthesis, and it lays the foundations for industrial production of L-Trp from DL-ATC and indole.

  2. The quorum-quenching N-acyl homoserine lactone acylase PvdQ is an Ntn-hydrolase with an unusual substrate-binding pocket

    NARCIS (Netherlands)

    Bokhove, Marcel; Jimenez, Pol Nadal; Quax, Wim J.; Dijkstra, Bauke W.

    2010-01-01

    In many Gram-negative pathogens, their virulent behavior is regulated by quorum sensing, in which diffusible signals such as N-acyl homoserine lactones (AHLs) act as chemical messaging compounds. Enzymatic degradation of these diffusible signals by, e. g., lactonases or amidohydrolases abolishes AHL

  3. Phage lysin LysK can be truncated to its CHAP domain and retain lytic activity against live antibiotic-resistant staphylococci.

    Science.gov (United States)

    Horgan, Marianne; O'Flynn, Gary; Garry, Jennifer; Cooney, Jakki; Coffey, Aidan; Fitzgerald, Gerald F; Ross, R Paul; McAuliffe, Olivia

    2009-02-01

    A truncated derivative of the phage endolysin LysK containing only the CHAP (cysteine- and histidine-dependent amidohydrolase/peptidase) domain exhibited lytic activity against live clinical staphylococcal isolates, including methicillin-resistant Staphylococcus aureus. This is the first known report of a truncated phage lysin which retains high lytic activity against live staphylococcal cells.

  4. Sequence Classification: 389974 [

    Lifescience Database Archive (English)

    Full Text Available Non-TMB Non-TMH Non-TMB Non-TMB Non-TMB Non-TMB >gi|31794485|ref|NP_856978.1| POSSIBLE N-ACYL-L-AMI...NO ACID AMIDOHYDROLASE AMIA1 (N-ACYL-L-AMINO ACID AMIDASE) || http://www.ncbi.nlm.nih.gov/protein/31794485 ...

  5. Sequence Classification: 399744 [

    Lifescience Database Archive (English)

    Full Text Available Non-TMB Non-TMH Non-TMB Non-TMB Non-TMB Non-TMB >gi|57117086|ref|YP_177955.1| POSSIBLE N-ACYL-L-AMI...NO ACID AMIDOHYDROLASE AMIA1 (N-ACYL-L-AMINO ACID AMINOHYDROLASE) || http://www.ncbi.nlm.nih.gov/protein/57117086 ...

  6. Cyanase-mediated utilization of cyanate in Pseudomonas fluorescens NCIB 11764.

    OpenAIRE

    Kunz, D A; Nagappan, O

    1989-01-01

    Pseudomonas fluorescens NCIB 11764 was capable of utilizing cyanate (OCN-) as a sole nitrogen source for growth. Crude cell extracts from cells grown on cyanate, but not on ammonium sulfate, were induced for an enzyme catalyzing cyanate conversion to ammonia. Enzymatic activity was shown to be bicarbonate dependent and specific for cyanate as a substrate, suggesting that cyanate utilization in this organism is facilitated by an enzyme resembling cyanase (cyanate amidohydrolase; EC 3.5.5.3), a...

  7. Cyanase-mediated utilization of cyanate in Pseudomonas fluorescens NCIB 11764.

    Science.gov (United States)

    Kunz, D A; Nagappan, O

    1989-01-01

    Pseudomonas fluorescens NCIB 11764 was capable of utilizing cyanate (OCN-) as a sole nitrogen source for growth. Crude cell extracts from cells grown on cyanate, but not on ammonium sulfate, were induced for an enzyme catalyzing cyanate conversion to ammonia. Enzymatic activity was shown to be bicarbonate dependent and specific for cyanate as a substrate, suggesting that cyanate utilization in this organism is facilitated by an enzyme resembling cyanase (cyanate amidohydrolase; EC 3.5.5.3), as described previously in Escherichia coli and Flavobacterium sp.

  8. Purification, characterization, and genetic analysis of Mycobacterium tuberculosis urease, a potentially critical determinant of host-pathogen interaction.

    OpenAIRE

    Clemens, D L; Lee, B. Y.; Horwitz, M A

    1995-01-01

    Mycobacterium tuberculosis urease (urea amidohydrolase [EC 3.5.1.5]) was purified and shown to contain three subunits: two small subunits, each approximately 11,000 Da, and a large subunit of 62,000 Da. The N-terminal sequences of the three subunits were homologous to those of the A, B, and C subunits, respectively, of other bacterial ureases. M. tuberculosis urease was specific for urea, with a Km of 0.3 mM, and did not hydrolyze thiourea, hydroxyurea, arginine, or asparagine. The enzyme was...

  9. Potentials for Soil Enzyme as Indicators of Ecological Management

    Science.gov (United States)

    Senwo, Z. N.; Manu, A.; Coleman, T. L.

    1997-01-01

    Activity measurements of selected soil enzymes (cellulase, glucosidase, amidohydrolase, phosphatase, arylsulfatase) involved in carbon, nitrogen, phosphorus, and sulfur cycling in the biosphere, hold potential as early and sensitive indicators of soil ecological stress and restoration, These measurements are advantageous because the procedures are simple, rapid, and reproducible over time. Enzyme activities are sensitive to short-term changes in soil and kind-use management. Enzyme activities have also been observed to be closely related to soil organic matter proposed as an index of soil quality.

  10. The ygeW encoded protein from Escherichia coli is a knotted ancestral catabolic transcarbamylase

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yongdong; Jin, Zhongmin; Yu, Xiaolin; Allewell, Norma M.; Tuchman, Mendel; Shi, Dashuang (Maryland); (GWU); (Georgia)

    2012-06-28

    Purine degradation plays an essential role in nitrogen metabolism in most organisms. Uric acid is the final product of purine catabolism in humans, anthropoid apes, birds, uricotelic reptiles, and almost all insects. Elevated levels of uric acid in blood (hyperuricemia) cause human diseases such as gout, kidney stones, and renal failure. Although no enzyme has been identified that further degrades uric acid in humans, it can be oxidized to produce allantoin by free-radical attack. Indeed, elevated levels of allantoin are found in patients with rheumatoid arthritis, chronic lung disease, bacterial meningitis, and noninsulin-dependent diabetes mellitus. In other mammals, some insects and gastropods, uric acid is enzymatically degraded to the more soluble allantoin through the sequential action of three enzymes: urate oxidase, 5-hydroxyisourate (HIU) hydrolase and 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) decarboxylase. Therefore, an elective treatment for acute hyperuricemia is the administration of urate oxidase. Many organisms, including plants, some fungi and several bacteria, are able to catabolize allantoin to release nitrogen, carbon, and energy. In Arabidopsis thaliana and Eschrichia coli, S-allantoin has recently been shown to be degraded to glycolate and urea by four enzymes: allantoinase, allantoate amidohydrolase, ureidoglycine aminohydrolase, and ureidoglycolate amidohydrolase.

  11. AepA of Pectobacterium is not involved in the regulation of extracellular plant cell wall degrading enzymes production.

    Science.gov (United States)

    Kõiv, Viia; Andresen, Liis; Mäe, Andres

    2010-06-01

    Plant cell wall degrading enzymes (PCWDE) are the major virulence determinants in phytopathogenic Pectobacterium, and their production is controlled by many regulatory factors. In this study, we focus on the role of the AepA protein, which was previously described to be a global regulator of PCWDE production in Pectobacterium carotovorum (Murata et al. in Mol Plant Microbe Interact 4:239-246, 1991). Our results show that neither inactivation nor overexpression of aepA affects PCWDE production in either Pectobacterium atrosepticum SCRI1043 or Pectobacterium carotovorum subsp. carotovorum SCC3193. The previously published observation based on the overexpression of aepA could be explained by the presence of the adjacent regulatory rsmB gene in the constructs used. Our database searches indicated that AepA belongs to the YtcJ subfamily of amidohydrolases. YtcJ-like amidohydrolases are present in bacteria, archaea, plants and some fungi. Although AepA has 28% identity with the formamide deformylase NfdA in Arthrobacter pascens F164, AepA was unable to catalyze the degradation of NdfA-specific N-substituted formamides. We conclude that AepA is a putative aminohydrolase not involved in regulation of PCWDE production.

  12. A new phosphotriesterase from Sulfolobus acidocaldarius and its comparison with the homologue from Sulfolobus solfataricus.

    Science.gov (United States)

    Porzio, Elena; Merone, Luigia; Mandrich, Luigi; Rossi, Mosè; Manco, Giuseppe

    2007-05-01

    The phosphotriesterase PTE, identified in the soil bacterium Pseudomonas diminuta, is thought to have evolved in the last several decades to degrade the pesticide paraoxon with proficiency approaching the limit of substrate diffusion (k(cat)/K(M) of 4 x 10(7)M(-1)s(-1)). It belongs to the amidohydrolase superfamily, but its evolutionary origin remains obscure. The enzyme has important potentiality in the field of the organophosphate decontamination. Recently we reported on the characterization of an archaeal member of the amidohydrolase superfamily, namely Sulfolobus solfataricus, showing low but significant and extremely thermostable paraoxonase activity (k(cat)/K(M) of 4 x 10(3)M(-1)s(-1)). Looking for other thermostable phosphotriesterases we assayed, among others, crude extracts of Sulfolobus acidocaldarius and detected activity. Since the genome of S. acidocaldarius has been recently reported, we identified there an open reading frame highly related to the S. solfataricus enzyme. The gene was cloned, the protein overexpressed in Escherichia coli, purified, and proven to have paraoxonase activity. A comparative analysis detected some significant differences between the two archaeal enzymes.

  13. Structural and Mechanistic Characterization of L-Histidinol Phosphate Phosphatase from the PHP Family of Proteins

    Science.gov (United States)

    Ghodge, Swapnil V.; Fedorov, Alexander A.; Fedorov, Elena V.; Hillerich, Brandan; Seidel, Ronald; Almo, Steven C.; Raushel, Frank M.

    2013-01-01

    l-Histidinol phosphate phosphatase (HPP) catalyzes the hydrolysis of L-histidinol phosphate to L-histidinol and inorganic phosphate, the penultimate step in the biosynthesis of L-histidine. HPP from the polymerase and histidinol phosphatase (PHP) family of proteins possesses a trinuclear active site and a distorted (β/α)7-barrel protein fold. This group of enzymes is closely related to the amidohydrolase superfamily of enzymes. The mechanism of phosphomonoester bond hydrolysis by the PHP family of HPP enzymes was addressed. Recombinant HPP from Lactococcus lactis subsp. lactis that was expressed in Escherichia coli contained a mixture of iron and zinc in the active site and had a catalytic efficiency of ~103 M−1 s−1. Expression of the protein under iron-free conditions resulted in the production of enzyme with a two orders of magnitude improvement in catalytic efficiency and a mixture of zinc and manganese in the active site. Solvent isotope and viscosity effects demonstrated that proton transfer steps and product dissociation steps are not rate-limiting. X-ray structures of HPP were determined with sulfate, L-histidinol/phosphate, and a complex of L-histidinol and arsenate bound in the active site. These crystal structures and the catalytic properties of variants were used to identify the structural elements required for catalysis and substrate recognition by the HPP family of enzymes within the amidohydrolase superfamily. PMID:23327428

  14. Structural and mechanistic characterization of L-histidinol phosphate phosphatase from the polymerase and histidinol phosphatase family of proteins.

    Science.gov (United States)

    Ghodge, Swapnil V; Fedorov, Alexander A; Fedorov, Elena V; Hillerich, Brandan; Seidel, Ronald; Almo, Steven C; Raushel, Frank M

    2013-02-12

    L-Histidinol phosphate phosphatase (HPP) catalyzes the hydrolysis of L-histidinol phosphate to L-histidinol and inorganic phosphate, the penultimate step in the biosynthesis of L-histidine. HPP from the polymerase and histidinol phosphatase (PHP) family of proteins possesses a trinuclear active site and a distorted (β/α)(7)-barrel protein fold. This group of enzymes is closely related to the amidohydrolase superfamily of enzymes. The mechanism of phosphomonoester bond hydrolysis by the PHP family of HPP enzymes was addressed. Recombinant HPP from Lactococcus lactis subsp. lactis that was expressed in Escherichia coli contained a mixture of iron and zinc in the active site and had a catalytic efficiency of ~10(3) M(-1) s(-1). Expression of the protein under iron-free conditions resulted in the production of an enzyme with a 2 order of magnitude improvement in catalytic efficiency and a mixture of zinc and manganese in the active site. Solvent isotope and viscosity effects demonstrated that proton transfer steps and product dissociation steps are not rate-limiting. X-ray structures of HPP were determined with sulfate, L-histidinol phosphate, and a complex of L-histidinol and arsenate bound in the active site. These crystal structures and the catalytic properties of variants were used to identify the structural elements required for catalysis and substrate recognition by the HPP family of enzymes within the amidohydrolase superfamily.

  15. Crystal structure of dihydropyrimidinase from Pseudomonas aeruginosa PAO1: Insights into the molecular basis of formation of a dimer.

    Science.gov (United States)

    Tzeng, Ching-Ting; Huang, Yen-Hua; Huang, Cheng-Yang

    2016-09-23

    Dihydropyrimidinase, a tetrameric metalloenzyme, is a member of the cyclic amidohydrolase family, which also includes allantoinase, dihydroorotase, hydantoinase, and imidase. In this paper, we report the crystal structure of dihydropyrimidinase from Pseudomonas aeruginosa PAO1 at 2.1 Å resolution. The structure of P. aeruginosa dihydropyrimidinase reveals a classic (β/α)8-barrel structure core embedding the catalytic dimetal center and a β-sandwich domain, which is commonly found in the architecture of dihydropyrimidinases. In contrast to all dihydropyrimidinases, P. aeruginosa dihydropyrimidinase forms a dimer, rather than a tetramer, both in the crystalline state and in the solution. Basing on sequence analysis and structural comparison of the C-terminal region and the dimer-dimer interface between P. aeruginosa dihydropyrimidinase and Thermus sp. dihydropyrimidinase, we propose a working model to explain why this enzyme cannot be a tetramer.

  16. Genetic diversity of Leptospira in northwestern Colombia: first report of Leptospira santarosai as a recognised leptospirosis agent

    Directory of Open Access Journals (Sweden)

    Ronald Guillermo Peláez Sanchez

    Full Text Available The region of Antioquia in northeastern Colombia has the highest number of reported leptospirosis cases in the country. It also shows high seroprevalence indexes in the general population and socio-environmental conditions favourable for the transmission of the disease between humans and animals. In this study, 25 Leptospira isolates from Colombia’s Antioquia department were identified to the species level as L. santarosai (12, L. interrogans (9 and L. meyeri (4 using phylogenetic analysis of the Amidohydrolase gene. Typing at the serovar level was performed using multilocus sequence typing (MLST and monoclonal antibodies. The serovars Canalzonae, Babudieri, Alice, Beye, and Copenhageni have been identified as causing human or animal infections in Antioquia, Colombia. The four environmental isolates were not identified to the serovar level. L. santarosai serovar Canalzonae and Alice were identified as new etiologic agents of human leptospirosis in Antioquia, Colombia. This paper reports species and serovars that were previously unknown in the region.

  17. Genetic diversity of Leptospira in northwestern Colombia: first report of Leptospira santarosai as a recognised leptospirosis agent

    Science.gov (United States)

    Peláez Sanchez, Ronald Guillermo; Lopez, Juan Álvaro; Pereira, Martha María; Arboleda Naranjo, Margarita; Agudelo-Flórez, Piedad

    2016-01-01

    The region of Antioquia in northeastern Colombia has the highest number of reported leptospirosis cases in the country. It also shows high seroprevalence indexes in the general population and socio-environmental conditions favourable for the transmission of the disease between humans and animals. In this study, 25 Leptospira isolates from Colombia’s Antioquia department were identified to the species level as L. santarosai (12), L. interrogans (9) and L. meyeri (4) using phylogenetic analysis of the Amidohydrolase gene. Typing at the serovar level was performed using multilocus sequence typing (MLST) and monoclonal antibodies. The serovars Canalzonae, Babudieri, Alice, Beye, and Copenhageni have been identified as causing human or animal infections in Antioquia, Colombia. The four environmental isolates were not identified to the serovar level. L. santarosai serovar Canalzonae and Alice were identified as new etiologic agents of human leptospirosis in Antioquia, Colombia. This paper reports species and serovars that were previously unknown in the region. PMID:27982303

  18. X-Ray Structure and Mutagenesis Studies of the N-Isopropylammelide Isopropylaminohydrolase, AtzC.

    Directory of Open Access Journals (Sweden)

    Sahil Balotra

    Full Text Available The N-isopropylammelide isopropylaminohydrolase from Pseudomonas sp. strain ADP, AtzC, provides the third hydrolytic step in the mineralization of s-triazine herbicides, such as atrazine. We obtained the X-ray crystal structure of AtzC at 1.84 Å with a weak inhibitor bound in the active site and then used a combination of in silico docking and site-directed mutagenesis to understand the interactions between AtzC and its substrate, isopropylammelide. The substitution of an active site histidine residue (His249 for an alanine abolished the enzyme's catalytic activity. We propose a plausible catalytic mechanism, consistent with the biochemical and crystallographic data obtained that is similar to that found in carbonic anhydrase and other members of subtype III of the amidohydrolase family.

  19. Finding homes for orphan enzymes

    Directory of Open Access Journals (Sweden)

    Frank M. Raushel

    2016-12-01

    Full Text Available The rate at which new genes are being sequenced greatly exceeds our ability to correctly annotate the functional properties of the corresponding proteins. Annotations based primarily on sequence identity to experimentally characterized proteins are often misleading because closely related sequences may have different functions, while highly divergent sequences may have identical functions. Our understanding of the principles that dictate the catalytic properties of enzymes, based on protein sequence alone, is often insufficient to correctly annotate proteins of unknown function. To address these problems, we are working to develop a comprehensive strategy for the functional annotation of newly sequenced genes using a combination of structural biology, bioinformatics, computational biology, and molecular enzymology. The power of this multidisciplinary approach for discovering new reactions catalyzed by uncharacterized enzymes has been tested using the amidohydrolase superfamily as a model system.

  20. Genome mining of the hitachimycin biosynthetic gene cluster: involvement of a phenylalanine-2,3-aminomutase in biosynthesis.

    Science.gov (United States)

    Kudo, Fumitaka; Kawamura, Koichi; Uchino, Asuka; Miyanaga, Akimasa; Numakura, Mario; Takayanagi, Ryuichi; Eguchi, Tadashi

    2015-04-13

    Hitachimycin is a macrolactam antibiotic with (S)-β-phenylalanine (β-Phe) at the starter position of its polyketide skeleton. To understand the incorporation mechanism of β-Phe and the modification mechanism of the unique polyketide skeleton, the biosynthetic gene cluster for hitachimycin in Streptomyces scabrisporus was identified by genome mining. The identified gene cluster contains a putative phenylalanine-2,3-aminomutase (PAM), five polyketide synthases, four β-amino-acid-carrying enzymes, and a characteristic amidohydrolase. A hitA knockout mutant showed no hitachimycin production, but antibiotic production was restored by feeding with (S)-β-Phe. We also confirmed the enzymatic activity of the HitA PAM. The results suggest that the identified gene cluster is responsible for the biosynthesis of hitachimycin. A plausible biosynthetic pathway for hitachimycin, including a unique polyketide skeletal transformation mechanism, is proposed.

  1. Structural basis for thermostability revealed through the identification and characterization of a highly thermostable phosphotriesterase-like lactonase from Geobacillus stearothermophilus

    Energy Technology Data Exchange (ETDEWEB)

    Hawwa, Renda; Aikens, John; Turner, Robert J.; Santarsiero, Bernard D.; Mescar, Andrew D.; (Lybradyn Inc.); (UIC)

    2009-08-31

    A new enzyme homologous to phosphotriesterase was identified from the bacterium Geobacillus stearothermophilus (GsP). This enzyme belongs to the amidohydrolase family and possesses the ability to hydrolyze both lactone and organophosphate (OP) compounds, making it a phosphotriesterase-like lactonase (PLL). GsP possesses higher OP-degrading activity than recently characterized PLLs, and it is extremely thermostable. GsP is active up to 100 C with an energy of activation of 8.0 kcal/mol towards ethyl paraoxon, and it can withstand an incubation temperature of 60 C for two days. In an attempt to understand the thermostability of PLLs, the X-ray structure of GsP was determined and compared to those of existing PLLs. Based upon a comparative analysis, a new thermal advantage score and plot was developed and reveals that a number of different factors contribute to the thermostability of PLLs.

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

    Science.gov (United States)

    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 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  4. [The mechanism of action of cannabis and cannabinoids].

    Science.gov (United States)

    Scholten, W K

    2006-01-21

    The effect ofcannabis can be explained on the basis of the function of the cannabinoid receptor system, which consists of CB receptors (CB1, CB2), endoligands to activate these receptors and an enzyme--fatty acid amidohydrolase--to metabolize the endoligands. The endoligands of the cannabinoid receptor system are arachidonic acid-like substances, and are called endocannabinoids. Indications exist that the body also contains arachidonic acid-like substances that inhibit fatty acid amido hydrolase. Various cannabinoids have diverse effects on the receptors, functioning as agonists, antagonists or partial antagonists, as well as affecting the vanilloid receptor. Many known effects ofcannabis can be explained on the basis of this mechanism of action as can the use ofcannabis in various conditions including multiple sclerosis, Parkinson's disease, glaucoma, nausea, vomiting and rheumatoid arthritis.

  5. Eukaryotic beta-alanine synthases are functionally related but have a high degree of structural diversity

    DEFF Research Database (Denmark)

    Gojkovic, Zoran; Sandrini, Michael; Piskur, Jure

    2001-01-01

    beta -Alanine synthase (EC 3.5.1.6), which catalyzes the final step of pyrimidine catabolism, has only been characterized in mammals. A Saccharomyces kluyveri pyd3 mutant that is unable to grow on N-carbamy-beta -alanine as the sole nitrogen source and exhibits diminished beta -alanine synthase...... no pyrimidine catabolic pathway, it enabled growth on N-carbamyl- beta -alanine as the sole nitrogen source. The D. discoideum and D. melanogaster PYD3 gene products are similar to mammalian beta -alanine synthases. In contrast, the S. kluyveri protein is quite different from these and more similar to bacterial...... N- carbamyl amidohydrolases. All three beta -alanine synthases are to some degree related to various aspartate transcarbamylases, which catalyze the second step of the de novo pyrimidine biosynthetic pathway. PYD3 expression in yeast seems to be inducible by dihydrouracil and N-carbamyl-beta...

  6. The role of two families of bacterial enzymes in putrescine synthesis from agmatine via agmatine deiminase.

    Science.gov (United States)

    Landete, José M; Arena, Mario E; Pardo, Isabel; Manca de Nadra, María C; Ferrer, Sergi

    2010-12-01

    Putrescine, one of the main biogenic amines associated to microbial food spoilage, can be formed by bacteria from arginine via ornithine decarboxylase (ODC), or from agmatine via agmatine deiminase (AgDI). This study aims to correlate putrescine production from agmatine to the pathway involving N-carbamoylputrescine formation via AdDI (the aguA product) and N-carbamoylputrescine amidohydrolase (the aguB product), or putrescine carbamoyltransferase (the ptcA product) in bacteria. PCR methods were developed to detect the two genes involved in putrescine production from agmatine. Putrescine production from agmatine could be linked to the aguA and ptcA genes in Lactobacillus hilgardii X1B, Enterococcus faecalis ATCC 11700, and Bacillus cereus ATCC 14579. By contrast Lactobacillus sakei 23K was unable to produce putrescine, and although a fragment of DNA corresponding to the gene aguA was amplified, no amplification was observed for the ptcA gene. Pseudomonas aeruginosa PAO1 produces putrescine and is reported to harbour aguA and aguB genes, responsible for agmatine deiminase and N-carbamoylputrescine amidohydrolase activities. The enzyme from P. aeruginosa PAO1 that converts N-carbamoylputrescine to putrescine (the aguB product) is different from other microorganisms studied (the ptcA product). Therefore, the aguB gene from P. aeruginosa PAO1 could not be amplified with ptcA-specific primers. The aguB and ptcA genes have frequently been erroneously annotated in the past, as in fact these two enzymes are neither homologous nor analogous. Furthermore, the aguA, aguB and ptcA sequences available from GenBank were subjected to phylogenetic analysis, revealing that gram-positive bacteria harboured ptcA, whereas gram-negative bacteria harbour aguB. This paper also discusses the role of the agmatine deiminase system (AgDS) in acid stress resistance.

  7. Function Discovery and Structural Characterization of a Methylphosphonate Esterase

    Energy Technology Data Exchange (ETDEWEB)

    Xiang, Dao Feng [Texas A & M Univ., College Station, TX (United States); Patskovsky, Yury [Einstein College of Medicine, Bronx, NY (United States); Nemmara, Venkatesh V. [Texas A & M Univ., College Station, TX (United States); Toro, Rafael [Einstein College of Medicine, Bronx, NY (United States); Almo, Steven C. [Einstein College of Medicine, Bronx, NY (United States); Raushel, Frank M. [Texas A & M Univ., College Station, TX (United States)

    2015-05-12

    Pmi1525, an enzyme of unknown function from Proteus mirabilis HI4320 and the amidohydrolase superfamily, was cloned, purified to homogeneity, and functionally characterized. The three-dimensional structure of Pmi1525 was determined with zinc and cacodylate bound in the active site (PDB id: 3RHG). We also determined the structure with manganese and butyrate in the active site (PDB id: 4QSF). Pmi1525 folds as a distorted (β/α)8-barrel that is typical for members of the amidohydrolase superfamily and cog1735. Moreover, the substrate profile for Pmi1525 was determined via a strategy that marshaled the utilization of bioinformatics, structural characterization, and focused library screening. The protein was found to efficiently catalyze the hydrolysis of organophosphonate and carboxylate esters. The best substrates identified for Pmi1525 are ethyl 4-nitrophenylmethyl phosphonate (kcat and kcat /Km values of 580 s–1 and 1.2 × 105 M–1 s–1, respectively) and 4-nitrophenyl butyrate (kcat and kcat /Km values of 140 s–1 and 1.4 × 105 M–1 s–1, respectively). Pmi1525 is stereoselective for the hydrolysis of chiral methylphosphonate esters. The enzyme hydrolyzes the (SP)-enantiomer of isobutyl 4-nitrophenyl methylphosphonate 14 times faster than the corresponding (RP)-enantiomer. The catalytic properties of this enzyme make it an attractive template for the evolution of novel enzymes for the detection, destruction, and detoxification of organophosphonate nerve agents.

  8. Metabolism of allantoin in soybeans

    Energy Technology Data Exchange (ETDEWEB)

    Winkler, R.G.; Polacco, J.C.; Blevins, D.G.; Randall, D.D.

    1986-04-01

    The metabolism of (2-7 /sup 14/C) and (4-5 /sup 14/C) allantoin has been studied in intact leaf tissue to elucidate the pathway of allantoin catabolism and its regulation. 1.3 mM (2-7 /sup 14/C) and 1.3 mM (4-5 /sup 14/C) DL allantoin release /sup 14/CO/sub 2/ from intact leaf discs at 0.9 and 0.08 ..mu..moles.hr/sup -1/.g fresh wt/sup -1/ respectively. The most potent urease inhibitor known, phenyl phosphordiamidate (PPD), inhibited urease in intact tissue at concentrations from 0.1 mM to 10 mM. In contrast /sup 14/CO/sub 2/ release from (2-7 /sup 14/C) and (4-5 /sup 14/C) allantoin was not inhibited by 1 mM PPD in 0.5 h and 1 h assays. These data are consistent with allantoate amido-hydrolase action. Intact tissue discs were incubated with (4-5 /sup 14/C) allantoin and analyzed for catabolites by ion exclusion HPLC. Allantoate, ureidoglycolate, and glyoxylate were identified by their retention time. Two /sup 14/C labelled peaks did not migrate with known standards. They are being characterized to identify their structure. These data are consistent with a pathway of catabolism including allantoate, ureidoglycolate and glyoxylate, NH/sub 3/ and CO/sub 2/.

  9. Interactions of "bora-penicilloates" with serine β-lactamases and DD-peptidases.

    Science.gov (United States)

    Dzhekieva, Liudmila; Adediran, S A; Pratt, R F

    2014-10-21

    Specific boronic acids are generally powerful tetrahedral intermediate/transition state analogue inhibitors of serine amidohydrolases. This group of enzymes includes bacterial β-lactamases and DD-peptidases where there has been considerable development of boronic acid inhibitors. This paper describes the synthesis, determination of the inhibitory activity, and analysis of the results from two α-(2-thiazolidinyl) boronic acids that are closer analogues of particular tetrahedral intermediates involved in β-lactamase and DD-peptidase catalysis than those previously described. One of them, 2-[1-(dihydroxyboranyl)(2-phenylacetamido)methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid, is a direct analogue of the deacylation tetrahedral intermediates of these enzymes. These compounds are micromolar inhibitors of class C β-lactamases but, very unexpectedly, not inhibitors of class A β-lactamases. We rationalize the latter result on the basis of a new mechanism of boronic acid inhibition of the class A enzymes. A stable inhibitory complex is not accessible because of the instability of an intermediate on its pathway of formation. The new boronic acids also do not inhibit bacterial DD-peptidases (penicillin-binding proteins). This result strongly supports a central feature of a previously proposed mechanism of action of β-lactam antibiotics, where deacylation of β-lactam-derived acyl-enzymes is not possible because of unfavorable steric interactions.

  10. Characterization of a novel cell wall binding domain-containing Staphylococcus aureus endolysin LysSA97.

    Science.gov (United States)

    Chang, Yoonjee; Ryu, Sangryeol

    2017-01-01

    Endolysin from Staphylococcus aureus phage SA97 (LysSA97) was cloned and investigated. LysSA97 specifically lyse the staphylococcal strains and effectively disrupted staphylococcal biofilms. Bioinformatic analysis of LysSA97 revealed a novel putative cell wall binding domain (CBD) as well as two enzymatically active domains (EADs) containing cysteine, histidine-dependent amidohydrolases/peptidases (CHAP, PF05257) and N-acetylmuramoyl-L-alanine amidase (Amidase-3, PF01520) domains. Comparison of 98 endolysin genes of S. aureus phages deposited in GenBank showed that they can be classified into six groups based on their domain composition. Interestingly, approximately 80.61 % of the staphylococcal endolysins have a src-homology 3 (SH3, PF08460) domain as CBD, but the remaining 19.39 %, including LysSA97, has a putative C-terminal CBD with no homology to the known CBD. The fusion protein containing green fluorescent protein and the putative CBD of LysSA97 showed a specific binding spectrum against staphylococcal cells comparable to SH3 domain (PF08460), suggesting that the C-terminal domain of LysSA97 is a novel CBD of staphylococcal endolysins.

  11. Discovery of a cAMP deaminase that quenches cyclic AMP-dependent regulation.

    Science.gov (United States)

    Goble, Alissa M; Feng, Youjun; Raushel, Frank M; Cronan, John E

    2013-12-20

    An enzyme of unknown function within the amidohydrolase superfamily was discovered to catalyze the hydrolysis of the universal second messenger, cyclic-3',5'-adenosine monophosphate (cAMP). The enzyme, which we have named CadD, is encoded by the human pathogenic bacterium Leptospira interrogans. Although CadD is annotated as an adenosine deaminase, the protein specifically deaminates cAMP to cyclic-3',5'-inosine monophosphate (cIMP) with a kcat/Km of 2.7 ± 0.4 × 10(5) M(-1) s(-1) and has no activity on adenosine, adenine, or 5'-adenosine monophosphate (AMP). This is the first identification of a deaminase specific for cAMP. Expression of CadD in Escherichia coli mimics the loss of adenylate cyclase in that it blocks growth on carbon sources that require the cAMP-CRP transcriptional activator complex for expression of the cognate genes. The cIMP reaction product cannot replace cAMP as the ligand for CRP binding to DNA in vitro and cIMP is a very poor competitor of cAMP activation of CRP for DNA binding. Transcriptional analyses indicate that CadD expression represses expression of several cAMP-CRP dependent genes. CadD adds a new activity to the cAMP metabolic network and may be a useful tool in intracellular study of cAMP-dependent processes.

  12. A novel endolysin disrupts Streptococcus suis with high efficiency.

    Science.gov (United States)

    Ji, Wenhui; Huang, Qingqing; Sun, Liang; Wang, Hengan; Yan, Yaxian; Sun, Jianhe

    2015-12-01

    Streptococcus suis serotype 2 (S. suis 2) is a zoonotic pathogen that exhibits high-level resistance and multi-drug resistance to classic antibiotics and causes serious human casualties and heavy economic losses in the swine industry worldwide. Therefore, alternative therapies or novel antibacterial agents need to be developed to combat this pathogen. A novel endolysin derived from the S. suis temperate phage phi7917, termed Ly7917, was identified, which had broad lytic activity against S. suis type 1, 2, 7 and 9. Ly7917 consisted of an N-terminal cysteine, histidine-dependent amidohydrolases/peptidase catalytic domain and C-terminal SH3b cell wall binding domain. The endolysin maintained activity at high pH and its catalytic activity could be improved by addition of 10 μM 1.5 mM Ca(2+). In animal studies, 90% of BALB/c mice challenged with typical virulent strain HA9801 of S. suis 2 were protected by Ly7917 treatment. The bacterial load in the blood of HA9801-challenged mice was efficiently reduced almost 50% by Ly7917 while that of penicillin-G-treated mice kept almost unchanged. Our data suggest that Ly7917 may be an alternative therapeutic agent for infections caused by virulent S. suis strains.

  13. Synthesis and biochemical analysis of 2,2,3,3,4,4,5,5,6,6,7,7-dodecafluoro-N-hydroxy-octanediamides as inhibitors of human histone deacetylases.

    Science.gov (United States)

    Henkes, Leonhard M; Haus, Patricia; Jäger, Felix; Ludwig, Joachim; Meyer-Almes, Franz-Josef

    2012-01-15

    Inhibition of human histone deacetylases (HDACs) has emerged as a novel concept in the chemotherapeutic treatment of cancer. Two chemical entities, SAHA (ZOLINZA, Merck) and romidepsin (Istodax, Celgene) have been recently approved by the FDA as first-in-class drugs against cutaneous T-cell lymphoma. Clinical use of these drugs revealed several side effects including gastro-intestinal symptoms, fatigue, thrombocytopenia, thrombosis. Romidepsin is associated with an yet unresolved cardiotoxicity issue. A general hypothesis for the diminishment of unwanted adverse effects and an improved therapeutical window suggests the development of more isotype selective inhibitors. In this study the first time HDAC inhibitors with perfluorinated spacers between the zinc chelating moiety and the aromatic capping group were synthesized and tested against representatives of HDAC classes I, IIa and IIb. Competitive binding assays and a combined approach by using blind docking and molecular dynamics support binding of the perfluorinated analogs of SAHA to the active site of the HDAC-like amidohydrolase from Bordetella/Alcaligenes and presumably also to human HDACs. In contrast to the alkyl spacer of SAHA and derivatives, the perfluorinated alkyl spacer seems to contribute to or facilitate the induction of selectivity for class II, particularly class IIa, HDACs even though the overall potency of the perfluorinated SAHA analogs in this study against human HDACs remained still rather moderate in the micromolar range.

  14. Expanding targets for a metabolic therapy of cancer: L-asparaginase.

    Science.gov (United States)

    Covini, Daniele; Tardito, Saverio; Bussolati, Ovidio; Chiarelli, Laurent R; Pasquetto, Maria V; Digilio, Rita; Valentini, Giovanna; Scotti, Claudia

    2012-01-01

    The antitumour enzyme L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1, ASNase), which catalyses the deamidation of L-asparagine (Asn) to L-aspartic acid and ammonia, has been used for many years in the treatment of acute lymphoblastic leukaemia. Also NK tumours, subtypes of myeloid leukaemias and T-cell lymphomas respond to ASNase, and ovarian carcinomas and other solid tumours have been proposed as additional targets for ASNase, with a potential role for its glutaminase activity. The increasing attention devoted to the antitumour activity of ASNase prompted us to analyse recent patents specifically concerning this enzyme. Here, we first give an overview of metabolic pathways affected by Asn and Gln depletion and, hence, potential targets of ASNase. We then discuss recent published patents concerning ASNases. In particular, we pay attention to novel ASNases, such as the recently characterised ASNase produced by Helicobacter pylori, and those presenting amino acid substitutions aimed at improving enzymatic activity of the classical Escherichia coli enzyme. We detail modifications, such as natural glycosylation or synthetic conjugation with other molecules, for therapeutic purposes. Finally, we analyse patents concerning biotechnological protocols and strategies applied to production of ASNase as well as to its administration and delivery in organisms.

  15. Phage endolysins with broad antimicrobial activity against Enterococcus faecalis clinical strains.

    Science.gov (United States)

    Proença, Daniela; Fernandes, Sofia; Leandro, Clara; Silva, Filipa Antunes; Santos, Sofia; Lopes, Fátima; Mato, Rosario; Cavaco-Silva, Patrícia; Pimentel, Madalena; São-José, Carlos

    2012-06-01

    Increasing antibiotic resistance of bacterial pathogens has drawn the attention to the potential use of bacteriophage endolysins as alternative antibacterial agents. Here we have identified, characterized, and studied the lytic potential of two endolysins, Lys168 and Lys170, from phages infecting Enterococcus faecalis. Lys168 and Lys170 belong to the cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) and amidase-2 protein families, respectively. Lys168 is quite a unique enterococcal phage endolysin. It shares 95% amino acidic identity with the endolysin of Staphylococcus aureus phage SAP6, which in turn is distantly related to all known CHAP endolysins of S. aureus phages. Lys170 seems to be a natural chimera assembling catalytic and cell-wall-binding domains of different origin. Both endolysins showed a clear preference to act against E. faecalis and they were able to lyse a high proportion of clinical isolates of this species. Specifically, Lys168 and Lys170 lysed more than 70% and 90% of the tested isolates, respectively, which included a panel of diverse and typed strains representative of highly prevalent clonal complexes. Lys170 was active against all tested E. faecalis VRE strains. The quasi specificity toward E. faecalis is discussed considering the nature of the enzymes' functional domains and the structure of the cell wall peptidoglycan.

  16. Three-Dimensional Structure and Catalytic Mechanism of Cytosine Deaminase

    Energy Technology Data Exchange (ETDEWEB)

    R Hall; A Fedorov; C Xu; E Fedorov; S Almo; F Raushel

    2011-12-31

    Cytosine deaminase (CDA) from E. coli is a member of the amidohydrolase superfamily. The structure of the zinc-activated enzyme was determined in the presence of phosphonocytosine, a mimic of the tetrahedral reaction intermediate. This compound inhibits the deamination of cytosine with a K{sub i} of 52 nM. The zinc- and iron-containing enzymes were characterized to determine the effect of the divalent cations on activation of the hydrolytic water. Fe-CDA loses activity at low pH with a kinetic pKa of 6.0, and Zn-CDA has a kinetic pKa of 7.3. Mutation of Gln-156 decreased the catalytic activity by more than 5 orders of magnitude, supporting its role in substrate binding. Mutation of Glu-217, Asp-313, and His-246 significantly decreased catalytic activity supporting the role of these three residues in activation of the hydrolytic water molecule and facilitation of proton transfer reactions. A library of potential substrates was used to probe the structural determinants responsible for catalytic activity. CDA was able to catalyze the deamination of isocytosine and the hydrolysis of 3-oxauracil. Large inverse solvent isotope effects were obtained on k{sub cat} and k{sub cat}/K{sub m}, consistent with the formation of a low-barrier hydrogen bond during the conversion of cytosine to uracil. A chemical mechanism for substrate deamination by CDA was proposed.

  17. Inhibition of Urease by Disulfiram, an FDA-Approved Thiol Reagent Used in Humans

    Directory of Open Access Journals (Sweden)

    Ángel Gabriel Díaz-Sánchez

    2016-11-01

    Full Text Available Urease is a nickel-dependent amidohydrolase that catalyses the decomposition of urea into carbamate and ammonia, a reaction that constitutes an important source of nitrogen for bacteria, fungi and plants. It is recognized as a potential antimicrobial target with an impact on medicine, agriculture, and the environment. The list of possible urease inhibitors is continuously increasing, with a special interest in those that interact with and block the flexible active site flap. We show that disulfiram inhibits urease in Citrullus vulgaris (CVU, following a non-competitive mechanism, and may be one of this kind of inhibitors. Disulfiram is a well-known thiol reagent that has been approved by the FDA for treatment of chronic alcoholism. We also found that other thiol reactive compounds (l-captopril and Bithionol and quercetin inhibits CVU. These inhibitors protect the enzyme against its full inactivation by the thiol-specific reagent Aldrithiol (2,2′-dipyridyl disulphide, DPS, suggesting that the three drugs bind to the same subsite. Enzyme kinetics, competing inhibition experiments, auto-fluorescence binding experiments, and docking suggest that the disulfiram reactive site is Cys592, which has been proposed as a “hinge” located in the flexible active site flap. This study presents the basis for the use of disulfiram as one potential inhibitor to control urease activity.

  18. The Three-Dimensional Structure and Catalytic Mechanism of Cytosine Deaminase†

    Science.gov (United States)

    Hall, Richard S.; Fedorov, Alexander A.; Xu, Chengfu; Fedorov, Elena V.; Almo, Steven C.; Raushel, Frank M.

    2011-01-01

    Cytosine deaminase (CDA) from E. coli is a member of the amidohydrolase superfamily. The structure of the zinc-activated enzyme was determined in the presence of phosphonocytosine, a mimic of the tetrahedral reaction intermediate. This compound inhibits the deamination of cytosine with a Ki of 52 nM. The zinc and iron containing enzymes were characterized to determine the effect of the divalent cations on activation of the hydrolytic water. Fe-CDA loses activity at low pH with a kinetic pKa of 6.0 and Zn-CDA has a kinetic pKa of 7.3. Mutation of Gln-156 decreased the catalytic activity by more than 5 orders of magnitude, supporting its role in substrate binding. Mutation of Glu-217, Asp-313, and His-246 significantly decreased catalytic activity supporting the role of these three residues in activation of the hydrolytic water molecule and facilitation of proton transfer reactions. A library of potential substrates was used to probe the structural determinants responsible for catalytic activity. CDA was able to catalyze the deamination of isocytosine and the hydrolysis of 3-oxauracil. Large inverse solvent isotope effects were obtained on kcat and kcat/Km, consistent with the formation of a low-barrier hydrogen bond during the conversion of cytosine to uracil. A chemical mechanism for substrate deamination by CDA was proposed. PMID:21545144

  19. Characterization of the Biosynthetic Gene Cluster for Benzoxazole Antibiotics A33853 Reveals Unusual Assembly Logic.

    Science.gov (United States)

    Lv, Meinan; Zhao, Junfeng; Deng, Zixin; Yu, Yi

    2015-10-22

    A33853, which shows excellent bioactivity against Leishmania, is a benzoxazole-family compound formed from two moieties of 3-hydroxyanthranilic acid and one 3-hydroxypicolinic acid. In this study, we have identified the gene cluster responsible for the biosynthesis of A33853 in Streptomyces sp. NRRL12068 through genome mining and heterologous expression. Bioinformatics analysis and functional characterization of the orfs contained in the gene cluster revealed that the biosynthesis of A33853 is directed by a group of unusual enzymes. In particular, BomK, annotated as a ketosynthase, was found to catalyze the amide bond formation between 3-hydroxypicolinic and 3-hydroxyanthranilic acid during the assembly of A33853. BomJ, a putative ATP-dependent coenzyme A ligase, and BomN, a putative amidohydrolase, were further proposed to be involved in the benzoxazole formation in A33853 according to gene deletion experiments. Finally, we have successfully utilized mutasynthesis to generate two analogs of A33853, which were reported previously to possess excellent anti-leishmanial activity.

  20. Screening for N-AHSL-Based-Signaling Interfering Enzymes.

    Science.gov (United States)

    Uroz, Stéphane; Oger, Phil M

    2017-01-01

    Quorum sensing (QS)-based signaling is a widespread pathway used by bacteria for the regulation of functions involved in their relation to the environment or their host. QS relies upon the production, accumulation and perception of small diffusable molecules by the bacterial population, hence linking high gene expression with high cell population densities. Among the different QS signal molecules, an important class of signal molecules is the N-acyl homoserine lactone (N-AHSL). In pathogens such as Erwinia or Pseudomonas, N-AHSL based QS is crucial to overcome the host defenses and ensure a successful infection. Interfering with QS-regulation allows the algae Delisea pulcra to avoid surface colonization by bacteria. Thus, interfering the QS-regulation of pathogenic bacteria is a promising antibiotic-free antibacterial therapeutic strategy. To date, two N-AHSL lactonases and one amidohydrolase families of N-ASHL degradation enzymes have been characterized and have proven to be efficient in vitro to control N-AHSL-based QS-regulated functions in pathogens. In this chapter, we provide methods to screen individual clones or bacterial strains as well as pool of clones for genomic and metagenomic libraries, that can be used to identify strains or clones carrying N-ASHL degradation enzymes.

  1. X-ray crystal structure of the streptococcal specific phage lysin PlyC

    Science.gov (United States)

    McGowan, Sheena; Buckle, Ashley M.; Mitchell, Michael S.; Hoopes, James T.; Gallagher, D. Travis; Heselpoth, Ryan D.; Shen, Yang; Reboul, Cyril F.; Law, Ruby H. P.; Fischetti, Vincent A.; Whisstock, James C.; Nelson, Daniel C.

    2012-01-01

    Bacteriophages deploy lysins that degrade the bacterial cell wall and facilitate virus egress from the host. When applied exogenously, these enzymes destroy susceptible microbes and, accordingly, have potential as therapeutic agents. The most potent lysin identified to date is PlyC, an enzyme assembled from two components (PlyCA and PlyCB) that is specific for streptococcal species. Here the structure of the PlyC holoenzyme reveals that a single PlyCA moiety is tethered to a ring-shaped assembly of eight PlyCB molecules. Structure-guided mutagenesis reveals that the bacterial cell wall binding is achieved through a cleft on PlyCB. Unexpectedly, our structural data reveal that PlyCA contains a glycoside hydrolase domain in addition to the previously recognized cysteine, histidine-dependent amidohydrolases/peptidases catalytic domain. The presence of eight cell wall-binding domains together with two catalytic domains may explain the extraordinary potency of the PlyC holoenyzme toward target bacteria. PMID:22807482

  2. Plant growth regulators induced urease activity in Cucurbita pepo L. cotyledons.

    Science.gov (United States)

    El Shora, Hamed M; Ali, Awatif S

    2016-03-01

    This study is aimed to investigate the activity of urease (EC 3.5.1.5, urea amidohydrolase) that catalyzes the hydrolysis of urea in 5-day-old Cucurbita pepo cotyledons subjected to various concentrations of different growth regulators. The treatment of C. pepo cotyledons with different concentrations (100-600 μmol) of different auxins [indole-3-acetic acid (IAA), indole butyric acid (IBA), indole propionic acid (IPA) and naphthalene acetic acid (NAA)]; or with different concentrations (100-300 μmol) of different cytokinins [kinetin, zeatin and benzyladenine (6-BA)] resulted in a significant increase of urease activity, compared to control. The optimal effects were recorded for each of 500 μmol of IAA and 300 μmol of zeatin treatments. A gradual increase in urease activity was detected in cotyledons treated with various concentrations (0.2-1.0 mM) of 28-homobrassinolide (HBL), in relative to control. A substantial increase in urease activity was observed in cotyledons subjected to different concentrations of triazole (10-60 mg L(-1)), containing either triadimefon (TDM) or hexaconazole (HEX), compared to control. The combination of 300 μmol zeatin with any of protein inhibitors, namely 5-fluorouridine (FUrd), cordycepin and α-amanitin, resulted in the alleviation of their inhibitory effect on the urease activity.

  3. Proteins from Erwinia asparaginase Erwinase ® and E. coli asparaginase 2 MEDAC ® for treatment of human leukemia, show a multitude of modifications for which the consequences are completely unclear.

    Science.gov (United States)

    Bae, Narkhyun; Pollak, Arnold; Lubec, Gert

    2011-07-01

    L-Asparaginase from Erwinia chrysanthemi (ASPG_ERWCH; UniProtKB accession number P06608 (Erwinase(®))) and L-asparaginase 2 from Escherichia coli (ASPG2_ECOLI; UniProtKB accession number P00805 (Medac(®))), both L-asparagine amidohydrolases, are widely used for the treatment of acute lymphoblastic leukemia. A series of serious side effects have been reported and this warrants studies into the protein chemistry of the medical products sold. Mass spectrometry (MS) data on ASPG_ERWCH and ASPG2_ECOLI have not been published so far and herein a gel-based proteomics study was performed to provide information about sequence and modifications of the commercially available medical products. ASPG_ERWCH and ASPG2_ECOLI were applied onto two-dimensional gel electrophoresis, spots were in-gel digested with several proteases and resulting peptides and protein modifications were analysed by nano-ESI-LC-MS/MS. Four spots were observed for ASPG_ERWCH, six spots were observed for ASPG2_ECOLI and the identified proteins showed high sequence coverage without sequence conflicts. Several protein modifications including technical and posttranslational modifications were demonstrated. Protein modifications are known to change physicochemical, immunochemical, biological and pharmacological properties and results from this work may challenge re-designing of the product including possible removal of the modifications by the manufacturer because it is not known whether they are contributing to the serious adverse effects of the protein drug.

  4. Complex structure of a bacterial class 2 histone deacetylase homologue with a trifluoromethylketone inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    Nielsen, Tine Kragh [Abteilung für Molekulare Strukturbiologie, Institut für Mikrobiologie und Genetik and GZMB, Justus-von-Liebig Weg 11, 37077 Göttingen (Germany); Hildmann, Christian; Riester, Daniel; Wegener, Dennis; Schwienhorst, Andreas [Abteilung für Molekulare Genetik und Präparative Molekularbiologie, Institut für Mikrobiologie und Genetik, Grisebachstrasse 8, 37077 Göttingen (Germany); Ficner, Ralf, E-mail: rficner@gwdg.de [Abteilung für Molekulare Strukturbiologie, Institut für Mikrobiologie und Genetik and GZMB, Justus-von-Liebig Weg 11, 37077 Göttingen (Germany)

    2007-04-01

    The crystal structure of HDAH FB188 in complex with a trifluoromethylketone at 2.2 Å resolution is reported and compared to a previously determined inhibitor complex. Histone deacetylases (HDACs) have emerged as attractive targets in anticancer drug development. To date, a number of HDAC inhibitors have been developed and most of them are hydroxamic acid derivatives, typified by suberoylanilide hydroxamic acid (SAHA). Not surprisingly, structural information that can greatly enhance the design of novel HDAC inhibitors is so far only available for hydroxamic acids in complex with HDAC or HDAC-like enzymes. Here, the first structure of an enzyme complex with a nonhydroxamate HDAC inhibitor is presented. The structure of the trifluoromethyl ketone inhibitor 9,9,9-trifluoro-8-oxo-N-phenylnonanamide in complex with bacterial FB188 HDAH (histone deacetylase-like amidohydrolase from Bordetella/Alcaligenes strain FB188) has been determined. HDAH reveals high sequential and functional homology to human class 2 HDACs and a high structural homology to human class 1 HDACs. Comparison with the structure of HDAH in complex with SAHA reveals that the two inhibitors superimpose well. However, significant differences in binding to the active site of HDAH were observed. In the presented structure the O atom of the trifluoromethyl ketone moiety is within binding distance of the Zn atom of the enzyme and the F atoms participate in interactions with the enzyme, thereby involving more amino acids in enzyme–inhibitor binding.

  5. Tackling Critical Catalytic Residues in Helicobacter pylori L-Asparaginase

    Directory of Open Access Journals (Sweden)

    Maristella Maggi

    2015-03-01

    Full Text Available Bacterial asparaginases (amidohydrolases, EC 3.5.1.1 are important enzymes in cancer therapy, especially for Acute Lymphoblastic Leukemia. They are tetrameric enzymes able to catalyze the deamination of L-ASN and, to a variable extent, of L-GLN, on which leukemia cells are dependent for survival. In contrast to other known L-asparaginases, Helicobacter pylori CCUG 17874 type II enzyme (HpASNase is cooperative and has a low affinity towards L-GLN. In this study, some critical amino acids forming the active site of HpASNase (T16, T95 and E289 have been tackled by rational engineering in the attempt to better define their role in catalysis and to achieve a deeper understanding of the peculiar cooperative behavior of this enzyme. Mutations T16E, T95D and T95H led to a complete loss of enzymatic activity. Mutation E289A dramatically reduced the catalytic activity of the enzyme, but increased its thermostability. Interestingly, E289 belongs to a loop that is very variable in L-asparaginases from the structure, sequence and length point of view, and which could be a main determinant of their different catalytic features.

  6. Production and optimization of L-glutaminase enzyme from Hypocrea jecorina pure culture.

    Science.gov (United States)

    Bülbül, Dilara; Karakuş, Emine

    2013-01-01

    L-Glutaminase (L-glutamine amidohydrolase, EC 3.5.1.2) is the important enzyme that catalyzes the deamination of L-glutamine to L-glutamic acid and ammonium ions. Recently, L-glutaminase has received much attention with respect to its therapeutic and industrial applications. It acts as a potent antileukemic agent and shows flavor-enhancing capacity in the production of fermented foods. Glutaminase activity is widely distributed in plants, animal tissues, and microorganisms, including bacteria, yeasts, and fungi. This study presents microbial production of glutaminase enzyme from Hypocrea jecorina pure culture and determination of optimum conditions and calculation of kinetic parameters of the produced enzyme. The optimum values were determined by using sa Nesslerization reaction for our produced glutaminase enzyme. The optimum pH value was determined as 8.0 and optimum temperature as 50°C for the glutaminase enzyme. The Km and Vmax values, the kinetic parameters, of enzyme produced from Hypocrea jecorina, pure culture were determined as 0.491 mM for Km and 13.86 U/L for Vmax by plotted Lineweaver-Burk graphing, respectively. The glutaminase enzyme from H. jecorina microorganism has very high thermal and storage stability.

  7. Degradation of atrazine by Frankia alni ACN14a: gene regulation, dealkylation, and dechlorination.

    Science.gov (United States)

    Rehan, Medhat; Kluge, Martin; Fränzle, Stefan; Kellner, Harald; Ullrich, René; Hofrichter, Martin

    2014-07-01

    Atrazine is transformed to N-isopropylammelide through hydroxyatrazine as an intermediate as indicated by high-performance liquid chromatography/mass spectroscopy in culture filtrates of Frankia alni ACN14a and Frankia sp. EuI1c. Both Frankia strains have the ability to degrade atrazine via dechlorination and dealkylation and, subsequently, may be using it as a nitrogen and carbon source as detected here by increasing their growth patterns. Bioinformatic analysis of the Frankia genomes revealed that a potential gene cluster involved in atrazine decomposition contains three genes, namely, trzN (FRAAL1474 and FraEuI1c_5874), atzB (FRAAL1473 and FraEuI1c_5875), and atzR (FRAAL1471). The relative messenger RNA gene expression of the former genes was examined by qRT-PCR. The LysR-type transcriptional regulator atzR (FRAAL1471), which is expected to control the cluster expression, showed a 13-fold increase in the expression level under atrazine stress. Moreover, the putative adenosine aminohydrolase 3 atzB (FRAAL1473), which is expected to dealkylate the N-ethyl group of atrazine, showed also an increased expression by factor 16 with increased exposure. Eventually, the trzN (FRAAL1474) gene, which is predicted to encode a putative amidohydrolase catalyzing atrazine dechlorination, exhibited 31-fold increased expression. To our best knowledge, this is the first report about adenosine aminohydrolase 3 function in the dealkylation of the N-ethyl group from atrazine.

  8. Endocannabinoid System Contributes to Liver Injury and Inflammation by Activation of Bone Marrow-Derived Monocytes/Macrophages in a CB1-Dependent Manner.

    Science.gov (United States)

    Mai, Ping; Yang, Le; Tian, Lei; Wang, Lin; Jia, Shuangshuang; Zhang, Yuanyuan; Liu, Xin; Yang, Lin; Li, Liying

    2015-10-01

    Hepatic injury undergoes significant increases in endocannabinoidsand infiltrations of macrophages, yet the concrete mechanisms of changes in endocannabinoids and the functions of macrophage-expressed cannabinoid receptors (CBs) are unclear. Biosynthetic and degradative enzymes of endocannabinoids revealed a significant change in human fibrotic liver. Meanwhile, we showed dynamic changes of these enzymes and CBs (CB1 and CB2) from 1 to 56 d in carbon tetrachloride-induced murine liver injury. Biosynthetic enzymes (N-acylphosphatidyl-ethanolamine selective phospholipase D and diacylglycerol lipase-α) and CBs were markedly increased, whereas degradative enzymes (fatty acid amidohydrolase and monoacylglycerol lipase) were downregulated. Moreover, these enzymes intimately correlated with the fibrosis parameter [procollagen α1(III)]. Bone marrow-derived monocytes/macrophages (BMM) expressed CBs. Interestingly, CB1 but not CB2 mediated BMM migration through a Boyden chambers assay, and the effect depended on the G(α)i/o/RhoA/ROCK signaling pathway. ICR mice were lethally irradiated and received BM transplants from enhanced GFP transgenic mice. Four weeks later, mice of BM reconstruction were subjected to carbon tetrachloride-induced liver injury. In the chimeric murine model, we found that blockade of CB1 by administration of a CB1 antagonist inhibited the recruitment of BMM into injured liver using immunofluorescence staining and FACS, but it did not have effects on migration of T cells and dendritic cells without CB1 expression. Furthermore, activation of CB1 enhanced cytokine expression of BMM. In vivo, inhibition of CB1 attenuated the inflammatory cytokine level through real-time RT-PCR and cytometric bead array, ameliorating hepatic inflammation and fibrosis. In this study, we identify inactivation of BMM-expressed CB1 as a therapeutic strategy for reducing hepatic inflammation and fibrosis.

  9. Salmonella typhimurium's transthyretin-like protein is a host-specific factor important in fecal survival in chickens.

    Directory of Open Access Journals (Sweden)

    Sarah C Hennebry

    Full Text Available The transthyretin-like protein (TLP from Salmonella enterica subspecies I is a periplasmic protein with high level structural similarity to a protein found in mammals and fish. In humans, the protein homologue, transthyretin, binds and carries retinol and thyroxine, and a series of other, unrelated aromatic compounds. Here we show that the amino acid sequence of the TLP from different species, subspecies and serovars of the Salmonella genus is highly conserved and demonstrate that the TLP gene is constitutively expressed in S. Typhimurium and that copper and other divalent metal ions severely inhibit enzyme activity of the TLP, a cyclic amidohydrolase that hydrolyses 5-hydroxyisourate (5-HIU. In order to determine the in vivo role of the S. Typhimurium TLP, we constructed a strain of mouse-virulent S. Typhimurium SL1344 bearing a mutation in the TLP gene (SL1344 ΔyedX. We assessed the virulence of this strain via oral inoculation of mice and chickens. Whilst SL1344 ΔyedX induced a systemic infection in both organisms, the bacterial load detected in the faeces of infected chickens was significantly reduced when compared to the load of S. Typhimurium SL1344. These data demonstrate that the TLP gene is required for survival of S. Typhimurium in a high uric acid environment such as chicken faeces, and that metabolic traits of Salmonellae in natural and contrived hosts may be fundamentally different. Our data also highlight the importance of using appropriate animal models for the study of bacterial pathogenesis especially where host-specific virulence factors or traits are the subject of the study.

  10. Structure-Based Function Discovery of an Enzyme for the Hydrolysis of Phosphorylated Sugar Lactones

    Science.gov (United States)

    Xiang, Dao Feng; Kolb, Peter; Fedorov, Alexander A.; Xu, Chengfu; Fedorov, Elena V.; Narindoshivili, Tamari; Williams, Howard J.; Shoichet, Brian K.; Almo, Steven C.; Raushel, Frank M.

    2012-01-01

    Two enzymes of unknown function from the cog1735 subset of the amidohydrolase superfamily (AHS), LMOf2365_2620 (Lmo2620) from Listeria monocytogenes str. 4b F2365 and Bh0225 from Bacillus halodurans C-125, were cloned, expressed and purified to homogeneity. The catalytic functions of these two enzymes were interrogated by an integrated strategy encompassing bioinformatics, computational docking to three-dimensional crystal structures, and library screening. The three-dimensional structure of Lmo2620 was determined at a resolution of 1.6 Å with two phosphates and a binuclear zinc center in the active site. The proximal phosphate bridges the binuclear metal center and is 7.1 Å away from the distal phosphate. The distal phosphate hydrogen bonds with Lys-242, Lys-244, Arg-275 and Tyr-278. Enzymes within cog1735 of the AHS have previously been shown to catalyze the hydrolysis of substituted lactones. Computational docking of the high energy intermediate (HEI) form of the KEGG database to the three-dimensional structure of Lmo2620 highly enriched anionic lactones versus other candidate substrates. The active site structure and the computational docking results suggested that probable substrates would likely include phosphorylated sugar lactones. A small library of diacid sugar lactones and phosphorylated sugar lactones was synthesized and tested for substrate activity with Lmo2620 and Bh0225. Two substrates were identified for these enzymes, d-lyxono-1,4-lactone-5-phosphate and l-ribono-1,4-lactone-5-phosphate. The kcat/Km values for the cobalt-substituted enzymes with these substrates are ~105 M−1 s−1. PMID:22313111

  11. Mechanism of platelet activation induced by endocannabinoids in blood and plasma.

    Science.gov (United States)

    Brantl, S Annette; Khandoga, Anna L; Siess, Wolfgang

    2014-01-01

    Platelets play a central role in atherosclerosis and atherothrombosis, and circulating endocannabinoids might modulate platelet function. Previous studies concerning effects of anandamide (N-arachidonylethanolamide) and 2-arachidonoylglycerol (2-AG) on platelets, mainly performed on isolated cells, provided conflicting results. We therefore investigated the action of three main endocannabinoids [anandamide, 2-AG and virodhamine (arachidonoylethanolamine)] on human platelets in blood and platelet-rich plasma (PRP). 2-AG and virodhamine induced platelet aggregation in blood, and shape change, aggregation and adenosine triphosphate (ATP) secretion in PRP. The EC50 of 2-AG and virodhamine for platelet aggregation in blood was 97 and 160 µM, respectively. Lower concentrations of 2-AG (20 µM) and virodhamine (50 µM) synergistically induced aggregation with other platelet stimuli. Platelet activation induced by 2-AG and virodhamine resembled arachidonic acid (AA)-induced aggregation: shape change, the first platelet response, ATP secretion and aggregation induced by 2-AG and virodhamine were all blocked by acetylsalicylic acid (ASA) or the specific thromboxane A2 (TXA2) antagonist daltroban. In addition, platelet activation induced by 2-AG and virodhamine in blood and PRP were inhibited by JZL184, a selective inhibitor of monoacylglycerol lipase (MAGL). In contrast to 2-AG and virodhamine, anandamide, a substrate of fatty acid amidohydrolase, was inactive. Synthetic cannabinoid receptor subtype 1 (CB1) and 2 (CB2) agonists lacked stimulatory as well as inhibitory platelet activity. We conclude that 2-AG and virodhamine stimulate platelets in blood and PRP by a MAGL-triggered mechanism leading to free AA and its metabolism by platelet cyclooxygenase-1/thromboxane synthase to TXA2. CB1, CB2 or non-CB1/CB2 receptors are not involved. Our results imply that ASA and MAGL inhibitors will protect platelets from activation by high endocannabinoid levels, and that

  12. Cloning, expression and characterization of D-aminoacylase from Achromobacter xylosoxidans subsp. denitrificans ATCC 15173.

    Science.gov (United States)

    Wang, Wei; Xi, Huange; Bi, Qirui; Hu, Ying; Zhang, Yang; Ni, Mengxiang

    2013-07-19

    D-Aminoacylase catalyzes the conversion of N-acyl-D-amino acids to d-amino acids and fatty acids. The aim of this study was to identify the D-aminoacylase gene from Achromobacter xylosoxidans subsp. denitrificans ATCC 15173 and investigate the biochemical characterization of the enzyme. A previously uncharacterized D-aminoacylase gene (ADdan) from this organism was cloned and sequenced. The open reading frame (ORF) of ADdan was 1467 bp in size encoding a 488-amino acid polypeptide. ADdan, with a high amino acid similarity to N-acyl-D-aspartate amidohydrolase from Alcaligenes A6, showed relatively low sequence similarities to other characterized D-aminoacylases. The recombinant ADdan protein was expressed in Escherichia coli BL21 (DE3) using pET-28a with a T7 promoter. The enzyme was purified in a single chromatographic step using nickel affinity gel column. The molecular mass of the expressed protein, calculated by SDS-PAGE, was about 52 kDa. The purified ADdan showed optimal activity at pH 8.0 and 50°C, and was stable at pH 6.0-8.0 and up to 45°C. Its activity was inhibited by Cu(2+), Fe(2+), Ca(2+), Mn(2+), Ni(2+), Zn(2+) and Hg(2+), whereas Mg(2+) had no significant influence on this recombinant D-aminoacylase. This is the first report on the characterization of D-aminoacylase with activity towards both N-acyl derivatives of neutral D-amino acids and N-acyl-D-aspartate. The characteristics of ADdan could prove to be of interest in industrial production of D-amino acids.

  13. Discovery of a novel iota carrageenan sulfatase isolated from the marine bacterium Pseudoalteromonas carrageenovora

    Science.gov (United States)

    Genicot, Sabine; Groisillier, Agnès; Rogniaux, Hélène; Meslet-Cladière, Laurence; Barbeyron, Tristan; Helbert, William

    2014-08-01

    Carrageenans are sulfated polysaccharides extracted from the cell wall of some marine red algae. These polysaccharides are widely used as gelling, stabilizing, and viscosifying agents in the food and pharmaceutical industries. Since the rheological properties of these polysaccharides depend on their sulfate content, we screened several isolated marine bacteria for carrageenan specific sulfatase activity, in the aim of developing enzymatic bioconversion of carrageenans. As a result of the screening, an iota-carrageenan sulfatase was detected in the cell-free lysate of the marine bacterium Pseudoalteromonas carrageenovora strain PscT. It was purified through Phenyl Sepharose and Diethylaminoethyl Sepharose chromatography. The pure enzyme, Psc ?-CgsA, was characterized. It had a molecular weight of 115.9 kDaltons and exhibited an optimal activity/stability at pH ~8.3 and at 40°C ± 5°C. It was inactivated by phenylmethylsulfonyl fluoride but not by ethylene diamine tetraacetic acid. Psc ?-CgsA specifically catalyzes the hydrolysis of the 4-S sulfate of iota-carrageenan. The purified enzyme could transform iota-carrageenan into hybrid iota-/alpha- or pure alpha-carrageenan under controlled conditions. The gene encoding Psc ?-CgsA, a protein of 1038 amino acids, was cloned into Escherichia coli, and the sequence analysis revealed that Psc ?-CgsA has more than 90% sequence identity with a putative uncharacterized protein Q3IKL4 from the marine strain Pseudoalteromonas haloplanktis TAC 125, but besides this did not share any homology to characterized sulfatases. Phylogenetic studies show that P. carrageenovora sulfatase thus represents the first characterized member of a new sulfatase family, with a C-terminal domain having strong similarity with the superfamily of amidohydrolases, highlighting the still unexplored diversity of marine polysaccharide modifying enzymes.

  14. Expression of GPI-80, a beta2-integrin-associated glycosylphosphatidylinositol-anchored protein, requires neutrophil differentiation with dimethyl sulfoxide in HL-60 cells.

    Science.gov (United States)

    Takeda, Yuji; Fu, Junfen; Suzuki, Kichiya; Sendo, Dai; Nitto, Takeaki; Sendo, Fujiro; Araki, Yoshihiko

    2003-06-10

    GPI-80 is a member of the amidohydrolase family that has been proposed as a potential regulator of beta2-integrin-dependent leukocyte adhesion. GPI-80 is expressed mainly in human neutrophils. Our previous studies suggested that GPI-80 expression might be associated with myeloid differentiation. To verify this, we examined whether GPI-80 is expressed on the human promyelocytic leukemia cell line HL-60 following treatment with differentiation inducers. GPI-80 expression was induced in cells treated with dimethyl sulfoxide (DMSO) to stimulate differentiation down the neutrophil pathway. On the other hand, all-trans-retinoic acid (ATRA), another neutrophil-inducing reagent, induced no clear GPI-80 expression. Potent monocyte-inducing reagents such as 1alpha,25-dihydroxyvitamin D(3) or phorbol 12-myristate 13-acetate also had no significant effect on the protein expression. GPI-80-positive cells were found in the well-differentiated CD11b-positive and transferrin-receptor-negative cell population. Granulocyte colony-stimulating factor, which augments neutrophil differentiation of HL-60 cells, up-regulated GPI-80 expression in the presence of DMSO. Granulocyte/macrophage colony-stimulating factor, which is known to suppress the neutrophil maturation of cells, inhibited expression. Adhesion of DMSO-induced cells was regulated by anti-GPI-80 monoclonal antibody, similar to the regulation observed in neutrophils. These results suggest that use of DMSO to induce neutrophil differentiation provides suitable conditions for GPI-80 expression, and that this culture system may be a helpful model for further study of the regulation of GPI-80 expression during myeloid differentiation.

  15. Proteome analysis of soybean roots under waterlogging stress at an early vegetative stage

    Indian Academy of Sciences (India)

    Iftekhar Alam; Dong-Gi Lee; Kyung-Hee Kim; Choong-Hoon Park; Shamima Akhtar Sharmin; Hyoshin Lee; Ki-Won Oh; Byung-Wook Yun; Byung-Hyun Lee

    2010-03-01

    and by regulating programmed cell death. The identification of novel proteins such as a translation initiation factor, apyrase, auxin-amidohydrolase and coproporphyrinogen oxidase in response to waterlogging stress may provide new insight into the molecular basis of the waterlogging-stress response of soybean.

  16. Phylogenetic distribution and evolutionary pattern of an α-proteobacterial small RNA gene that controls polyhydroxybutyrate accumulation in Sinorhizobium meliloti.

    Science.gov (United States)

    Lagares, Antonio; Roux, Indra; Valverde, Claudio

    2016-06-01

    It has become clear that sRNAs play relevant regulatory functions in bacteria. However, a comprehensive understanding of their biological roles considering evolutionary aspects has not been achieved for most of them. Thus, we have characterized the evolutionary and phylogenetic aspects of the Sinorhizobium meliloti mmgR gene encoding the small RNA MmgR, which has been recently reported to be involved in the regulation of polyhydroxybutyrate accumulation in this bacterium. We constructed a covariance model from a multiple sequence and structure alignment of mmgR close homologs that allowed us to extend the search and to detect further remote homologs of the sRNA gene. From our results, mmgR seemed to evolve from a common ancestor of the α-proteobacteria that diverged from the order of Rickettsiales. We have found mmgR homologs in most current species of α-proteobacteria, with a few exceptions in which genomic reduction events or gene rearrangements seem to explain its absence. Furthermore, a strong microsyntenic relationship was found between a large set of mmgR homologs and homologs of a gene encoding a putative N-formyl glutamate amidohydrolase (NFGAH) that allowed us to trace back the evolutionary path of this group of mmgR orthologs. Among them, structure and sequence traits have been completely conserved throughout evolution, namely a Rho-independent terminator and a 10-mer (5'-UUUCCUCCCU-3') that is predicted to remain in a single-stranded region of the sRNA. We thus propose the definition of the new family of α-proteobacterial sRNAs αr8, as well as the subfamily αr8s1 which encompass S. meliloti mmgR orthologs physically linked with the downstream open reading frame encoding a putative NFGAH. So far, mmgR is the trans-encoded small RNA with the widest phylogenetic distribution of well recognized orthologs among α-proteobacteria. Expression of the expected MmgR transcript in rhizobiales other than S. meliloti (Sinorhizobium fredii, Rhizobium

  17. The Hunt for 8-Oxoguanine Deaminase

    Energy Technology Data Exchange (ETDEWEB)

    Hall, R.; Fedorov, A; Marti-Arbona, R; Fedorov, E; Kolb, P; Sauder, J; Burley, S; Shoichet, B; Almo, S; et. al.

    2010-01-01

    An enzyme from Pseudomonas aeruginosa, Pa0142 (gi|9945972), that is able to catalyze the deamination of 8-oxoguanine (8-oxoG) to uric acid has been identified for the first time. 8-Oxoguanine is formed by the oxidation of guanine residues within DNA by reactive oxygen species, and this lesion results in G:C to T:A transversions. The value of k{sub cat}/K{sub m} for the deamination of 8-oxoG by Pa0142 at pH 8.0 and 30 C is 2.0 x 10{sup 4} M{sup -1} s{sup -1}. This enzyme can also catalyze the deamination of isocystosine and guanine at rates that are approximately an order of magnitude lower. The three-dimensional structure of a homologous enzyme (gi|44264246) from the Sargasso Sea has been determined by X-ray diffraction methods to a resolution of 2.2 {angstrom} (PDB entry ). The enzyme folds as a ({beta}/{alpha}){sub 8} barrel and is a member of the amidohydrolase superfamily with a single zinc in the active site. This enzyme catalyzes the deamination of 8-oxoG with a k{sub cat}/K{sub m} value of 2.7 x 10{sup 5} M{sup -1} s{sup -1}. Computational docking of potential high-energy intermediates for the deamination reaction to the X-ray crystal structure suggests that active-site binding of 8-oxoG is facilitated by hydrogen-bond interactions from a conserved glutamine that follows {beta}-strand 1 with the carbonyl group at C6, a conserved tyrosine that follows {beta}-strand 2 with N7, and a conserved cysteine residue that follows {beta}-strand 4 with the carbonyl group at C8. A bioinformatic analysis of available protein sequences suggests that {approx}200 other bacteria possess an enzyme capable of catalyzing the deamination of 8-oxoG.

  18. Molecular characterization of a fungal gene paralogue of the penicillin penDE gene of Penicillium chrysogenum

    Directory of Open Access Journals (Sweden)

    Bovenberg Roel AL

    2009-05-01

    Full Text Available Abstract Background Penicillium chrysogenum converts isopenicillin N (IPN into hydrophobic penicillins by means of the peroxisomal IPN acyltransferase (IAT, which is encoded by the penDE gene. In silico analysis of the P. chrysogenum genome revealed the presence of a gene, Pc13g09140, initially described as paralogue of the IAT-encoding penDE gene. We have termed this gene ial because it encodes a protein with high similarity to IAT (IAL for IAT-Like. We have conducted an investigation to characterize the ial gene and to determine the role of the IAL protein in the penicillin biosynthetic pathway. Results The IAL contains motifs characteristic of the IAT such as the processing site, but lacks the peroxisomal targeting sequence ARL. Null ial mutants and overexpressing strains indicated that IAL lacks acyltransferase (penicillin biosynthetic and amidohydrolase (6-APA forming activities in vivo. When the canonical ARL motif (leading to peroxisomal targeting was added to the C-terminus of the IAL protein (IALARL by site-directed mutagenesis, no penicillin biosynthetic activity was detected. Since the IAT is only active after an accurate self-processing of the preprotein into α and β subunits, self-processing of the IAL was tested in Escherichia coli. Overexpression experiments and SDS-PAGE analysis revealed that IAL is also self-processed in two subunits, but despite the correct processing, the enzyme remained inactive in vitro. Conclusion No activity related to the penicillin biosynthesis was detected for the IAL. Sequence comparison among the P. chrysogenum IAL, the A. nidulans IAL homologue and the IAT, revealed that the lack of enzyme activity seems to be due to an alteration of the essential Ser309 in the thioesterase active site. Homologues of the ial gene have been found in many other ascomycetes, including non-penicillin producers. Our data suggest that like in A. nidulans, the ial and penDE genes might have been formed from a single

  19. ACEA (a highly selective cannabinoid CB1 receptor agonist) stimulates hippocampal neurogenesis in mice treated with antiepileptic drugs.

    Science.gov (United States)

    Andres-Mach, Marta; Haratym-Maj, Agnieszka; Zagaja, Miroslaw; Rola, Radoslaw; Maj, Maciej; Chrościńska-Krawczyk, Magdalena; Luszczki, Jarogniew J

    2015-10-22

    Hippocampal neurogenesis plays a very important role in learning and memory functions. In a search for best neurological drugs that protect neuronal cells and stimulate neurogenesis with no side effects, cannabinoids proved to be a strong group of substances having many beneficial properties. The aim of this study was to evaluate the impact of ACEA (arachidonyl-2'-chloroethylamide--a highly selective cannabinoid CB1 receptor agonist) combined with a classical antiepileptic drug sodium valproate (VPA) on neural precursor cells' proliferation and differentiation in the mouse brain. All experiments were performed on adolescent CB57/BL male mice injected i.p. with VPA (10mg/kg), ACEA (10mg/kg) and PMSF (30 mg/kg) (phenylmethylsulfonyl fluoride--a substance protecting ACEA against degradation by the fatty-acid amidohydrolase) for 10 days. Next an acute response of proliferating neural precursor cells to ACEA and VPA administration was evaluated with Ki-67 staining (Time point 1). Next, in order to determine whether acute changes translated into long-term alterations in neurogenesis, proliferating cells were labeled with 5-bromo-2deoxyuridine (BrdU) followed by confocal microscopy used to determine the percentage of BrdU-labeled cells that showed mature cell phenotypes (Time point 2). Results indicate that ACEA with PMSF significantly increase the total number of Ki-67-positive cells when compared to the control group. Moreover, ACEA in combination with VPA increased the number of Ki-67-positive cells, whereas VPA administered alone had no impact on proliferating cells' population. Accordingly, neurogenesis study results indicate that the combination of ACEA+PMSF administered alone and in combination with VPA considerably increases the total number of BrdU-positive cells in comparison to the control group while ACEA+PMSF alone and in combination with VPA increased total numbers of BrdU-positive cells, newly born neurons and astrocytes as compared to VPA group but not to

  20. The effects of cannabinoids on the brain.

    Science.gov (United States)

    Ameri, A

    1999-07-01

    Cannabinoids have a long history of consumption for recreational and medical reasons. The primary active constituent of the hemp plant Cannabis sativa is delta9-tetrahydrocannabinol (delta9-THC). In humans, psychoactive cannabinoids produce euphoria, enhancement of sensory perception, tachycardia, antinociception, difficulties in concentration and impairment of memory. The cognitive deficiencies seem to persist after withdrawal. The toxicity of marijuana has been underestimated for a long time, since recent findings revealed delta9-THC-induced cell death with shrinkage of neurons and DNA fragmentation in the hippocampus. The acute effects of cannabinoids as well as the development of tolerance are mediated by G protein-coupled cannabinoid receptors. The CB1 receptor and its splice variant CB1A, are found predominantly in the brain with highest densities in the hippocampus, cerebellum and striatum. The CB2 receptor is found predominantly in the spleen and in haemopoietic cells and has only 44% overall nucleotide sequence identity with the CB1 receptor. The existence of this receptor provided the molecular basis for the immunosuppressive actions of marijuana. The CB1 receptor mediates inhibition of adenylate cyclase, inhibition of N- and P/Q-type calcium channels, stimulation of potassium channels, and activation of mitogen-activated protein kinase. The CB2 receptor mediates inhibition of adenylate cyclase and activation of mitogen-activated protein kinase. The discovery of endogenous cannabinoid receptor ligands, anandamide (N-arachidonylethanolamine) and 2-arachidonylglycerol made the notion of a central cannabinoid neuromodulatory system plausible. Anandamide is released from neurons upon depolarization through a mechanism that requires calcium-dependent cleavage from a phospholipid precursor in neuronal membranes. The release of anandamide is followed by rapid uptake into the plasma and hydrolysis by fatty-acid amidohydrolase. The psychoactive cannabinoids

  1. The catalase activity of diiron adenine deaminase

    Energy Technology Data Exchange (ETDEWEB)

    Kamat S. S.; Swaminathan S.; Holmes-Hampton, G. P.; Bagaria, A.; Kumaran, D.; Tichy, S. E.; Gheyi, T.; Zheng, X.; Bain, K.; Groshong, C.; Emtage, S.; Sauder, J. M.; Burley, S. K.; Lindahl, P. A.; Raushel, F. M.

    2011-12-01

    Adenine deaminase (ADE) from the amidohydrolase superfamily (AHS) of enzymes catalyzes the conversion of adenine to hypoxanthine and ammonia. Enzyme isolated from Escherichia coli was largely inactive toward the deamination of adenine. Molecular weight determinations by mass spectrometry provided evidence that multiple histidine and methionine residues were oxygenated. When iron was sequestered with a metal chelator and the growth medium supplemented with Mn{sup 2+} before induction, the post-translational modifications disappeared. Enzyme expressed and purified under these conditions was substantially more active for adenine deamination. Apo-enzyme was prepared and reconstituted with two equivalents of FeSO{sub 4}. Inductively coupled plasma mass spectrometry and Moessbauer spectroscopy demonstrated that this protein contained two high-spin ferrous ions per monomer of ADE. In addition to the adenine deaminase activity, [Fe{sup II}/Fe{sup II}]-ADE catalyzed the conversion of H{sub 2}O{sub 2} to O{sub 2} and H{sub 2}O. The values of k{sub cat} and k{sub cat}/K{sub m} for the catalase activity are 200 s{sup -1} and 2.4 x 10{sup 4} M{sup -1} s{sup -1}, respectively. [Fe{sup II}/Fe{sup II}]-ADE underwent more than 100 turnovers with H{sub 2}O{sub 2} before the enzyme was inactivated due to oxygenation of histidine residues critical for metal binding. The iron in the inactive enzyme was high-spin ferric with g{sub ave} = 4.3 EPR signal and no evidence of anti-ferromagnetic spin-coupling. A model is proposed for the disproportionation of H{sub 2}O{sub 2} by [Fe{sup II}/Fe{sup II}]-ADE that involves the cycling of the binuclear metal center between the di-ferric and di-ferrous oxidation states. Oxygenation of active site residues occurs via release of hydroxyl radicals. These findings represent the first report of redox reaction catalysis by any member of the AHS.

  2. Proteome analysis of soybean roots under waterlogging stress at an early vegetative stage.

    Science.gov (United States)

    Alam, Iftekhar; Lee, Dong-Gi; Kim, Kyung-Hee; Park, Choong-Hoon; Sharmin, Shamima Akhtar; Lee, Hyoshin; Oh, Ki-Won; Yun, Byung-Wook; Lee, Byung-Hyun

    2010-03-01

    and by regulating programmed cell death. The identification of novel proteins such as a translation initiation factor, apyrase, auxin-amidohydrolase and coproporphyrinogen oxidase in response to waterlogging stress may provide new insight into the molecular basis of the waterlogging-stress response of soybean.

  3. Lytic activity of the virion-associated peptidoglycan hydrolase HydH5 of Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88

    Directory of Open Access Journals (Sweden)

    Donovan David M

    2011-06-01

    Full Text Available Abstract Background Staphylococcus aureus is a food-borne pathogen and the most common cause of infections in hospitalized patients. The increase in the resistance of this pathogen to antibacterials has made necessary the development of new anti-staphylococcal agents. In this context, bacteriophage lytic enzymes such as endolysins and structural peptidoglycan (PG hydrolases have received considerable attention as possible antimicrobials against gram-positive bacteria. Results S. aureus bacteriophage vB_SauS-phiIPLA88 (phiIPLA88 contains a virion-associated muralytic enzyme (HydH5 encoded by orf58, which is located in the morphogenetic module. Comparative bioinformatic analysis revealed that HydH5 significantly resembled other peptidoglycan hydrolases encoded by staphylococcal phages. The protein consists of 634 amino acid residues. Two putative lytic domains were identified: an N-terminal CHAP (cysteine, histidine-dependent amidohydrolase/peptidase domain (135 amino acid residues, and a C-terminal LYZ2 (lysozyme subfamily 2 domain (147 amino acid residues. These domains were also found when a predicted three-dimensional structure of HydH5 was made which provided the basis for deletion analysis. The complete HydH5 protein and truncated proteins containing only each catalytic domain were overproduced in E. coli and purified from inclusion bodies by subsequent refolding. Truncated and full-length HydH5 proteins were all able to bind and lyse S. aureus Sa9 cells as shown by binding assays, zymogram analyses and CFU reduction analysis. HydH5 demonstrated high antibiotic activity against early exponential cells, at 45°C and in the absence of divalent cations (Ca2+, Mg2+, Mn2+. Thermostability assays showed that HydH5 retained 72% of its activity after 5 min at 100°C. Conclusions The virion-associated PG hydrolase HydH5 has lytic activity against S. aureus, which makes it attractive as antimicrobial for food biopreservation and anti

  4. Dynamics of Jasmonate Metabolism upon Flowering and across Leaf Stress Responses in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Emilie Widemann

    2016-01-01

    Full Text Available The jasmonic acid (JA signaling pathway plays important roles in adaptation of plants to environmental cues and in specific steps of their development, particularly in reproduction. Recent advances in metabolic studies have highlighted intricate mechanisms that govern enzymatic conversions within the jasmonate family. Here we analyzed jasmonate profile changes upon Arabidopsis thaliana flower development and investigated the contribution of catabolic pathways that were known to turnover the active hormonal compound jasmonoyl-isoleucine (JA-Ile upon leaf stress. We report a rapid decline of JA-Ile upon flower opening, concomitant with the massive accumulation of its most oxidized catabolite, 12COOH-JA-Ile. Detailed genetic analysis identified CYP94C1 as the major player in this process. CYP94C1 is one out of three characterized cytochrome P450 enzymes that define an oxidative JA-Ile turnover pathway, besides a second, hydrolytic pathway represented by the amido-hydrolases IAR3 and ILL6. Expression studies combined with reporter gene analysis revealed the dominant expression of CYP94C1 in mature anthers, consistent with the established role of JA signaling in male fertility. Significant CYP94B1 expression was also evidenced in stamen filaments, but surprisingly, CYP94B1 deficiency was not associated with significant changes in JA profiles. Finally, we compared global flower JA profiles with those previously reported in leaves reacting to mechanical wounding or submitted to infection by the necrotrophic fungus Botrytis cinerea. These comparisons revealed distinct dynamics of JA accumulation and conversions in these three biological systems. Leaf injury boosts a strong and transient JA and JA-Ile accumulation that evolves rapidly into a profile dominated by ω-oxidized and/or Ile-conjugated derivatives. In contrast, B. cinerea-infected leaves contain mostly unconjugated jasmonates, about half of this content being ω-oxidized. Finally, developing

  5. Dynamics of Jasmonate Metabolism upon Flowering and across Leaf Stress Responses in Arabidopsis thaliana

    Science.gov (United States)

    Widemann, Emilie; Smirnova, Ekaterina; Aubert, Yann; Miesch, Laurence; Heitz, Thierry

    2016-01-01

    The jasmonic acid (JA) signaling pathway plays important roles in adaptation of plants to environmental cues and in specific steps of their development, particularly in reproduction. Recent advances in metabolic studies have highlighted intricate mechanisms that govern enzymatic conversions within the jasmonate family. Here we analyzed jasmonate profile changes upon Arabidopsis thaliana flower development and investigated the contribution of catabolic pathways that were known to turnover the active hormonal compound jasmonoyl-isoleucine (JA-Ile) upon leaf stress. We report a rapid decline of JA-Ile upon flower opening, concomitant with the massive accumulation of its most oxidized catabolite, 12COOH-JA-Ile. Detailed genetic analysis identified CYP94C1 as the major player in this process. CYP94C1 is one out of three characterized cytochrome P450 enzymes that define an oxidative JA-Ile turnover pathway, besides a second, hydrolytic pathway represented by the amido-hydrolases IAR3 and ILL6. Expression studies combined with reporter gene analysis revealed the dominant expression of CYP94C1 in mature anthers, consistent with the established role of JA signaling in male fertility. Significant CYP94B1 expression was also evidenced in stamen filaments, but surprisingly, CYP94B1 deficiency was not associated with significant changes in JA profiles. Finally, we compared global flower JA profiles with those previously reported in leaves reacting to mechanical wounding or submitted to infection by the necrotrophic fungus Botrytis cinerea. These comparisons revealed distinct dynamics of JA accumulation and conversions in these three biological systems. Leaf injury boosts a strong and transient JA and JA-Ile accumulation that evolves rapidly into a profile dominated by ω-oxidized and/or Ile-conjugated derivatives. In contrast, B. cinerea-infected leaves contain mostly unconjugated jasmonates, about half of this content being ω-oxidized. Finally, developing flowers present an

  6. Glutamic acid gamma-monohydroxamate and hydroxylamine are alternate substrates for Escherichia coli asparagine synthetase B.

    Science.gov (United States)

    Boehlein, S K; Schuster, S M; Richards, N G

    1996-03-01

    Escherichia coli asparagine synthetase B (AS-B) catalyzes the synthesis of asparagine from aspartic acid and glutamine in an ATP-dependent reaction. The ability of this enzyme to employ hydroxylamine and L-glutamic acid gamma-monohydroxamate (LGH) as alternative substrates in place of ammonia and L-glutamine, respectively, has been investigated. The enzyme is able to function as an amidohydrolase, liberating hydroxylamine from LGH with high catalytic efficiency, as measured by k(cat)/K(M). In addition, the kinetic parameters determined for hydroxylamine in AS-B synthetase activity are very similar to those of ammonia. Nitrogen transfer from LGH to yield aspartic acid beta-monohydroxamate is also catalyzed by AS-B. While such an observation has been made for a few members of the trpG amidotransferase family, our results appear to be the first demonstration that nitrogen transfer can occur from glutamine analogs in a purF amidotransferase. However, k(cat)/K(M) for the ATP-dependent transfer of hydroxylamine from LGH to aspartic acid is reduced 3-fold relative to that for glutamine-dependent asparagine synthesis. Further, the AS-B mutant in which asparagine is replaced by alanine (N74A) can also use hydroxylamine as an alternate substrate to ammonia and catalyze the hydrolysis of LGH. The catalytic efficiencies (k(cat)/K(M)) of nitrogen transfer from LGH and L-glutamine to beta-aspartyl-AMP are almost identical for the N74A AS-B mutant. These observations support the proposal that Asn-74 plays a role in catalyzing glutamine-dependent nitrogen transfer. We interpret our kinetic data as further evidence against ammonia-mediated nitrogen transfer from glutamine in the purF amidotransferase AS-B. These results are consistent with two alternate chemical mechanisms that have been proposed for this reaction [Boehlein, S. K., Richards, N. G. J., Walworth, E. S., & Schuster, S. M. (1994) J. Biol. Chem. 269, 26789-26795].

  7. 免耕留茬覆盖对土壤呼吸和土壤酶活性及养分的影响%No-tillage Stubble with Residues on Soil Respiration and the Soil Enzyme Activity and Nutrient Influence

    Institute of Scientific and Technical Information of China (English)

    崔凤娟; 李立军; 刘景辉; 高婕; 王永强

    2011-01-01

    Stubble height was studied with different leave covered the soil respiration and the quantity combination of change, the soil enzyme activity of Inner Mongolia for clear protective cultivation on the Shangtuhaixiang climate change and the influence of soil productivity as well as local farms provide theoretical basis for scientific management. Tests for 2009, a total of three treatment: traditional farming (CT), leave low stubbly half quantity coverage (DH), leave low stubbly total quantity coverage (DW), leave high stubbly half quantity coverage (GH), leave high stubbly total quantity coverage (GW). Static boxes of soil respiration rate determination of farmland, and the hierarchical soil enzyme activity and soil nutrient were determined to clear the region's soil fertility status. Soil respiration rate in jointing stage seasonal variation peak, no-till leave compared with conventional tillage crop cover soil respiration rate was significantly lower stubble heightno-tillage same leave covered significant difference, different quantity of no-tillage same cover quantity and leave the difference between different crop height of soil respiration rate was not significant, with straw mulching quantity decreased. Diurnal variation of single-peak curve changes in no-tillage leave high crop total content covers processing and atmospheric temperature and the correlation coefficient was 0.9239. Conventional tillage soil respiration rate and the correlation coefficient for atmospheric temperature, 0.8652 significant related level. No-till leave stubbly cover treatment could greatly improve the 0-5 cm and 5-10 cm soil organic matter, total amount of nutrients, available nutrient content and soil amidohydrolase and oxidation and reduction of the enzyme activity. Soil respiration and total nitrogen and hydrogen peroxide, soil nutrient and outside the enzyme hydrolysis enzyme was a significant or very significant positive correlation between soil organic matter, nutrient and

  8. 青刺果油对神经酰胺合成及神经酰胺酶表达的影响%Effects of Prinsepia utilis Royle oil on the synthesis of ceramide and expression of ceramidase

    Institute of Scientific and Technical Information of China (English)

    涂颖; 顾华; 李娜; 庞勤; 何黎

    2012-01-01

    Objective To evaluate the effects of Prinsepia utilis Royle oil (PURO) on the synthesis of ceramide and expression of acid ceramidase N-acylsphingosine amidohydrolase 1 (ASH1),and to explore the mechanisms underlying its moisturizing and skin barrier-repairing effects.Methods Keratinocytes from human foreskin tissue were classified into 2 groups to be cultured in keratinocyte-serum free medium (K-SFM) with or without the presence of PURO.Enzyme linked immunosorbent assay (ELISA) was performed to measure the level of ceramide in the culture supernatant of keratinocytes at 0,3,8,24 and 48 hours.The back of nude mice was divided into 4 areas,i.e.,test area,matrix area,blank control area and negative control area.Acetone and ether were used to destroy the epidermal barrier in the test,matrix,and blank control areas,then,the former 2 areas were topically treated with emulsions containing 1% PURO and matrix,respectively,and the blank control area remained untreated.The epidermal barrier remained intact and untreated in the negative control area.Noninvasive methods were used to determine transepidermal water loss (TEWL),epidermal moisture content and skin lipid content in these areas on day 0,1,3,and 7.Skin tissue was obtained from these areas on day 0 and 7 followed by an immunohistochemical study for the quantification of ASH1 expression.Results The level of supernatant ceramide increased with time in the PURO-treated keratinocytes,which was significantly higher at 24 hours and 48 hours than at 0 hour (1.3817 ± 0.100 and 1.3737 ± 0.047 vs.0.7630 ± 0.143,both P < 0.05).The supernatant ceramide was also elevated in the PURO-treated keratinocytes compared with untreated keratinocytes at 24 and 48 hours (both P < 0.05).Noninvasive skin tests showed a gradual decrease in the TEWL,but an increase in the epidermal moisture content and skin lipid content with time in the 3 epidermal barrier-destroyed areas.As far as the test area was concerned,TEWL value was