Science.gov

Sample records for acylpeptide hydrolase aph

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

    PubMed

    Fu, Ping; Sun, Wei; Zhang, Ze

    2016-04-10

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

  2. Exploration of the chlorpyrifos escape pathway from acylpeptide hydrolases using steered molecular dynamics simulations.

    PubMed

    Wang, Dongmei; Jin, Hanyong; Wang, Junling; Guan, Shanshan; Zhang, Zuoming; Han, Weiwei

    2016-04-01

    Acylpeptide hydrolases (APH) catalyze the removal of an N-acylated amino acid from blocked peptides. APH is significantly more sensitive than acetylcholinesterase, a target of Alzheimer's disease, to inhibition by organophosphorus (OP) compounds. Thus, OP compounds can be used as a tool to probe the physiological functions of APH. Here, we report the results of a computational study of molecular dynamics simulations of APH bound to the OP compounds and an exploration of the chlorpyrifos escape pathway using steered molecular dynamics (SMD) simulations. In addition, we apply SMD simulations to identify potential escape routes of chlorpyrifos from hydrolase hydrophobic cavities in the APH-inhibitor complex. Two previously proposed APH pathways were reliably identified by CAVER 3.0, with the estimated relative importance of P1 > P2 for its size. We identify the major pathway, P2, using SMD simulations, and Arg526, Glu88, Gly86, and Asn65 are identified as important residues for the ligand leaving via P2. These results may help in the design of APH-targeting drugs with improved efficacy, as well as in understanding APH selectivity of the inhibitor binding in the prolyl oligopeptidase family. PMID:26155973

  3. Molecular Dynamics Simulations of Acylpeptide Hydrolase Bound to Chlorpyrifosmethyl Oxon and Dichlorvos

    PubMed Central

    Jin, Hanyong; Zhou, Zhenhuan; Wang, Dongmei; Guan, Shanshan; Han, Weiwei

    2015-01-01

    Acylpeptide hydrolases (APHs) catalyze the removal of N-acylated amino acids from blocked peptides. Like other prolyloligopeptidase (POP) family members, APHs are believed to be important targets for drug design. To date, the binding pose of organophosphorus (OP) compounds of APH, as well as the different OP compounds binding and inducing conformational changes in two domains, namely, α/β hydrolase and β-propeller, remain poorly understood. We report a computational study of APH bound to chlorpyrifosmethyl oxon and dichlorvos. In our docking study, Val471 and Gly368 are important residues for chlorpyrifosmethyl oxon and dichlorvos binding. Molecular dynamics simulations were also performed to explore the conformational changes between the chlorpyrifosmethyl oxon and dichlorvos bound to APH, which indicated that the structural feature of chlorpyrifosmethyl oxon binding in APH permitted partial opening of the β-propeller fold and allowed the chlorpyrifosmethyl oxon to easily enter the catalytic site. These results may facilitate the design of APH-targeting drugs with improved efficacy. PMID:25794283

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

    PubMed

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

    2005-08-01

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

  5. Expression, purification, crystallization and preliminary X-ray diffraction analysis of acylpeptide hydrolase from Deinococcus radiodurans

    PubMed Central

    Are, Venkata Narayana; Ghosh, Biplab; Kumar, Ashwani; Yadav, Pooja; Bhatnagar, Deepak; Jamdar, Sahayog N.; Makde, Ravindra D.

    2014-01-01

    Acylpeptide hydrolase (APH; EC 3.4.19.1), which belongs to the S9 family of serine peptidases (MEROPS), catalyzes the removal of an N-acylated amino acid from a blocked peptide. The role of this enzyme in mammalian cells has been suggested to be in the clearance of oxidatively damaged proteins as well as in the degradation of the β-amyloid peptides implicated in Alzheimer’s disease. Detailed structural information for the enzyme has been reported from two thermophilic archaea; both of the archaeal APHs share a similar monomeric structure. However, the mechanisms of substrate selectivity and active-site accessibility are totally different and are determined by inter-domain flexibility or the oligomeric structure. An APH homologue from a bacterium, Deinococcus radiodurans (APHdr), has been crystallized using microbatch-under-oil employing the random microseed matrix screening method. The protein crystallized in space group P21, with unit-cell parameters a = 77.6, b = 189.6, c = 120.4 Å, β = 108.4°. A Matthews coefficient of 2.89 Å3 Da−1 corresponds to four monomers, each with a molecular mass of ∼73 kDa, in the asymmetric unit. The APHdr structure will reveal the mechanisms of substrate selectivity and active-site accessibility in the bacterial enzyme. It will also be helpful in elucidating the functional role of this enzyme in D. radiodurans. PMID:25195912

  6. Acylpeptide hydrolase: inhibitors and some active site residues of the human enzyme.

    PubMed

    Scaloni, A; Jones, W M; Barra, D; Pospischil, M; Sassa, S; Popowicz, A; Manning, L R; Schneewind, O; Manning, J M

    1992-02-25

    Acylpeptide hydrolase may be involved in N-terminal deacetylation of nascent polypeptide chains and of bioactive peptides. The activity of this enzyme from human erythrocytes is sensitive to anions such as chloride, nitrate, and fluoride. Furthermore, blocked amino acids act as competitive inhibitors of the enzyme. Acetyl leucine chloromethyl ketone has been employed to identify one active site residue as His-707. Diisopropylfluorophosphate has been used to identify a second active site residue as Ser-587. Chemical modification studies with a water-soluble carbodiimide implicate a carboxyl group in catalytic activity. These results and the sequence around these active site residues, especially near Ser-587, suggest that acylpeptide hydrolase contains a catalytic triad. The presence of a cysteine residue in the vicinity of the active site is suggested by the inactivation of the enzyme by sulfhydryl-modifying agents and also by a low amount of modification by the peptide chloromethyl ketone inhibitor. Ebelactone A, an inhibitor of the formyl aminopeptidase, the bacterial counterpart of eukaryotic acylpeptide hydrolase, was found to be an effective inhibitor of this enzyme. These findings suggest that acylpeptidase hydrolase is a member of a family of enzymes with extremely diverse functions. PMID:1740429

  7. Identification of oxidized protein hydrolase of human erythrocytes as acylpeptide hydrolase.

    PubMed

    Fujino, T; Watanabe, K; Beppu, M; Kikugawa, K; Yasuda, H

    2000-03-16

    Partial amino acid sequence of 80 kDa oxidized protein hydrolase (OPH), a serine protease present in human erythrocyte cytosol (Fujino et al., J. Biochem. 124 (1998) 1077-1085) that is adherent to oxidized erythrocyte membranes and preferentially degrades oxidatively damaged proteins (Beppu et al., Biochim. Biophys. Acta 1196 (1994) 81-87; Fujino et al., Biochim. Biophys. Acta 1374 (1998) 47-55) was determined. The N-terminal amino acid of diisopropyl fluorophosphate (DFP)-labeled OPH was suggested to be masked. Six peptide fragments of OPH obtained by digestion of DFP-labeled OPH with lysyl endopeptidase were isolated by use of reverse-phase high-performance liquid chromatography, and the sequence of more than eight amino acids from the N-terminal position of each peptide was determined. Results of homology search of amino acid sequence of each peptide strongly suggested that the protein was identical with human liver acylpeptide hydrolase (ACPH). OPH showed ACPH activity when N-acetyl-L-alanine p-nitroanilide and N-acetylmethionyl L-alanine were used as substrates. Glutathione S-transferase (GST)-tagged recombinant ACPH (rACPH) was prepared by use of baculovirus expression system as a 107-kDa protein from cDNA of human erythroleukemic cell line K-562. rACPH reacted with anti-OPH antiserum from rabbit. rACPH showed OPH activity when hydrogen peroxide-oxidized or glycated bovine serum albumin was used as substrates. As well as the enzyme activities of OPH, those of rACPH were inhibited by DFP. The results clearly demonstrate that ACPH, whose physiological function has not yet been well characterized, can play an important role as OPH in destroying oxidatively damaged proteins in living cells. PMID:10719179

  8. Acylpeptide Hydrolase Inhibition as Targeted Strategy to Induce Proteasomal Down-Regulation

    PubMed Central

    Luini, Alberto; Ruvo, Menotti; Gogliettino, Marta; Langella, Emma; Saviano, Michele; Hegde, Ramanath N.; Sandomenico, Annamaria; Rossi, Mose

    2011-01-01

    Acylpeptide hydrolase (APEH), one of the four members of the prolyl oligopeptidase class, catalyses the removal of N-acylated amino acids from acetylated peptides and it has been postulated to play a key role in protein degradation machinery. Disruption of protein turnover has been established as an effective strategy to down-regulate the ubiquitin-proteasome system (UPS) and as a promising approach in anticancer therapy. Here, we illustrate a new pathway modulating UPS and proteasome activity through inhibition of APEH. To find novel molecules able to down-regulate APEH activity, we screened a set of synthetic peptides, reproducing the reactive-site loop of a known archaeal inhibitor of APEH (SsCEI), and the conjugated linoleic acid (CLA) isomers. A 12-mer SsCEI peptide and the trans10-cis12 isomer of CLA, were identified as specific APEH inhibitors and their effects on cell-based assays were paralleled by a dose-dependent reduction of proteasome activity and the activation of the pro-apoptotic caspase cascade. Moreover, cell treatment with the individual compounds increased the cytoplasm levels of several classic hallmarks of proteasome inhibition, such as NFkappaB, p21, and misfolded or polyubiquitinylated proteins, and additive effects were observed in cells exposed to a combination of both inhibitors without any cytotoxicity. Remarkably, transfection of human bronchial epithelial cells with APEH siRNA, promoted a marked accumulation of a mutant of the cystic fibrosis transmembrane conductance regulator (CFTR), herein used as a model of misfolded protein typically degraded by UPS. Finally, molecular modeling studies, to gain insights into the APEH inhibition by the trans10-cis12 CLA isomer, were performed. Our study supports a previously unrecognized role of APEH as a negative effector of proteasome activity by an unknown mechanism and opens new perspectives for the development of strategies aimed at modulation of cancer progression. PMID:22016782

  9. Effect of short-term exposure to dichlorvos on synaptic plasticity of rat hippocampal slices: Involvement of acylpeptide hydrolase and {alpha}{sub 7} nicotinic receptors

    SciTech Connect

    Olmos, Cristina; Sandoval, Rodrigo; Rozas, Carlos; Navarro, Sebastian; Wyneken, Ursula; Zeise, Marc; Morales, Bernardo; Pancetti, Floria

    2009-07-01

    Dichlorvos is the active molecule of the pro-drug metrifonate used to revert the cognitive deficits associated with Alzheimer's disease. A few years ago it was reported that dichlorvos inhibits the enzyme acylpeptide hydrolase at lower doses than those necessary to inhibit acetylcholinesterase to the same extent. Therefore, the aim of our investigation was to test the hypothesis that dichlorvos can enhance synaptic efficacy through a mechanism that involves acylpeptide hydrolase instead of acetylcholinesterase inhibition. We used long-term potentiation induced in rat hippocampal slices as a model of synaptic plasticity. Our results indicate that short-term exposures (20 min) to 50 {mu}M dichlorvos enhance long-term potentiation in about 200% compared to the control condition. This effect is correlated with approximately 60% inhibition of acylpeptide hydrolase activity, whereas acetylcholinesterase activity remains unaffected. Paired-pulse facilitation and inhibition experiments indicate that dichlorvos does not have any presynaptic effect in the CA3 {yields} CA1 pathway nor affect gabaergic interneurons. Interestingly, the application of 100 nM methyllicaconitine, an {alpha}{sub 7} nicotinic receptor antagonist, blocked the enhancing effect of dichlorvos on long-term potentiation. These results indicate that under the exposure conditions described above, dichlorvos enhances long-term potentiation through a postsynaptic mechanism that involves (a) the inhibition of the enzyme acylpeptide hydrolase and (b) the modulation of {alpha}{sub 7} nicotinic receptors.

  10. Advanced Plant Habitat (APH)

    NASA Technical Reports Server (NTRS)

    Richards, Stephanie E. (Compiler); Levine, Howard G.; Reed, David W.

    2016-01-01

    The Advanced Plant Habitat (APH) hardware will be a large growth volume plant habitat, capable of hosting multigenerational studies, in which environmental variables (e.g., temperature, relative humidity, carbon dioxide level light intensity and spectral quality) can be tracked and controlled in support of whole plant physiological testing and Bio-regenerative Life Support System investigations.

  11. Click-generated triazole ureas as ultrapotent in vivo-active serine hydrolase inhibitors.

    PubMed

    Adibekian, Alexander; Martin, Brent R; Wang, Chu; Hsu, Ku-Lung; Bachovchin, Daniel A; Niessen, Sherry; Hoover, Heather; Cravatt, Benjamin F

    2011-07-01

    Serine hydrolases are a diverse enzyme class representing ∼1% of all human proteins. The biological functions of most serine hydrolases remain poorly characterized owing to a lack of selective inhibitors to probe their activity in living systems. Here we show that a substantial number of serine hydrolases can be irreversibly inactivated by 1,2,3-triazole ureas, which show negligible cross-reactivity with other protein classes. Rapid lead optimization by click chemistry-enabled synthesis and competitive activity-based profiling identified 1,2,3-triazole ureas that selectively inhibit enzymes from diverse branches of the serine hydrolase class, including peptidases (acyl-peptide hydrolase, or APEH), lipases (platelet-activating factor acetylhydrolase-2, or PAFAH2) and uncharacterized hydrolases (α,β-hydrolase-11, or ABHD11), with exceptional potency in cells (sub-nanomolar) and mice (<1 mg kg(-1)). We show that APEH inhibition leads to accumulation of N-acetylated proteins and promotes proliferation in T cells. These data indicate 1,2,3-triazole ureas are a pharmacologically privileged chemotype for serine hydrolase inhibition, combining broad activity across the serine hydrolase class with tunable selectivity for individual enzymes. PMID:21572424

  12. Click-generated triazole ureas as ultrapotent, in vivo-active serine hydrolase inhibitors

    PubMed Central

    Adibekian, Alexander; Martin, Brent R.; Wang, Chu; Hsu, Ku-Lung; Bachovchin, Daniel A.; Niessen, Sherry; Hoover, Heather; Cravatt, Benjamin F.

    2011-01-01

    Serine hydrolases (SHs) are a diverse enzyme class representing > 1% of all human proteins. The biological functions for most SHs remain poorly characterized due to a lack of selective inhibitors to probe their activity in living systems. Here, we show that a substantial number of SHs can be irreversibly inactivated by 1,2,3-triazole ureas, which exhibit negligible cross-reactivity with other protein classes. Rapid lead optimization by click chemistry-enabled synthesis and competitive activity-based profiling identified 1,2,3-triazole ureas that selectively inhibit enzymes from diverse branches of the SH superfamily, including peptidases (acyl-peptide hydrolase or APEH), lipases (platelet-activating factor acetylhyrolase-2 or PAFAH2), and uncharacterized hydrolases (α, β-hydrolase 11 or ABHD11), with exceptional potency in cells (sub-nM) and mice (< 1 mg/kg). We show that APEH inhibition leads to accumulation of N-acetylated proteins and promotes proliferation in T-cells. These data designate 1,2,3-triazole ureas as a pharmacologically privileged chemotype for SH inhibition that shows broad activity across the SH class coupled with tunable selectivity for individual enzymes. PMID:21572424

  13. Structure and Function of APH(4)-Ia, a Hygromycin B Resistance Enzyme

    SciTech Connect

    Stogios, Peter J.; Shakya, Tushar; Evdokimova, Elena; Savchenko, Alexei; Wright, Gerard D.

    2011-11-18

    The aminoglycoside phosphotransferase (APH) APH(4)-Ia is one of two enzymes responsible for bacterial resistance to the atypical aminoglycoside antibiotic hygromycin B (hygB). The crystal structure of APH(4)-Ia enzyme was solved in complex with hygB at 1.95 {angstrom} resolution. The APH(4)-Ia structure adapts a general two-lobe architecture shared by other APH enzymes and eukaryotic kinases, with the active site located at the interdomain cavity. The enzyme forms an extended hydrogen bond network with hygB primarily through polar and acidic side chain groups. Individual alanine substitutions of seven residues involved in hygB binding did not have significant effect on APH(4)-Ia enzymatic activity, indicating that the binding affinity is spread across a distributed network. hygB appeared as the only substrate recognized by APH(4)-Ia among the panel of 14 aminoglycoside compounds. Analysis of the active site architecture and the interaction with the hygB molecule demonstrated several unique features supporting such restricted substrate specificity. Primarily the APH(4)-Ia substrate-binding site contains a cluster of hydrophobic residues that provides a complementary surface to the twisted structure of the substrate. Similar to APH(2{double_prime}) enzymes, the APH(4)-Ia is able to utilize either ATP or GTP for phosphoryl transfer. The defined structural features of APH(4)-Ia interactions with hygB and the promiscuity in regard to ATP or GTP binding could be exploited for the design of novel aminoglycoside antibiotics or inhibitors of this enzyme.

  14. Palmitoyl acyltransferase Aph2 in cardiac function and the development of cardiomyopathy

    PubMed Central

    Zhou, Tielin; Li, Jing; Zhao, Peiquan; Liu, Huijuan; Jia, Deyong; Jia, Hao; He, Lin; Cang, Yong; Boast, Sharon; Chen, Yi-Han; Thibault, Hélène; Scherrer-Crosbie, Marielle; Goff, Stephen P.; Li, Baojie

    2015-01-01

    Protein palmitoylation regulates many aspects of cell function and is carried out by acyl transferases that contain zf-DHHC motifs. The in vivo physiological function of protein palmitoylation is largely unknown. Here we generated mice deficient in the acyl transferase Aph2 (Ablphilin 2 or zf-DHHC16) and demonstrated an essential role for Aph2 in embryonic/postnatal survival, eye development, and heart development. Aph2−/− embryos and pups showed cardiomyopathy and cardiac defects including bradycardia. We identified phospholamban, a protein often associated with human cardiomyopathy, as an interacting partner and a substrate of Aph2. Aph2-mediated palmitoylation of phospholamban on cysteine 36 differentially alters its interaction with PKA and protein phosphatase 1 α, augmenting serine 16 phosphorylation, and regulates phospholamban pentamer formation. Aph2 deficiency results in phospholamban hypophosphorylation, a hyperinhibitory form. Ablation of phospholamban in Aph2−/− mice histologically and functionally alleviated the heart defects. These findings establish Aph2 as a critical in vivo regulator of cardiac function and reveal roles for protein palmitoylation in the development of other organs including eyes. PMID:26644582

  15. Palmitoyl acyltransferase Aph2 in cardiac function and the development of cardiomyopathy.

    PubMed

    Zhou, Tielin; Li, Jing; Zhao, Peiquan; Liu, Huijuan; Jia, Deyong; Jia, Hao; He, Lin; Cang, Yong; Boast, Sharon; Chen, Yi-Han; Thibault, Hélène; Scherrer-Crosbie, Marielle; Goff, Stephen P; Li, Baojie

    2015-12-22

    Protein palmitoylation regulates many aspects of cell function and is carried out by acyl transferases that contain zf-DHHC motifs. The in vivo physiological function of protein palmitoylation is largely unknown. Here we generated mice deficient in the acyl transferase Aph2 (Ablphilin 2 or zf-DHHC16) and demonstrated an essential role for Aph2 in embryonic/postnatal survival, eye development, and heart development. Aph2(-/-) embryos and pups showed cardiomyopathy and cardiac defects including bradycardia. We identified phospholamban, a protein often associated with human cardiomyopathy, as an interacting partner and a substrate of Aph2. Aph2-mediated palmitoylation of phospholamban on cysteine 36 differentially alters its interaction with PKA and protein phosphatase 1 α, augmenting serine 16 phosphorylation, and regulates phospholamban pentamer formation. Aph2 deficiency results in phospholamban hypophosphorylation, a hyperinhibitory form. Ablation of phospholamban in Aph2(-/-) mice histologically and functionally alleviated the heart defects. These findings establish Aph2 as a critical in vivo regulator of cardiac function and reveal roles for protein palmitoylation in the development of other organs including eyes. PMID:26644582

  16. 40 CFR 174.526 - Hygromycin B phosphotransferase (APH4) marker protein in all plants; exemption from the...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Hygromycin B phosphotransferase (APH4) marker protein in all plants; exemption from the requirement of a tolerance. 174.526 Section 174.526... phosphotransferase (APH4) marker protein in all plants; exemption from the requirement of a tolerance. Residues...

  17. 40 CFR 174.526 - Hygromycin B phosphotransferase (APH4) marker protein in all plants; exemption from the...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Hygromycin B phosphotransferase (APH4) marker protein in all plants; exemption from the requirement of a tolerance. 174.526 Section 174.526... phosphotransferase (APH4) marker protein in all plants; exemption from the requirement of a tolerance. Residues...

  18. 40 CFR 174.526 - Hygromycin B phosphotransferase (APH4) marker protein in all plants; exemption from the...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Hygromycin B phosphotransferase (APH4) marker protein in all plants; exemption from the requirement of a tolerance. 174.526 Section 174.526... phosphotransferase (APH4) marker protein in all plants; exemption from the requirement of a tolerance. Residues...

  19. 40 CFR 174.526 - Hygromycin B phosphotransferase (APH4) marker protein in all plants; exemption from the...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Hygromycin B phosphotransferase (APH4) marker protein in all plants; exemption from the requirement of a tolerance. 174.526 Section 174.526... phosphotransferase (APH4) marker protein in all plants; exemption from the requirement of a tolerance. Residues...

  20. 40 CFR 174.526 - Hygromycin B phosphotransferase (APH4) marker protein in all plants; exemption from the...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Hygromycin B phosphotransferase (APH4) marker protein in all plants; exemption from the requirement of a tolerance. 174.526 Section 174.526... phosphotransferase (APH4) marker protein in all plants; exemption from the requirement of a tolerance. Residues...

  1. National Plant Diagnostic Network, Taxonomic training videos: Introduction to AphID

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Training is a critical part of aphid (Hemiptera: Aphididae) identification. This video provides visual instruction on the use of the expert system, AphID, for aphid examination and identification. The video demonstrates the use of different training modules that allow the user to gain familiarity wi...

  2. Purification and Characterization of Aminoglycoside Phosphotransferase APH(6)-Id, a Streptomycin Inactivating Enzyme

    PubMed Central

    Ashenafi, Meseret; Ammosova, Tatiana; Nekhai, Sergei; Byrnes, W. Malcolm

    2014-01-01

    As part of an overall project to characterize the streptomycin phosphotransferase enzyme APH(6)-Id, which confers bacterial resistance to streptomycin, we cloned, expressed, purified and characterized the enzyme. When expressed in E. coli, the recombinant enzyme increased by up to 70-fold the minimum inhibitory concentration (MIC) needed to inhibit cell growth. Size exclusion chromatography gave a molecular mass of 31.4 ± 1.3 kDa for the enzyme, showing that it functions as a monomer. Activity was assayed using three methods: (1) an HPLC-based method that measures the consumption of streptomycin over time; (2) a spectrophotometric method that utilizes a coupled assay; and (3) a radioenzymatic method that detects production of 32P-labeled streptomycin phosphate. Altogether, the three methods demonstrated that streptomycin was consumed in the APH(6)-Id-catalyzed reaction, ATP was hydrolyzed, and streptomycin phosphate was produced in a substrate-dependent manner, demonstrating that APH(6)-Id is a streptomycin phosphotransferase. Steady state kinetic analysis gave the following results: Km(streptomycin) of 0.38 ± 0.13 mM, Km(ATP) of 1.03 ± 0.1 mM, Vmax of 3.2 ± 1.1 μmol/min/mg and kcat of 1.7 ± 0.6 s−1. Our study demonstrates that APH(6)-Id is a bona fide streptomycin phosphotransferase, functions as a monomer, and confers resistance to streptomycin. PMID:24248535

  3. Involvement of aph(3')-IIa in the formation of mosaic aminoglycoside resistance genes in natural environments.

    PubMed

    Woegerbauer, Markus; Kuffner, Melanie; Domingues, Sara; Nielsen, Kaare M

    2015-01-01

    Intragenic recombination leading to mosaic gene formation is known to alter resistance profiles for particular genes and bacterial species. Few studies have examined to what extent aminoglycoside resistance genes undergo intragenic recombination. We screened the GenBank database for mosaic gene formation in homologs of the aph(3')-IIa (nptII) gene. APH(3')-IIa inactivates important aminoglycoside antibiotics. The gene is widely used as a selectable marker in biotechnology and enters the environment via laboratory discharges and the release of transgenic organisms. Such releases may provide opportunities for recombination in competent environmental bacteria. The retrieved GenBank sequences were grouped in three datasets comprising river water samples, duck pathogens and full-length variants from various bacterial genomes and plasmids. Analysis for recombination in these datasets was performed with the Recombination Detection Program (RDP4), and the Genetic Algorithm for Recombination Detection (GARD). From a total of 89 homologous sequences, 83% showed 99-100% sequence identity with aph(3')-IIa originally described as part of transposon Tn5. Fifty one were unique sequence variants eligible for recombination analysis. Only a single recombination event was identified with high confidence and indicated the involvement of aph(3')-IIa in the formation of a mosaic gene located on a plasmid of environmental origin in the multi-resistant isolate Pseudomonas aeruginosa PA96. The available data suggest that aph(3')-IIa is not an archetypical mosaic gene as the divergence between the described sequence variants and the number of detectable recombination events is low. This is in contrast to the numerous mosaic alleles reported for certain penicillin or tetracycline resistance determinants. PMID:26042098

  4. Variants of glycoside hydrolases

    SciTech Connect

    Teter, Sarah; Ward, Connie; Cherry, Joel; Jones, Aubrey; Harris, Paul; Yi, Jung

    2013-02-26

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

  5. Variants of glycoside hydrolases

    DOEpatents

    Teter, Sarah; Ward, Connie; Cherry, Joel; Jones, Aubrey; Harris, Paul; Yi, Jung

    2011-04-26

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

  6. Origin in Acinetobacter guillouiae and Dissemination of the Aminoglycoside-Modifying Enzyme Aph(3′)-VI

    PubMed Central

    Yoon, Eun-Jeong; Goussard, Sylvie; Touchon, Marie; Krizova, Lenka; Cerqueira, Gustavo; Murphy, Cheryl; Lambert, Thierry; Grillot-Courvalin, Catherine; Nemec, Alexandr

    2014-01-01

    ABSTRACT The amikacin resistance gene aphA6 was first detected in the nosocomial pathogen Acinetobacter baumannii and subsequently in other genera. Analysis of 133 whole-genome sequences covering the taxonomic diversity of Acinetobacter spp. detected aphA6 in the chromosome of 2 isolates of A. guillouiae, which is an environmental species, 1 of 8 A. parvus isolates, and 5 of 34 A. baumannii isolates. The gene was also present in 29 out of 36 A. guillouiae isolates screened by PCR, indicating that it is ancestral to this species. The Pnative promoter for aphA6 in A. guillouiae and A. parvus was replaced in A. baumannii by PaphA6, which was generated by use of the insertion sequence ISAba125, which brought a −35 sequence. Study of promoter strength in Escherichia coli and A. baumannii indicated that PaphA6 was four times more potent than Pnative. There was a good correlation between aminoglycoside MICs and aphA6 transcription in A. guillouiae isolates that remained susceptible to amikacin. The marked topology differences of the phylogenetic trees of aphA6 and of the hosts strongly support its recent direct transfer within Acinetobacter spp. and also to evolutionarily remote bacterial genera. Concomitant expression of aphA6 must have occurred because, contrary to the donors, it can confer resistance to the new hosts. Mobilization and expression of aphA6 via composite transposons and the upstream IS-generating hybrid PaphA6, followed by conjugation, seems the most plausible mechanism. This is in agreement with the observation that, in the recipients, aphA6 is carried by conjugative plasmids and flanked by IS that are common in Acinetobacter spp. Our data indicate that resistance genes can also be found in susceptible environmental bacteria.   PMID:25336457

  7. Structural Basis of APH(3)-IIIa-Mediated Resistance to N1-Substituted Aminoglycoside Antibiotics

    SciTech Connect

    Fong, D.; Berghuis, A

    2009-01-01

    Butirosin is unique among the naturally occurring aminoglycosides, having a substituted amino group at position 1 (N1) of the 2-deoxystreptamine ring with an (S)-4-amino-2-hydroxybutyrate (AHB) group. While bacterial resistance to aminoglycosides can be ascribed chiefly to drug inactivation by plasmid-encoded aminoglycoside-modifying enzymes, the presence of an AHB group protects the aminoglycoside from binding to many resistance enzymes, and hence, the antibiotic retains its bactericidal properties. Consequently, several semisynthetic N1-substituted aminoglycosides, such as amikacin, isepamicin, and netilmicin, were developed. Unfortunately, butirosin, amikacin, and isepamicin are not resistant to inactivation by 3'-aminoglycoside O-phosphotransferase type IIIa [APH(3')-IIIa]. We report here the crystal structure of APH(3')-IIIa in complex with an ATP analog, AMPPNP [adenosine 5'-(?,{gamma}-imido)triphosphate], and butirosin A to 2.4-A resolution. The structure shows that butirosin A binds to the enzyme in a manner analogous to other 4,5-disubstituted aminoglycosides, and the flexible antibiotic-binding loop is key to the accommodation of structurally diverse substrates. Based on the crystal structure, we have also constructed a model of APH(3')-IIIa in complex with amikacin, a commonly used semisynthetic N1-substituted 4,6-disubstituted aminoglycoside. Together, these results suggest a strategy to further derivatize the AHB group in order to generate new aminoglycoside derivatives that can elude inactivation by resistance enzymes while maintaining their ability to bind to the ribosomal A site.

  8. Antibiotic Binding Drives Catalytic Activation of Aminoglycoside Kinase APH(2″)-Ia.

    PubMed

    Caldwell, Shane J; Huang, Yue; Berghuis, Albert M

    2016-06-01

    APH(2″)-Ia is a widely disseminated resistance factor frequently found in clinical isolates of Staphylococcus aureus and pathogenic enterococci, where it is constitutively expressed. APH(2″)-Ia confers high-level resistance to gentamicin and related aminoglycosides through phosphorylation of the antibiotic using guanosine triphosphate (GTP) as phosphate donor. We have determined crystal structures of the APH(2″)-Ia in complex with GTP analogs, guanosine diphosphate, and aminoglycosides. These structures collectively demonstrate that aminoglycoside binding to the GTP-bound kinase drives conformational changes that bring distant regions of the protein into contact. These changes in turn drive a switch of the triphosphate cofactor from an inactive, stabilized conformation to a catalytically competent active conformation. This switch has not been previously reported for antibiotic kinases or for the structurally related eukaryotic protein kinases. This catalytic triphosphate switch presents a means by which the enzyme can curtail wasteful hydrolysis of GTP in the absence of aminoglycosides, providing an evolutionary advantage to this enzyme. PMID:27161980

  9. Soluble Epoxide Hydrolase Dimerization Is Required for Hydrolase Activity*

    PubMed Central

    Nelson, Jonathan W.; Subrahmanyan, Rishi M.; Summers, Sol A.; Xiao, Xiangshu; Alkayed, Nabil J.

    2013-01-01

    Soluble epoxide hydrolase (sEH) plays a key role in the metabolic conversion of the protective eicosanoid 14,15-epoxyeicosatrienoic acid to 14,15-dihydroxyeicosatrienoic acid. Accordingly, inhibition of sEH hydrolase activity has been shown to be beneficial in multiple models of cardiovascular diseases, thus identifying sEH as a valuable therapeutic target. Recently, a common human polymorphism (R287Q) was identified that reduces sEH hydrolase activity and is localized to the dimerization interface of the protein, suggesting a relationship between sEH dimerization and activity. To directly test the hypothesis that dimerization is essential for the proper function of sEH, we generated mutations within the sEH protein that would either disrupt or stabilize dimerization. We quantified the dimerization state of each mutant using a split firefly luciferase protein fragment-assisted complementation system. The hydrolase activity of each mutant was determined using a fluorescence-based substrate conversion assay. We found that mutations that disrupted dimerization also eliminated hydrolase enzymatic activity. In contrast, a mutation that stabilized dimerization restored hydrolase activity. Finally, we investigated the kinetics of sEH dimerization and found that the human R287Q polymorphism was metastable and capable of swapping dimer partners faster than the WT enzyme. These results indicate that dimerization is required for sEH hydrolase activity. Disrupting sEH dimerization may therefore serve as a novel therapeutic strategy for reducing sEH hydrolase activity. PMID:23362272

  10. Soluble epoxide hydrolase dimerization is required for hydrolase activity.

    PubMed

    Nelson, Jonathan W; Subrahmanyan, Rishi M; Summers, Sol A; Xiao, Xiangshu; Alkayed, Nabil J

    2013-03-15

    Soluble epoxide hydrolase (sEH) plays a key role in the metabolic conversion of the protective eicosanoid 14,15-epoxyeicosatrienoic acid to 14,15-dihydroxyeicosatrienoic acid. Accordingly, inhibition of sEH hydrolase activity has been shown to be beneficial in multiple models of cardiovascular diseases, thus identifying sEH as a valuable therapeutic target. Recently, a common human polymorphism (R287Q) was identified that reduces sEH hydrolase activity and is localized to the dimerization interface of the protein, suggesting a relationship between sEH dimerization and activity. To directly test the hypothesis that dimerization is essential for the proper function of sEH, we generated mutations within the sEH protein that would either disrupt or stabilize dimerization. We quantified the dimerization state of each mutant using a split firefly luciferase protein fragment-assisted complementation system. The hydrolase activity of each mutant was determined using a fluorescence-based substrate conversion assay. We found that mutations that disrupted dimerization also eliminated hydrolase enzymatic activity. In contrast, a mutation that stabilized dimerization restored hydrolase activity. Finally, we investigated the kinetics of sEH dimerization and found that the human R287Q polymorphism was metastable and capable of swapping dimer partners faster than the WT enzyme. These results indicate that dimerization is required for sEH hydrolase activity. Disrupting sEH dimerization may therefore serve as a novel therapeutic strategy for reducing sEH hydrolase activity. PMID:23362272

  11. Peptidoglycan Hydrolases of Escherichia coli

    PubMed Central

    van Heijenoort, Jean

    2011-01-01

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

  12. Near infrared fluorescent biliproteins generated from bacteriophytochrome AphB of Nostoc sp. PCC 7120.

    PubMed

    Yuan, Che; Li, Hui-Zhen; Tang, Kun; Gärtner, Wolfgang; Scheer, Hugo; Zhou, Ming; Zhao, Kai-Hong

    2016-04-13

    The genome of the cyanobacterium Nostoc sp. PCC 7120 encodes a large number of putative bacteriophytochrome and cyanobacteriochrome photoreceptors that, due to their long-wavelength absorption and fluorescence emission, might serve as fluorescent tags in intracellular investigations. We show that the PAS-GAF domain of the bacteriophytochrome, AphB, binds biliverdin covalently and exhibits, besides its reversible photochemistry, a moderate fluorescence in the near infrared (NIR) spectral region. It was selected for further increasing the brightness while retaining the NIR fluorescence. In the first step, amino acids assumed to improve fluorescence were selectively mutated. The resulting variants were then subjected to several rounds of random mutagenesis and screened for enhanced fluorescence in the NIR. The brightness of optimized PAS-GAF variants increased more than threefold compared to that of wt AphB(1-321), with only insignificant spectral shifts (Amax around 695 nm, and Fmax around 720 nm). In general, the brightness increases with decreasing wavelengths, which allows for a selection of the fluorophore depending on the optical properties of the tissue. A spectral heterogeneity was observed when residue His260, located in close proximity to the chromophore, was mutated to Tyr, emphasizing the strong effects of the environment on the electronic properties of the bound biliverdin chromophore. PMID:27004456

  13. Polyglycine hydrolases secreted by pathogenic fungi

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  14. Involvement of aph(3′)-IIa in the formation of mosaic aminoglycoside resistance genes in natural environments

    PubMed Central

    Woegerbauer, Markus; Kuffner, Melanie; Domingues, Sara; Nielsen, Kaare M.

    2015-01-01

    Intragenic recombination leading to mosaic gene formation is known to alter resistance profiles for particular genes and bacterial species. Few studies have examined to what extent aminoglycoside resistance genes undergo intragenic recombination. We screened the GenBank database for mosaic gene formation in homologs of the aph(3′)-IIa (nptII) gene. APH(3′)-IIa inactivates important aminoglycoside antibiotics. The gene is widely used as a selectable marker in biotechnology and enters the environment via laboratory discharges and the release of transgenic organisms. Such releases may provide opportunities for recombination in competent environmental bacteria. The retrieved GenBank sequences were grouped in three datasets comprising river water samples, duck pathogens and full-length variants from various bacterial genomes and plasmids. Analysis for recombination in these datasets was performed with the Recombination Detection Program (RDP4), and the Genetic Algorithm for Recombination Detection (GARD). From a total of 89 homologous sequences, 83% showed 99–100% sequence identity with aph(3′)-IIa originally described as part of transposon Tn5. Fifty one were unique sequence variants eligible for recombination analysis. Only a single recombination event was identified with high confidence and indicated the involvement of aph(3′)-IIa in the formation of a mosaic gene located on a plasmid of environmental origin in the multi-resistant isolate Pseudomonas aeruginosa PA96. The available data suggest that aph(3′)-IIa is not an archetypical mosaic gene as the divergence between the described sequence variants and the number of detectable recombination events is low. This is in contrast to the numerous mosaic alleles reported for certain penicillin or tetracycline resistance determinants. PMID:26042098

  15. Individual and combined roles of the master regulators AphA and LuxR in control of the Vibrio harveyi quorum-sensing regulon.

    PubMed

    van Kessel, Julia C; Rutherford, Steven T; Shao, Yi; Utria, Alan F; Bassler, Bonnie L

    2013-02-01

    Bacteria use a chemical communication process called quorum sensing to control transitions between individual and group behaviors. In the Vibrio harveyi quorum-sensing circuit, two master transcription factors, AphA and LuxR, coordinate the quorum-sensing response. Here we show that AphA regulates 167 genes, LuxR regulates 625 genes, and they coregulate 77 genes. LuxR strongly controls genes at both low cell density and high cell density, suggesting that it is the major quorum-sensing regulator. In contrast, AphA is absent at high cell density and acts to fine-tune quorum-sensing gene expression at low cell density. We examined two loci as case studies of coregulation by AphA and LuxR. First, AphA and LuxR directly regulate expression of the genes encoding the quorum-regulatory small RNAs Qrr2, Qrr3, and Qrr4, the consequence of which is a specifically timed transition between the individual and the group life-styles. Second, AphA and LuxR repress type III secretion system genes but at different times and to different extents. The consequence of this regulation is that type III secretion is restricted to a peak at mid-cell density. Thus, the asymmetric production of AphA and LuxR coupled with differences in their strengths and timing of target gene regulation generate a precise temporal pattern of gene expression. PMID:23204455

  16. Individual and Combined Roles of the Master Regulators AphA and LuxR in Control of the Vibrio harveyi Quorum-Sensing Regulon

    PubMed Central

    van Kessel, Julia C.; Rutherford, Steven T.; Shao, Yi; Utria, Alan F.

    2013-01-01

    Bacteria use a chemical communication process called quorum sensing to control transitions between individual and group behaviors. In the Vibrio harveyi quorum-sensing circuit, two master transcription factors, AphA and LuxR, coordinate the quorum-sensing response. Here we show that AphA regulates 167 genes, LuxR regulates 625 genes, and they coregulate 77 genes. LuxR strongly controls genes at both low cell density and high cell density, suggesting that it is the major quorum-sensing regulator. In contrast, AphA is absent at high cell density and acts to fine-tune quorum-sensing gene expression at low cell density. We examined two loci as case studies of coregulation by AphA and LuxR. First, AphA and LuxR directly regulate expression of the genes encoding the quorum-regulatory small RNAs Qrr2, Qrr3, and Qrr4, the consequence of which is a specifically timed transition between the individual and the group life-styles. Second, AphA and LuxR repress type III secretion system genes but at different times and to different extents. The consequence of this regulation is that type III secretion is restricted to a peak at mid-cell density. Thus, the asymmetric production of AphA and LuxR coupled with differences in their strengths and timing of target gene regulation generate a precise temporal pattern of gene expression. PMID:23204455

  17. Structural characterization of the novel aminoglycoside phosphotransferase AphVIII from Streptomyces rimosus with enzymatic activity modulated by phosphorylation.

    PubMed

    Boyko, Konstantin M; Gorbacheva, Marina A; Korzhenevskiy, Dmitry A; Alekseeva, Maria G; Mavletova, Dilara A; Zakharevich, Natalia V; Elizarov, Sergey M; Rudakova, Natalia N; Danilenko, Valery N; Popov, Vladimir O

    2016-09-01

    Aminoglycoside phosphotransferases represent a broad class of enzymes that promote bacterial resistance to aminoglycoside antibiotics via the phosphorylation of hydroxyl groups in the latter. Here we report the spatial structure of the 3'-aminoglycoside phosphotransferase of novel VIII class (AphVIII) solved by X-ray diffraction method with a resolution of 2.15 Å. Deep analysis of APHVIII structure and its comparison with known structures of aminoglycoside phosphotransferases of various types reveals that AphVIII has a typical two-domain fold and, however, possesses some unique characteristics that distinguish the enzyme from its known homologues. The most important difference is the presence of the activation loop with unique Ser146 residue. We demonstrate that in the apo-state of the enzyme the activation loop does not interact with other parts of the enzyme and seems to adopt catalytically competent state only after substrate binding. PMID:27338640

  18. Purification, Crystallization And Preliminary X-Ray Analysis of Aminoglycoside-2 ''-Phosphotransferase-Ic [APH(2 '')-Ic] From Enterococcus Gallinarum

    SciTech Connect

    Byrnes, L.J.; Badarau, A.; Vakulenko, S.B.; Smith, C.A.; /SLAC, SSRL

    2009-04-30

    Bacterial resistance to aminoglycoside antibiotics is primarily the result of deactivation of the drugs. Three families of enzymes are responsible for this activity, with one such family being the aminoglycoside phosphotransferases (APHs). The gene encoding one of these enzymes, aminoglycoside-2{double_prime}-phosphotransferase-Ic [APH(2{double_prime})-Ic] from Enterococcus gallinarum, has been cloned and the wild-type protein (comprising 308 amino-acid residues) and three mutants that showed elevated minimum inhibitory concentrations towards gentamicin (F108L, H258L and a double mutant F108L/H258L) were expressed in Escherichia coli and subsequently purified. All APH(2{double_prime})-Ic variants were crystallized in the presence of 14-20%(w/v) PEG 4000, 0.25 M MgCl{sub 2}, 0.1 M Tris-HCl pH 8.5 and 1 mM Mg{sub 2}GTP. The crystals belong to the monoclinic space group C2, with one molecule in the asymmetric unit. The approximate unit-cell parameters are a = 82.4, b = 54.2, c = 77.0 {angstrom}, {beta} = 108.8{sup o}. X-ray diffraction data were collected to approximately 2.15 {angstrom} resolution from an F108L crystal at beamline BL9-2 at SSRL, Stanford, California, USA.

  19. Hierarchical classification of glycoside hydrolases.

    PubMed

    Naumoff, D G

    2011-06-01

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

  20. Biomarkers of organophosphorus (OP) exposures in humans

    PubMed Central

    Marsillach, Judit; Richter, Rebecca J.; Kim, Jerry H.; Stevens, Richard C.; MacCoss, Michael J.; Tomazela, Daniela; Suzuki, Stephanie M.; Schopfer, Lawrence M; Lockridge, Oksana; Furlong, Clement E.

    2011-01-01

    There are ongoing events where aircraft engine lubricant containing tricresyl phosphates (TCPs) contaminates aircraft cabins. Some individuals have experienced tremors or other neurological symptoms that may last for many months following exposures. Mass spectrometric (MS) protocols are being developed to determine the percentage of “biomarker proteins” that are modified by such exposures, specifically on active site serines. Both plasma butyrylcholinesterase (BChE) and red cell acylpeptide hydrolase (APH) are readily inhibited by 2-(o-cresyl)-4H-1:3:2:benzodioxaphosphoran-2-one (CBDP) or phenyl saligenin cyclic phosphate (PSP) and have the potential to provide information about the level of exposure of an individual. We have developed immunomagnetic bead-based single-step purification protocols for both BChE and APH and have characterized the active site serine adducts of BChE by MS. PMID:21767566

  1. Increasing Prevalence of Aminoglycoside-Resistant Enterococcus faecalis Isolates Due to the aac(6’)-aph(2”) Gene: A Therapeutic Problem in Kermanshah, Iran

    PubMed Central

    Khani, Mitra; Fatollahzade, Mahdie; Pajavand, Hamid; Bakhtiari, Somaye; Abiri, Ramin

    2016-01-01

    Background: Enterococci are important pathogens in nosocomial infections. Various types of antibiotics, such as aminoglycosides, are used for treatment of these infections. Enterococci can acquire resistant traits, which can lead to therapeutic problems with aminoglycosides. Objectives: This study was designed to identify the prevalence of, and to compare, the aac(6’)-aph(2”) and aph(3)-IIIa genes and their antimicrobial resistance patterns among Enterococcus faecalis and E. faecium isolates from patients at Imam Reza hospital in Kermanshah in 2011 - 2012. Patients and Methods: One hundred thirty-eight clinical specimens collected from different wards of Imam Reza hospital were identified to the species level by biochemical tests. Antimicrobial susceptibility tests against kanamycin, teicoplanin, streptomycin, imipenem, ciprofloxacin, and ampicillin were performed by the disk diffusion method. The minimum inhibitory concentrations of gentamicin, streptomycin, kanamycin, and amikacin were evaluated with the microbroth dilution method. The aminoglycoside resistance genes aac(6’)-aph(2”) and aph(3”)-IIIa were analyzed with multiplex PCR. Results: The prevalence of isolates was 33 (24.1%) for E. faecium and 63 (46%) for E. faecalis. Eighty-nine percent of the isolates were high-level gentamicin resistant (HLGR), and 32.8% of E. faecium isolates and 67.2% of E. faecalis isolates carried aac(6’)-aph(2”). The prevalence of aph(3”)-IIIa among the E. faecalis and E. faecium isolates was 22.7% and 77.3%, respectively. Conclusions: Remarkably increased incidence of aac(6’)-aph(2”) among HLGR isolates explains the relationship between this gene and the high level of resistance to aminoglycosides. As the resistant gene among enterococci can be transferred, the use of new-generation antibiotics is necessary. PMID:27217920

  2. Prunus serotina Amygdalin Hydrolase and Prunasin Hydrolase 1

    PubMed Central

    Li, Chun Ping; Swain, Elisabeth; Poulton, Jonathan E.

    1992-01-01

    In black cherry (Prunus serotina Ehrh.) seed homogenates, amygdalin hydrolase (AH) participates with prunasin hydrolase (PH) and mandelonitrile lyase in the sequential degradation of (R)-amygdalin to HCN, benzaldehyde, and glucose. Four isozymes of AH (designated AH I, I′, II, II′) were purified from mature cherry seeds by concanavalin A-Sepharose 4B chromatography, ion-exchange chromatography, and chromatofocusing. All isozymes were monomeric glycoproteins with native molecular masses of 52 kD. They showed similar kinetic properties (pH optima, Km, Vmax) but differed in their isoelectric points and N-terminal amino acid sequences. Analytical isoelectric focusing revealed the presence of subisozymes of each isozyme. The relative abundance of these isozymes and/or subisozymes varied from seed to seed. Three isozymes of PH (designated PH I, IIa, and IIb) were purified to apparent homogeneity by affinity, ion-exchange, and hydroxyapatite chromatography and by nondenaturing polyacrylamide gel electrophoresis. PH I and PH IIb are 68-kD monomeric glycoproteins, whereas PH IIa is dimeric (140 kD). The N-terminal sequences of all PH and AH isozymes showed considerable similarity. Polyclonal antisera raised in rabbits against deglycosylated AH I or a mixture of the three deglycosylated PH isozymes were not monospecific as judged by immunoblotting analysis, but also cross-reacted with the opposing glucosidase. Monospecific antisera deemed suitable for immunocytochemistry and screening of expression libraries were obtained by affinity chromatography. Each antiserum recognized all known isozymes of the specific glucosidase used as antigen. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 9 PMID:16652959

  3. Assays for the classification of two types of esterases: carboxylic ester hydrolases and phosphoric triester hydrolases.

    PubMed

    Anspaugh, Douglas D; Roe, R Michael

    2002-11-01

    Assays for the Classification of Two Types of Esterases: Carboxylic Ester Hydrolase and Phosphoric Triester Hydrolase (Douglas D. Anspaugh and Michael Roe, North Carolina State University, Raleigh, North Carolina). This unit describes assays that quantitate two types of esterase the carboxylic ester hydrolases and the phosphoric triester hydrolases. Carboxylic ester hydrolases include the B-esterases, which are inhibited by organophosphorus compounds. Among the phosphoric triester hydrolases is aryldialkylphosphatase, which has been called A-esterase or paraoxonase due to its ability to oxidize paraoxon and other organophosphates. These assays are colorimetric and miniaturized for rapid simultaneous testing of multiple, small-volume samples in a microtiter plate format. There is also a discussion of the history of esterase nomenclature and the reasons why this large group of enzymes is so difficult to classify. PMID:20945297

  4. Novel interactions between the HTLV antisense proteins HBZ and APH-2 and the NFAR protein family: Implications for the HTLV lifecycles.

    PubMed

    Murphy, Jane; Hall, William W; Ratner, Lee; Sheehy, Noreen

    2016-07-01

    The human T-cell leukaemia virus type 1 and type 2 (HTLV-1/HTLV-2) antisense proteins HBZ and APH-2 play key roles in the HTLV lifecycles and persistence in the host. Nuclear Factors Associated with double-stranded RNA (NFAR) proteins NF90/110 function in the lifecycles of several viruses and participate in host innate immunity against infection and oncogenesis. Using GST pulldown and co-immunoprecipitation assays we demonstrate specific novel interactions between HBZ/APH-2 and NF90/110 and characterised the protein domains involved. Moreover we show that NF90/110 significantly enhance Tax mediated LTR activation, an effect that was abolished by HBZ but enhanced by APH-2. Additionally we found that HBZ and APH-2 modulate the promoter activity of survivin and are capable of antagonising NF110-mediated survivin activation. Thus interactions between HTLV antisense proteins and the NFAR protein family have an overall positive impact on HTLV infection. Hence NFARs may represent potential therapeutic targets in HTLV infected cells. PMID:27110706

  5. Novel interactions between the HTLV antisense proteins HBZ and APH-2 and the NFAR protein family: Implications for the HTLV lifecycles

    PubMed Central

    Murphy, Jane; Hall, William W.; Ratner, Lee; Sheehy, Noreen

    2016-01-01

    The human T-cell leukaemia virus type 1 and type 2 (HTLV-1/HTLV-2) antisense proteins HBZ and APH-2 play key roles in the HTLV lifecycles and persistence in the host. Nuclear Factors Associated with double-stranded RNA (NFAR) proteins NF90/110 function in the lifecycles of several viruses and participate in host innate immunity against infection and oncogenesis. Using GST pulldown and co-immunoprecipitation assays we demonstrate specific novel interactions between HBZ/APH-2 and NF90/110 and characterised the protein domains involved. Moreover we show that NF90/110 significantly enhance Tax mediated LTR activation, an effect that was abolished by HBZ but enhanced by APH-2. Additionally we found that HBZ and APH-2 modulate the promoter activity of survivin and are capable of antagonising NF110-mediated survivin activation. Thus interactions between HTLV antisense proteins and the NFAR protein family have an overall positive impact on HTLV infection. Hence NFARs may represent potential therapeutic targets in HTLV infected cells. PMID:27110706

  6. Purification, crystallization and preliminary X-ray analysis of Enterococcus faecium aminoglycoside-2′′-phosphotransferase-Ib [APH(2′′)-Ib

    SciTech Connect

    Walanj, Rupa; Young, Paul; Baker, Heather M.; Baker, Edward N.; Metcalf, Peter; Chow, Joseph W.; Lerner, Stephen; Vakulenko, Sergei; Smith, Clyde A.

    2005-04-01

    APH(2′′)-Ib is an enzyme responsible for high-level gentamicin resistance in E. faecium isolates. Native crystals of this enzyme have been prepared and preliminary X-ray diffraction experiments have been undertaken. Bacterial resistance to the aminoglycoside antibiotics is primarily the result of deactivation of the drugs. Three families of enzymes are responsible for this activity, with one such family being the aminoglycoside phosphotransferases (APHs). The gene encoding one of these enzymes, APH(2′′)-Ib, has been cloned and the protein (comprising 299 amino-acid residues) expressed in Escherichia coli, purified and crystallized in the presence of 16%(w/v) PEG 3350 and gentamicin. The crystals belong to the monoclinic space group P2{sub 1}, with approximate unit-cell parameters a = 79.7, b = 58.8, c = 81.4 Å, β = 98.4°, and preliminary X-ray diffraction analysis is consistent with the presence of two molecules in the asymmetric unit. Synchrotron diffraction data to approximately 2.65 Å resolution were collected from a native APH(2′′)-Ib crystal at beamline BL9-2 at SSRL (Stanford, CA, USA). Selenium-substituted crystals have also been produced and structure determination is proceeding.

  7. The effect of APH treatment on surface bonding and osseointegration of Ti-6Al-7Nb implants: an in vitro and in vivo study.

    PubMed

    Nguyen, Thuy-Duong Thi; Moon, So-Hee; Oh, Tae-Ju; Park, Il-Song; Lee, Min-Ho; Bae, Tae-Sung

    2015-04-01

    This study investigated the effects of anodization-cyclic precalcification-heat (APH) treatment on the bonding ability of Ca-P coating to the parent metal and osseointegration of Ti-6Al-7Nb implants. Eighteen Ti-6Al-7Nb discs, 9 untreated and 9 APH-treated, were cultured with osteoblast cells in vitro, and the cellular differentiation ability was assayed at 1, 2, and 3 weeks. For in vivo testing, 28 Ti-6Al-7Nb implants (14 implants of each group) were inserted to rat tibias, and after each 4 and 6 weeks of implantation, bone bonding, and osseointegration were evaluated through removal torque and histological analysis. Osteoblast-culturing showed twice as much of the alkaline phosphatase activity on the treated surface at 3 weeks than on the untreated surface (p < 0.05). The treated implants exhibited higher removal torque values than the untreated ones (15.5 vs. 1.8 Ncm at 4 weeks and 19.7 vs. 2.6 Ncm at 6 weeks, p < 0.05). Moreover, the excellent bonding quality of coats was confirmed by the existence of cohesive fractures on the surface of removed APH implants (field emission scanning electron microscopy and histological observation). Within the limits of this study, it can be concluded that the APH treatment significantly enhanced osseointegration of the Ti-6Al-7Nb implant, with the stable bonding between the coating and the implant surface. PMID:24976109

  8. Catabolism of endogenous and overexpressed APH1a and PEN2: evidence for artifactual involvement of the proteasome in the degradation of overexpressed proteins

    PubMed Central

    Dunys, Julie; Kawarai, Toshitaka; Wilk, Sherwin; St. George-Hyslop, Peter; Alves Da Costa, Cristine; Checler, Frédéric

    2005-01-01

    PS (presenilin)-dependent γ-secretase occurs as a high-molecular-mass complex composed of either PS1 or PS2 associated with Nct (nicastrin), PEN2 (presenilin enhancer 2 homologue) and APH1 (anterior pharynx defective 1 homologue). Numerous reports have documented the very complicated physical and functional cross-talk between these proteins that ultimately governs the biological activity of the γ-secretase, but very few studies examined the fate of the components of the complex. We show that, in both HEK-293 cells and the TSM1 neuronal cell line, the immunoreactivities of overexpressed myc-tagged-APH1a and -PEN2 were enhanced by the proteasome inhibitors ZIE and lactacystin, whereas a broad range of protease inhibitors had no effect. By contrast, proteasome inhibitors were totally unable to affect the cellular expression of endogenous APH1aL and PEN2 in HEK-293 cells, TSM1 and primary cultured cortical neurons. To explain this apparent discrepancy, we examined the degradation of myc-tagged-APH1a and -PEN2, in vitro, by cell extracts containing endogenous proteasome and by purified 20S proteasome. Strikingly, myc-tagged-APH1a and -PEN2 resist proteolysis by endogenous proteasome and purified 20S proteasome. We also show that endogenous PEN2 expression was drastically higher in wild-type than in PS- and Nct-deficient fibroblasts and was enhanced by proteasome inhibitors only in the two deficient cell systems. However, here again, purified 20S proteasome appeared unable to cleave endogenous PEN2 present in PS-deficient fibroblasts. The levels of endogenous APH1aL-like immunoreactivity were not modified by proteasome inhibitors and were unaffected by PS deficiency. Altogether, our results indicate that endogenous PEN2 and APH1aL do not undergo proteasomal degradation under physiological conditions in HEK-293 cells, TSM1 cells and fibroblasts and that the clearance of PEN2 in PS- and Nct-deficient fibroblasts is not mediated by 20S proteasome. Whether the 26S

  9. In vitro antibacterial activity of vertilmicin and its susceptibility to modifications by the recombinant AAC6'-APH2'' enzyme.

    PubMed

    Li, Cong-Ran; Yang, Xin-Yi; Lou, Ren-Hui; Zhang, Wei-Xin; Wang, Yue-Ming; Yuan, Min; Li, Yi; Chen, Hui-Zhen; Hong, Bin; Sun, Cheng-Hang; Zhao, Li-Xun; Li, Zhuo-Rong; Jiang, Jian-Dong; You, Xue-Fu

    2008-11-01

    Vertilmicin is a new semisynthetic aminoglycoside with a structure similar to that of netilmicin except for a methyl group at the C-6' position. In the present study, the in vitro antibacterial activity of vertilmicin was studied, and its susceptibility to modifications by the recombinant aminoglycoside bifunctional modifying enzyme AAC(6')-APH(2'') was compared with those of verdamicin and netilmicin. A total of 1,185 clinical isolates collected from hospitals in Beijing between 2000 and 2001 were subjected to the in vitro antibacterial activity evaluations, including MIC, minimum bactericidal concentration (MBC), and time-kill curve tests. The MICs were evaluated in non-gentamicin-resistant (gentamicin-susceptible and gentamicin-intermediate) strains and gentamicin-resistant strains, respectively. For most of the non-gentamicin-resistant bacteria (except for the isolates of Pseudomonas spp.), the MIC(90)s of vertilmicin were in the range of 0.5 to 8 microg/ml, comparable to those of the reference aminoglycosides. For the gentamicin-resistant isolates, the three semisynthetic aminoglycosides (vertilmicin, netilmicin, and amikacin) demonstrated low MIC(50)s and/or MIC(90)s, as well as high percent susceptibility values. Among the study drugs, vertilmicin showed the lowest MIC(90)s, 16 microg/ml, for the gram-positive gentamicin-resistant isolates of Staphylococcus aureus and Staphylococcus epidermidis. Meanwhile, vertilmicin was a potent bactericidal agent, with MBC/MIC ratios in the range of 1 to 2 for Escherichia coli, Klebsiella pneumoniae, and S. aureus and 1 to 4 for S. epidermidis. The time-kill curve determination further demonstrated that this effect was rapid and concentration dependent. In evaluations of susceptibility to modifications by the recombinant AAC(6')-APH(2'') with maximum rate of metabolism/K(m) measurements, vertilmicin exhibited susceptibilities to both acetylation and phosphorylation lower than those of netilmicin and verdamicin. PMID

  10. Reduced Alzheimer's disease β-amyloid deposition in transgenic mice expressing S-palmitoylation-deficient APH1aL and nicastrin

    PubMed Central

    Meckler, Xavier; Roseman, Jelita; Das, Pritam; Cheng, Haipeng; Pei, Susan; Keat, Marcia; Kassarjian, Breanne; Golde, Todd E.; Parent, Angèle T.; Thinakaran, Gopal

    2010-01-01

    Sequential cleavage of amyloid precursor protein by β- and γ-secretases generates β-amyloid peptides (Aβ), which accumulate in the brains of patients with Alzheimer's disease. We recently identified S-palmitoylation of two γ-secretase subunits, APH1 and nicastrin. S-palmitoylation is an essential post-translational modification for the proper trafficking and function of many neuronal proteins. In cultured cell lines, lack of S-palmitoylation causes instability of nascent APH1 and nicastrin, but does not affect γ-secretase processing of amyloid precursor protein. To determine the importance of γ-secretase S-palmitoylation for Aβ deposition in the brain, we generated transgenic mice co-expressing human wild-type or S-palmitoylation-deficient APH1aL and nicastrin in neurons in the forebrain. We found that lack of S-palmitoylation did not impair the ability of APH1aL and nicastrin to form enzymatically active protein complexes with endogenous presenilin 1 and PEN2, or affect the localization of γ-secretase subunits in dendrites and axons of cortical neurons. When we crossed these mice with 85Dbo transgenic mice, which co-express familial Alzheimer's disease-causing amyloid precursor protein and presenilin 1 variants, we found that co-expression of wild-type or mutant APH1aL and nicastrin led to marked stabilization of transgenic presenilin 1 in the brains of double transgenic mice. Interestingly, we observed a moderate, but significant, reduction in amyloid deposits in the forebrain of mice expressing S-palmitoylation-deficient γ-secretase subunits as compared with mice overexpressing wild-type subunits, as well as a reduction in the levels of insoluble Aβ40-42. These results indicate that γ-secretase S-palmitoylation modulates Aβ deposition in the brain. PMID:21123562

  11. Reduced Alzheimer's disease ß-amyloid deposition in transgenic mice expressing S-palmitoylation-deficient APH1aL and nicastrin.

    PubMed

    Meckler, Xavier; Roseman, Jelita; Das, Pritam; Cheng, Haipeng; Pei, Susan; Keat, Marcia; Kassarjian, Breanne; Golde, Todd E; Parent, Angèle T; Thinakaran, Gopal

    2010-12-01

    Sequential cleavage of amyloid precursor protein by β- and γ-secretases generates β-amyloid peptides (Aβ), which accumulate in the brains of patients with Alzheimer's disease. We recently identified S-palmitoylation of two γ-secretase subunits, APH1 and nicastrin. S-Palmitoylation is an essential posttranslational modification for the proper trafficking and function of many neuronal proteins. In cultured cell lines, lack of S-palmitoylation causes instability of nascent APH1 and nicastrin but does not affect γ-secretase processing of amyloid precursor protein. To determine the importance of γ-secretase S-palmitoylation for Aβ deposition in the brain, we generated transgenic mice coexpressing human wild-type or S-palmitoylation-deficient APH1aL and nicastrin in neurons in the forebrain. We found that lack of S-palmitoylation did not impair the ability of APH1aL and nicastrin to form enzymatically active protein complexes with endogenous presenilin 1 and PEN2 or affect the localization of γ-secretase subunits in dendrites and axons of cortical neurons. When we crossed these mice with 85Dbo transgenic mice, which coexpress familial Alzheimer's disease-causing amyloid precursor protein and presenilin 1 variants, we found that coexpression of wild-type or mutant APH1aL and nicastrin led to marked stabilization of transgenic presenilin 1 in the brains of double-transgenic mice. Interestingly, we observed a moderate, but significant, reduction in amyloid deposits in the forebrain of mice expressing S-palmitoylation-deficient γ-secretase subunits compared with mice overexpressing wild-type subunits, as well as a reduction in the levels of insoluble Aβ(40-42). These results indicate that γ-secretase S-palmitoylation modulates Aβ deposition in the brain. PMID:21123562

  12. Hydrolase-catalyzed biotransformations in deep eutectic solvents.

    PubMed

    Gorke, Johnathan T; Srienc, Friedrich; Kazlauskas, Romas J

    2008-03-14

    Hydrolases show good catalytic activity in deep eutectic solvents, despite the presence of urea, which can denature enzymes, or alcohols, which can interfere with hydrolase-catalyzed reactions. PMID:18309428

  13. Twisting of glycosidic bonds by hydrolases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Patterns of scissile bond twisting have been found in crystal structures of glycoside hydrolases (GHs) that are complexed with substrates and inhibitors. To estimate the increased potential energy in the substrates that results from this twisting, we have plotted torsion angles for the scissile bond...

  14. Structure and function of polyglycine hydrolases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyglycine hydrolases (PGH)s are secreted fungal endoproteases that cleave polyglycine linkers of targeted plant defense chitinases. Unlike typical endoproteases that cleave a specific peptide bond, these 640 amino acid glycoproteins selectively cleave one of multiple peptide bonds within polyglyci...

  15. PLANT FATTY ACID (ETHANOL) AMIDE HYDROLASES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fatty acid amide hydrolase (FAAH) plays a central role in modulating endogenous N-acylethanolamine (NAE) levels in vertebrates, and, in part, constitutes an “endocannabinoid” signaling pathway that regulates diverse physiological and behavioral processes in animals. Recently, an Arabidopsis FAAH hom...

  16. Purification, crystallization and preliminary X-ray analysis of aminoglycoside-2′′-phosphotransferase-Ic [APH(2′′)-Ic] from Enterococcus gallinarum

    SciTech Connect

    Byrnes, Laura J.; Badarau, Adriana; Vakulenko, Sergei B.; Smith, Clyde A.

    2008-02-01

    APH(2′′)-Ic is an enzyme that is responsible for high-level gentamicin resistance in E. gallinarum isolates. Crystals of the wild-type enzyme and three mutants have been prepared and a complete X-ray diffraction data set was collected to 2.15 Å resolution from an F108L crystal. Bacterial resistance to aminoglycoside antibiotics is primarily the result of deactivation of the drugs. Three families of enzymes are responsible for this activity, with one such family being the aminoglycoside phosphotransferases (APHs). The gene encoding one of these enzymes, aminoglycoside-2′′-phosphotransferase-Ic [APH(2′′)-Ic] from Enterococcus gallinarum, has been cloned and the wild-type protein (comprising 308 amino-acid residues) and three mutants that showed elevated minimum inhibitory concentrations towards gentamicin (F108L, H258L and a double mutant F108L/H258L) were expressed in Escherichia coli and subsequently purified. All APH(2′′)-Ic variants were crystallized in the presence of 14–20%(w/v) PEG 4000, 0.25 M MgCl{sub 2}, 0.1 M Tris–HCl pH 8.5 and 1 mM Mg{sub 2}GTP. The crystals belong to the monoclinic space group C2, with one molecule in the asymmetric unit. The approximate unit-cell parameters are a = 82.4, b = 54.2, c = 77.0 Å, β = 108.8°. X-ray diffraction data were collected to approximately 2.15 Å resolution from an F108L crystal at beamline BL9-2 at SSRL, Stanford, California, USA.

  17. Bacterial Cyanuric Acid Hydrolase for Water Treatment.

    PubMed

    Yeom, Sujin; Mutlu, Baris R; Aksan, Alptekin; Wackett, Lawrence P

    2015-10-01

    Di- and trichloroisocyanuric acids are widely used as water disinfection agents, but cyanuric acid accumulates with repeated additions and must be removed to maintain free hypochlorite for disinfection. This study describes the development of methods for using a cyanuric acid-degrading enzyme contained within nonliving cells that were encapsulated within a porous silica matrix. Initially, three different bacterial cyanuric acid hydrolases were compared: TrzD from Acidovorax citrulli strain 12227, AtzD from Pseudomonas sp. strain ADP, and CAH from Moorella thermoacetica ATCC 39073. Each enzyme was expressed recombinantly in Escherichia coli and tested for cyanuric acid hydrolase activity using freely suspended or encapsulated cell formats. Cyanuric acid hydrolase activities differed by only a 2-fold range when comparing across the different enzymes with a given format. A practical water filtration system is most likely to be used with nonviable cells, and all cells were rendered nonviable by heat treatment at 70°C for 1 h. Only the CAH enzyme from the thermophile M. thermoacetica retained significant activity under those conditions, and so it was tested in a flowthrough system simulating a bioreactive pool filter. Starting with a cyanuric acid concentration of 10,000 μM, more than 70% of the cyanuric acid was degraded in 24 h, it was completely removed in 72 h, and a respike of 10,000 μM cyanuric acid a week later showed identical biodegradation kinetics. An experiment conducted with water obtained from municipal swimming pools showed the efficacy of the process, although cyanuric acid degradation rates decreased by 50% in the presence of 4.5 ppm hypochlorite. In total, these experiments demonstrated significant robustness of cyanuric acid hydrolase and the silica bead materials in remediation. PMID:26187963

  18. Bacterial Cyanuric Acid Hydrolase for Water Treatment

    PubMed Central

    Yeom, Sujin; Mutlu, Baris R.; Aksan, Alptekin

    2015-01-01

    Di- and trichloroisocyanuric acids are widely used as water disinfection agents, but cyanuric acid accumulates with repeated additions and must be removed to maintain free hypochlorite for disinfection. This study describes the development of methods for using a cyanuric acid-degrading enzyme contained within nonliving cells that were encapsulated within a porous silica matrix. Initially, three different bacterial cyanuric acid hydrolases were compared: TrzD from Acidovorax citrulli strain 12227, AtzD from Pseudomonas sp. strain ADP, and CAH from Moorella thermoacetica ATCC 39073. Each enzyme was expressed recombinantly in Escherichia coli and tested for cyanuric acid hydrolase activity using freely suspended or encapsulated cell formats. Cyanuric acid hydrolase activities differed by only a 2-fold range when comparing across the different enzymes with a given format. A practical water filtration system is most likely to be used with nonviable cells, and all cells were rendered nonviable by heat treatment at 70°C for 1 h. Only the CAH enzyme from the thermophile M. thermoacetica retained significant activity under those conditions, and so it was tested in a flowthrough system simulating a bioreactive pool filter. Starting with a cyanuric acid concentration of 10,000 μM, more than 70% of the cyanuric acid was degraded in 24 h, it was completely removed in 72 h, and a respike of 10,000 μM cyanuric acid a week later showed identical biodegradation kinetics. An experiment conducted with water obtained from municipal swimming pools showed the efficacy of the process, although cyanuric acid degradation rates decreased by 50% in the presence of 4.5 ppm hypochlorite. In total, these experiments demonstrated significant robustness of cyanuric acid hydrolase and the silica bead materials in remediation. PMID:26187963

  19. Structures and Mechanisms of Nudix Hydrolases

    SciTech Connect

    Mildvan,A.; Xia, Z.; Azurmendi, H.; saraswat, V.; Legler, P.; Massiah, M.; Gabelli, S.; Bianchet, M.; Kang, L.; Amzel, L.

    2005-01-01

    Nudix hydrolases are a family of proteins that contain the characteristic sequence GX(5)EX(7)REUXEEXG(I/L/V), the Nudix box. They catalyze the hydrolysis of a variety of nucleoside diphosphate derivatives such as ADP-ribose, Ap(n)A (3 hydrolases from several species, ranging from bacteria to humans, have been characterized, including, in some cases, the determination of their three-dimensional structures. The product of the Rv1700 gene of M. tuberculosis is a Nudix hydrolase specific for ADP-ribose (ADPR). We have determined the crystal structures of MT-ADPRase alone, and in complex with substrate, with substrate and the nonactivating metal ion Gd(3+), and in complex with a nonhydrolyzable ADPR analog and the activating metal ion Mn(2+). These structures, refined with data extending to resolutions between 2.0 and 2.3 A, showed that there are sequence differences in binding site residues between MT-ADPRase and a human homolog that may be exploited for antituberculosis drug development.

  20. Structure of the bifunctional aminoglycoside-resistance enzyme AAC(6′)-Ie-APH(2′′)-Ia revealed by crystallographic and small-angle X-ray scattering analysis

    PubMed Central

    Smith, Clyde A.; Toth, Marta; Weiss, Thomas M.; Frase, Hilary; Vakulenko, Sergei B.

    2014-01-01

    Broad-spectrum resistance to aminoglycoside antibiotics in clinically important Gram-positive staphylococcal and entero­coccal pathogens is primarily conferred by the bifunctional enzyme AAC(6′)-Ie-APH(2′′)-Ia. This enzyme possesses an N-terminal coenzyme A-dependent acetyltransferase domain [AAC(6′)-Ie] and a C-terminal GTP-dependent phosphotransferase domain [APH(2′′)-Ia], and together they produce resistance to almost all known aminoglycosides in clinical use. Despite considerable effort over the last two or more decades, structural details of AAC(6′)-Ie-APH(2′′)-Ia have remained elusive. In a recent breakthrough, the structure of the isolated C-terminal APH(2′′)-Ia enzyme was determined as the binary Mg2GDP complex. Here, the high-resolution structure of the N-terminal AAC(6′)-Ie enzyme is reported as a ternary kanamycin/coenzyme A abortive complex. The structure of the full-length bifunctional enzyme has subsequently been elucidated based upon small-angle X-ray scattering data using the two crystallographic models. The AAC(6′)-Ie enzyme is joined to APH(2′′)-Ia by a short, predominantly rigid linker at the N-terminal end of a long α-helix. This α-helix is in turn intrinsically associated with the N-terminus of APH(2′′)-Ia. This structural arrangement supports earlier observations that the presence of the intact α-helix is essential to the activity of both functionalities of the full-length AAC(6′)-Ie-APH(2′′)-Ia enzyme. PMID:25286858

  1. γ-Secretase Modulators and APH1 Isoforms Modulate γ-Secretase Cleavage but Not Position of ε-Cleavage of the Amyloid Precursor Protein (APP)

    PubMed Central

    Lessard, Christian B.; Cottrell, Barbara A.; Maruyama, Hiroko; Suresh, Suraj; Golde, Todd E.; Koo, Edward H.

    2015-01-01

    The relative increase in Aβ42 peptides from familial Alzheimer disease (FAD) linked APP and PSEN mutations can be related to changes in both ε-cleavage site utilization and subsequent step-wise cleavage. Cleavage at the ε-site releases the amyloid precursor protein (APP) intracellular domain (AICD), and perturbations in the position of ε-cleavage are closely associated with changes in the profile of amyloid β-protein (Aβ) species that are produced and secreted. The mechanisms by which γ-secretase modulators (GSMs) or FAD mutations affect the various γ-secretase cleavages to alter the generation of Aβ peptides have not been fully elucidated. Recent studies suggested that GSMs do not modulate ε-cleavage of APP, but the data were derived principally from recombinant truncated epitope tagged APP substrate. Here, using full length APP from transfected cells, we investigated whether GSMs modify the ε-cleavage of APP under more native conditions. Our results confirmed the previous findings that ε-cleavage is insensitive to GSMs. In addition, fenofibrate, an inverse GSM (iGSM), did not alter the position or kinetics of ε-cleavage position in vitro. APH1A and APH1B, a subunit of the γ-secretase complex, also modulated Aβ42/Aβ40 ratio without any alterations in ε-cleavage, a result in contrast to what has been observed with PS1 and APP FAD mutations. Consequently, GSMs and APH1 appear to modulate γ-secretase activity and Aβ42 generation by altering processivity but not ε-cleavage site utilization. PMID:26678856

  2. Thermostable Cyanuric Acid Hydrolase from Moorella thermoacetica ATCC 39073▿

    PubMed Central

    Li, Qingyan; Seffernick, Jennifer L.; Sadowsky, Michael J.; Wackett, Lawrence P.

    2009-01-01

    Cyanuric acid, a metabolic intermediate in the degradation of many s-triazine compounds, is further metabolized by cyanuric acid hydrolase. Cyanuric acid also accumulates in swimming pools due to the breakdown of the sanitizing agents di- and trichloroisocyanuric acid. Structurally stable cyanuric acid hydrolases are being considered for usage in pool water remediation. In this study, cyanuric acid hydrolase from the thermophile Moorella thermoacetica ATCC 39073 was cloned, expressed in Escherichia coli, and purified to homogeneity. The recombinant enzyme was found to have a broader temperature range and greater stability, at both elevated and low temperatures, than previously described cyanuric acid hydrolases. The enzyme had a narrow substrate specificity, acting only on cyanuric acid and N-methylisocyanuric acid. The M. thermoacetica enzyme did not require metals or other discernible cofactors for activity. Cyanuric acid hydrolase from M. thermoacetica is the most promising enzyme to use for cyanuric acid remediation applications. PMID:19767460

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

    PubMed

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

    2016-08-01

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

  4. Aspergillus niger DLFCC-90 rhamnoside hydrolase, a new type of flavonoid glycoside hydrolase.

    PubMed

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

    2012-07-01

    A novel rutin-α-L-rhamnosidase hydrolyzing α-L-rhamnoside of rutin, naringin, and hesperidin was purified and characterized from Aspergillus niger DLFCC-90, and the gene encoding this enzyme, which is highly homologous to the α-amylase gene, was cloned and expressed in Pichia pastoris GS115. The novel enzyme was classified in glycoside-hydrolase (GH) family 13. PMID:22544243

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

    PubMed Central

    Oguro, Ami; Imaoka, Susumu

    2012-01-01

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

  6. Bacterial Expression and HTS Assessment of Soluble Epoxide Hydrolase Phosphatase.

    PubMed

    Klingler, Franca-Maria; Wolf, Markus; Wittmann, Sandra; Gribbon, Philip; Proschak, Ewgenij

    2016-08-01

    Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that possesses an epoxide hydrolase and lipid phosphatase activity (sEH-P) at two distinct catalytic domains. While the physiological role of the epoxide hydrolase domain is well understood, the consequences of the phosphatase activity remain unclear. Herein we describe the bacterial expression of the recombinant N-terminal domain of sEH-P and the development of a high-throughput screening protocol using a sensitive and commercially available substrate fluorescein diphosphate. The usability of the assay system was demonstrated and novel inhibitors of sEH-P were identified. PMID:27009944

  7. Miniaturization of hydrolase assays in thermocyclers.

    PubMed

    Lucena, Severino A; Moraes, Caroline S; Costa, Samara G; de Souza, Wanderley; Azambuja, Patrícia; Garcia, Eloi S; Genta, Fernando A

    2013-03-01

    We adapted the protocols of reducing sugar measurements with dinitrosalicylic acid and bicinchoninic acid for thermocyclers and their use in enzymatic assays for hydrolases such as amylase and β-1,3-glucanase. The use of thermocyclers for these enzymatic assays resulted in a 10 times reduction in the amount of reagent and volume of the sample needed when compared with conventional microplate protocols. We standardized absorbance readings from the polymerase chain reaction plates, which allowed us to make direct readings of the techniques above, and a β-glycosidase assay was also established under the same conditions. Standardization of the enzymatic reaction in thermocyclers resulted in less time-consuming temperature calibrations and without loss of volume through leakage or evaporation from the microplate. Kinetic parameters were successfully obtained, and the use of the thermocycler allowed the measurement of enzymatic activities in biological samples from the field with a limited amount of protein. PMID:23123426

  8. Orally Bioavailable Potent Soluble Epoxide Hydrolase Inhibitors

    PubMed Central

    Hwang, Sung Hee; Tsai, Hsing-Ju; Liu, Jun-Yan; Morisseau, Christophe; Hammock, Bruce D.

    2008-01-01

    A series of N,N′-disubstituted ureas having a conformationally restricted cis- or trans-1,4-cyclohexane α to the urea were prepared and tested as soluble epoxide hydrolase (sEH) inhibitors. This series of compounds showed low nanomolar to picomolar activities against recombinant human sEH. Both isomers showed similar potencies, but the trans isomers were more metabolically stable in human hepatic microsomes. Furthermore, these new potent inhibitors show a greater metabolic stability in vivo than previously described sEH inhibitors. We demonstrated that trans-4-[4-(3-adamantan-1-ylureido)cyclohexyloxy]benzoic acid 13g (t-AUCB, IC50 = 1.3 ± 0.05 nM) had excellent oral bioavailability (98%, n = 2) and blood area under the curve in dogs and was effective in vivo to treat hypotension in lipopolysaccharide challenged murine models. PMID:17616115

  9. A simplified electrostatic model for hydrolase catalysis.

    PubMed

    Pessoa Filho, Pedro de Alcantara; Prausnitz, John M

    2015-07-01

    Toward the development of an electrostatic model for enzyme catalysis, the active site of the enzyme is represented by a cavity whose surface (and beyond) is populated by electric charges as determined by pH and the enzyme's structure. The electric field in the cavity is obtained from electrostatics and a suitable computer program. The key chemical bond in the substrate, at its ends, has partial charges with opposite signs determined from published force-field parameters. The electric field attracts one end of the bond and repels the other, causing bond tension. If that tension exceeds the attractive force between the atoms, the bond breaks; the enzyme is then a successful catalyst. To illustrate this very simple model, based on numerous assumptions, some results are presented for three hydrolases: hen-egg white lysozyme, bovine trypsin and bovine ribonuclease. Attention is given to the effect of pH. PMID:25881958

  10. The pharmacological landscape and therapeutic potential of serine hydrolases.

    PubMed

    Bachovchin, Daniel A; Cravatt, Benjamin F

    2012-01-01

    Serine hydrolases perform crucial roles in many biological processes, and several of these enzymes are targets of approved drugs for indications such as type 2 diabetes, Alzheimer's disease and infectious diseases. Despite this, most of the human serine hydrolases (of which there are more than 200) remain poorly characterized with respect to their physiological substrates and functions, and the vast majority lack selective, in vivo-active inhibitors. Here, we review the current state of pharmacology for mammalian serine hydrolases, including marketed drugs, compounds that are under clinical investigation and selective inhibitors emerging from academic probe development efforts. We also highlight recent methodological advances that have accelerated the rate of inhibitor discovery and optimization for serine hydrolases, which we anticipate will aid in their biological characterization and, in some cases, therapeutic validation. PMID:22212679

  11. The Pharmacological Landscape and Therapeutic Potential of Serine Hydrolases

    PubMed Central

    Bachovchin, Daniel A.; Cravatt, Benjamin F.

    2013-01-01

    Serine hydrolases play critical roles in many biological processes, and several are targets of approved drugs for indications such as type 2 diabetes, Alzheimer’s disease, and infectious disease. Despite this, most of the 200+ human serine hydrolases remain poorly characterized with respect to their physiological substrates and functions, and the vast majority lack selective, in vivo-active inhibitors. Here, we review the current state of pharmacology for mammalian serine hydrolases, including marketed drugs, compounds under clinical investigation, and selective inhibitors emerging from academic probe development efforts. We also highlight recent methodological advances that have accelerated the rate of inhibitor discovery and optimization for serine hydrolases, which we anticipate will aid in their biological characterization and, in some cases, therapeutic validation. PMID:22212679

  12. Deletion of the γ-secretase subunits Aph1B/C impairs memory and worsens the deficits of knock-in mice modeling the Alzheimer-like familial Danish dementia

    PubMed Central

    Biundo, Fabrizio; Ishiwari, Keita; Del Prete, Dolores; D'Adamio, Luciano

    2016-01-01

    Mutations in BRI2/ITM2b genes cause Familial British and Danish Dementias (FBD and FDD), which are pathogenically similar to Familial Alzheimer Disease (FAD). BRI2 inhibits processing of Amyloid precursor protein (APP), a protein involved in FAD pathogenesis. Accumulation of a carboxyl-terminal APP metabolite –β-CTF- causes memory deficits in a knock-in mouse model of FDD, called FDDKI. We have investigated further the pathogenic function of β-CTF studying the effect of Aph1B/C deletion on FDDKI mice. This strategy is based on the evidence that deletion of Aph1B/C proteins, which are components of the γ-secretase that cleaves β-CTF, results in stabilization of β-CTF and a reduction of Aβ. We found that both the FDD mutation and the Aph1B/C deficiency mildly interfered with spatial long term memory, spatial working/short-term memory and long-term contextual fear memory. In addition, the Aph1BC deficiency induced deficits in long-term cued fear memory. Moreover, the two mutations have additive adverse effects as they compromise the accuracy of spatial long-term memory and induce spatial memory retention deficits in young mice. Overall, the data are consistent with a role for β-CTF in the genesis of memory deficits. PMID:26942869

  13. Quantum reactive scattering in three dimensions using hyperspherical (APH) coordinates. IV. Discrete variable representation (DVR) basis functions and the analysis of accurate results for F+H2

    NASA Astrophysics Data System (ADS)

    Bačić, Z.; Kress, J. D.; Parker, G. A.; Pack, R. T.

    1990-02-01

    Accurate 3D coupled channel calculations for total angular momentum J=0 for the reaction F+H2→HF+H using a realistic potential energy surface are analyzed. The reactive scattering is formulated using the hyperspherical (APH) coordinates of Pack and Parker. The adiabatic basis functions are generated quite efficiently using the discrete variable representation method. Reaction probabilities for relative collision energies of up to 17.4 kcal/mol are presented. To aid in the interpretation of the resonances and quantum structure observed in the calculated reaction probabilities, we analyze the phases of the S matrix transition elements, Argand diagrams, time delays and eigenlifetimes of the collision lifetime matrix. Collinear (1D) and reduced dimensional 3D bending corrected rotating linear model (BCRLM) calculations are presented and compared with the accurate 3D calculations.

  14. Structural and functional attributes of malaria parasite diadenosine tetraphosphate hydrolase.

    PubMed

    Sharma, Arvind; Yogavel, Manickam; Sharma, Amit

    2016-01-01

    Malaria symptoms are driven by periodic multiplication cycles of Plasmodium parasites in human red blood corpuscles (RBCs). Malaria infection still accounts for ~600,000 annual deaths, and hence discovery of both new drug targets and drugs remains vital. In the present study, we have investigated the malaria parasite enzyme diadenosine tetraphosphate (Ap4A) hydrolase that regulates levels of signalling molecules like Ap4A by hydrolyzing them to ATP and AMP. We have tracked the spatial distribution of parasitic Ap4A hydrolase in infected RBCs, and reveal its unusual localization on the infected RBC membrane in subpopulation of infected cells. Interestingly, enzyme activity assays reveal an interaction between Ap4A hydrolase and the parasite growth inhibitor suramin. We also present a high resolution crystal structure of Ap4A hydrolase in apo- and sulphate- bound state, where the sulphate resides in the enzyme active site by mimicking the phosphate of substrates like Ap4A. The unexpected infected erythrocyte localization of the parasitic Ap4A hydrolase hints at a possible role of this enzyme in purinerigic signaling. In addition, atomic structure of Ap4A hydrolase provides insights for selective drug targeting. PMID:26829485

  15. Structural and functional attributes of malaria parasite diadenosine tetraphosphate hydrolase

    PubMed Central

    Sharma, Arvind; Yogavel, Manickam; Sharma, Amit

    2016-01-01

    Malaria symptoms are driven by periodic multiplication cycles of Plasmodium parasites in human red blood corpuscles (RBCs). Malaria infection still accounts for ~600,000 annual deaths, and hence discovery of both new drug targets and drugs remains vital. In the present study, we have investigated the malaria parasite enzyme diadenosine tetraphosphate (Ap4A) hydrolase that regulates levels of signalling molecules like Ap4A by hydrolyzing them to ATP and AMP. We have tracked the spatial distribution of parasitic Ap4A hydrolase in infected RBCs, and reveal its unusual localization on the infected RBC membrane in subpopulation of infected cells. Interestingly, enzyme activity assays reveal an interaction between Ap4A hydrolase and the parasite growth inhibitor suramin. We also present a high resolution crystal structure of Ap4A hydrolase in apo- and sulphate- bound state, where the sulphate resides in the enzyme active site by mimicking the phosphate of substrates like Ap4A. The unexpected infected erythrocyte localization of the parasitic Ap4A hydrolase hints at a possible role of this enzyme in purinerigic signaling. In addition, atomic structure of Ap4A hydrolase provides insights for selective drug targeting. PMID:26829485

  16. Investigation of the mechanism of phosphonoacetaldehyde hydrolase

    SciTech Connect

    Hepburn, T.W.; Olsen, D.B.; Dunaway-Mariano, D.; Mariano, P.S.

    1986-05-01

    The authors are presently studying enzymes which catalyze the formation and cleavage of carbon phosphorous bonds. In 1970 LaNauze et. al. reported the isolation of one enzyme of interest - phosphonoacetaldehyde hydrolase from a mutant of Bacillus cereus. This enzyme catalyzes the hydrolysis of phosphonoaldehyde to acetaldehyde and inorganic phosphate. They have isolated phosphonatase from wild type B. cereus (grown on 2-aminoethylphosphonate as the P/sub i/ source) and have used /sup 1/H-NMR and /sup 31/P-NMR techniques to determine the products of the enzyme reaction as phosphate and acetaldehyde. The mechanism of the enzyme could involve the formation of a Schiff base between phosphonoacetaldehyde and lysine or it might only require Mg/sup + +/, an essential cofactor for activity. To distinguish between these possibilities they have begun to look at the Schiff base formation in more detail. NaBH/sub 4/ was found to inactivate the enzyme in the presence of substrate but not in its absence. This is consistent with results obtained for the enzyme isolated from the mutant bacteria. In addition treatment of the wild type enzyme with tritiated NaBH/sub 4/ resulted in significant incorporation of radiolabel into the protein as compared to the control. These results tentatively suggest that hydrolysis proceeds via a covalent imine intermediate.

  17. Human valacyclovir hydrolase/biphenyl hydrolase-like protein is a highly efficient homocysteine thiolactonase.

    PubMed

    Marsillach, Judit; Suzuki, Stephanie M; Richter, Rebecca J; McDonald, Matthew G; Rademacher, Peter M; MacCoss, Michael J; Hsieh, Edward J; Rettie, Allan E; Furlong, Clement E

    2014-01-01

    Homocysteinylation of lysine residues by homocysteine thiolactone (HCTL), a reactive homocysteine metabolite, results in protein aggregation and malfunction, and is a well-known risk factor for cardiovascular, autoimmune and neurological diseases. Human plasma paraoxonase-1 (PON1) and bleomycin hydrolase (Blmh) have been reported as the physiological HCTL detoxifying enzymes. However, the catalytic efficiency of HCTL hydrolysis by Blmh is low and not saturated at 20 mM HCTL. The catalytic efficiency of PON1 for HCTL hydrolysis is 100-fold lower than that of Blmh. A homocysteine thiolactonase (HCTLase) was purified from human liver and identified by mass spectrometry (MS) as the previously described human biphenyl hydrolase-like protein (BPHL). To further characterize this newly described HCTLase activity, BPHL was expressed in Escherichia coli and purified. The sequence of the recombinant BPHL (rBPHL) and hydrolytic products of the substrates HCTL and valacyclovir were verified by MS. We found that the catalytic efficiency (kcat/Km) of rBPHL for HCTL hydrolysis was 7.7 × 10(4) M(-1)s(-1), orders of magnitude higher than that of PON1 or Blmh, indicating a more significant physiological role for BPHL in detoxifying HCTL. PMID:25333274

  18. Human Valacyclovir Hydrolase/Biphenyl Hydrolase-Like Protein Is a Highly Efficient Homocysteine Thiolactonase

    PubMed Central

    McDonald, Matthew G.; Rademacher, Peter M.; MacCoss, Michael J.; Hsieh, Edward J.; Rettie, Allan E.; Furlong, Clement E.

    2014-01-01

    Homocysteinylation of lysine residues by homocysteine thiolactone (HCTL), a reactive homocysteine metabolite, results in protein aggregation and malfunction, and is a well-known risk factor for cardiovascular, autoimmune and neurological diseases. Human plasma paraoxonase-1 (PON1) and bleomycin hydrolase (Blmh) have been reported as the physiological HCTL detoxifying enzymes. However, the catalytic efficiency of HCTL hydrolysis by Blmh is low and not saturated at 20 mM HCTL. The catalytic efficiency of PON1 for HCTL hydrolysis is 100-fold lower than that of Blmh. A homocysteine thiolactonase (HCTLase) was purified from human liver and identified by mass spectrometry (MS) as the previously described human biphenyl hydrolase-like protein (BPHL). To further characterize this newly described HCTLase activity, BPHL was expressed in Escherichia coli and purified. The sequence of the recombinant BPHL (rBPHL) and hydrolytic products of the substrates HCTL and valacyclovir were verified by MS. We found that the catalytic efficiency (kcat/Km) of rBPHL for HCTL hydrolysis was 7.7 × 104 M−1s−1, orders of magnitude higher than that of PON1 or Blmh, indicating a more significant physiological role for BPHL in detoxifying HCTL. PMID:25333274

  19. TECHNOLOGY DEMONSTRATION UNDERWATER HYDROLASING PHASE 0 & 1 & 2 TECHNICAL REPORT

    SciTech Connect

    CHRONISTER, G.B.

    2005-06-08

    From September 10 through December 17th, 2003, S.A.Robotics executed Phases 0, I, and II of the Technology Demonstration - Underwater Hydrolasing. Phase 0 was performed at the S.A.Robotics facility in Loveland, Colorado, while Phases I and II were performed at the Hanford K-Basin East Site. The purpose of the demonstrations was to show (1) underwater hydrolasing is a feasible method of removing contaminated concrete underwater to a required depth, (2) the hydrolasing head could be controlled during operation, (3) the depth of contamination in the concrete structure could be accurately measured, and (4) a characterization of the waste stream during hydrolasing activities could be recorded. Video monitoring was also used during all demonstrations. All phases of the demonstration were completed and deemed a success by both the observers and the demonstration team. Single and multiple passes were made using variable cutting rates, different stand-off distances were tested, and stationary cuts were executed. Hot and cold hyrdolasing was performed with radiological and depth scans of the affected surfaces. Specially designed equipment was installed and operated within the contaminated environment of 100-K East Basin. Separate results are documented below by phase. The Phase II radiological demonstration was performed to determine the feasibility of underwater hydrolasing technology for decontamination of the DOE spent fuel basins at Hanford 100-K area. This project demonstration was conducted at 105 KE Basin with the expectation that, once proven, this technology can be implemented at Hanford and other DOE sites.

  20. Identification and characterization of biomarkers of organophosphorus exposures in humans.

    PubMed

    Kim, Jerry H; Stevens, Richard C; MacCoss, Michael J; Goodlett, David R; Scherl, Alex; Richter, Rebecca J; Suzuki, Stephanie M; Furlong, Clement E

    2010-01-01

    Over 1 billion pounds of organophosphorus (OP) chemicals are manufactured worldwide each year, including 70 million pounds of pesticides sprayed in the US. Current methods to monitor environmental and occupational exposures to OPs such as chlorpyrifos (CPS) have limitations, including low specificity and sensitivity, and short time windows for detection. Biomarkers for the OP tricresyl phosphate (TCP), which can contaminate bleed air from jet engines and cause an occupational exposure of commercial airline pilots, crewmembers and passengers, have not been identified. The aim of our work has been to identify, purify, and characterize new biomarkers of OP exposure. Butyrylcholinesterase (BChE) inhibition has been a standard for monitoring OP exposure. By identifying and characterizing molecular biomarkers with longer half-lives, we should be able to clinically detect TCP and OP insecticide exposure after longer durations of time than are currently possible. Acylpeptide hydrolase (APH) is a red blood cell (RBC) cytosolic serine proteinase that removes N-acetylated amino acids from peptides and cleaves oxidized proteins. Due to its properties, it is an excellent candidate for a biomarker of exposure. We have been able to purify APH and detect inhibition by both CPS and metabolites of TCP. The 120-day lifetime of the RBC offers a much longer window for detecting exposure. The OP-modified serine conjugate in the active site tryptic peptide has been characterized by mass spectrometry. This research uses functional proteomics and enzyme activities to identify and characterize useful biomarkers of neurotoxic environmental and occupational OP exposures. PMID:20221871

  1. Identification and Characterization of Biomarkers of Organophosphorus Exposures in Humans

    PubMed Central

    Kim, Jerry H.; Stevens, Richard C.; MacCoss, Michael J.; Goodlett, David R.; Scherl, Alex; Richter, Rebecca J.; Suzuki, Stephanie M.; Furlong, Clement E.

    2010-01-01

    Over 1 billion pounds of organophosphorus (OP) chemicals are manufactured worldwide each year, including 70 million pounds of pesticides sprayed in the US. Current methods to monitor environmental and occupational exposures to OPs such as chlorpyrifos (CPS) have limitations, including low specificity and sensitivity, and short time windows for detection. Biomarkers for the OP tricresyl phosphate (TCP), which can contaminate bleed air from jet engines and cause an occupational exposure of commercial airline pilots, crewmembers and passengers, have not been identified. The aim of our work has been to identify, purify, and characterize new biomarkers of OP exposure. Butyrylcholinesterase (BChE) inhibition has been a standard for monitoring OP exposure. By identifying and characterizing molecular biomarkers with longer half-lives, we should be able to clinically detect TCP and OP insecticide exposure after longer durations of time than are currently possible. Acylpeptide hydrolase (APH) is a red blood cell (RBC) cytosolic serine proteinase that removes N-acetylated amino acids from peptides and cleaves oxidized proteins. Due to its properties, it is an excellent candidate for a biomarker of exposure. We have been able to purify APH and detect inhibition by both CPS and metabolites of TCP. The 120-day lifetime of the RBC offers a much longer window for detecting exposure. The OP-modified serine conjugate in the active site tryptic peptide has been characterized by mass spectrometry. This research uses functional proteomics and enzyme activities to identify and characterize useful biomarkers of neurotoxic environmental and occupational OP exposures. PMID:20221871

  2. Chronic dietary exposure to chlorpyrifos causes behavioral impairments, low activity of brain membrane-bound acetylcholinesterase, and increased brain acetylcholinesterase-R mRNA.

    PubMed

    López-Granero, Caridad; Cardona, Diana; Giménez, Estela; Lozano, Rafael; Barril, José; Sánchez-Santed, Fernando; Cañadas, Fernando

    2013-06-01

    Chlorpyrifos (CPF) is an organophosphate (OP) insecticide that is metabolically activated to the highly toxic chlorpyrifos oxon. Dietary exposure is the main route of intoxication for non-occupational exposures. However, only limited behavioral effects of chronic dietary exposure have been investigated. Therefore, male Wistar rats were fed a dose of 5mg/kg/day of CPF for thirty-one weeks. Animals were evaluated in spatial learning and impulsivity tasks after 21 weeks of CPF dietary exposure and one week after exposure ended, respectively. In addition, the degree of inhibition of brain acetylcholinesterase (AChE) was evaluated for both the soluble and particulate forms of the enzyme, as well as AChE gene expression. Also, brain acylpeptide hydrolase (APH) was investigated as an alternative target for OP-mediated effects. All variables were evaluated at various time points in response to CPF diet and after exposure ended. Results from behavioral procedures suggest cognitive and emotional disorders. Moreover, low levels of activity representing membrane-bound oligomeric forms (tetramers) were also observed. In addition, increased brain AChE-R mRNA levels were detected after four weeks of CPF dietary exposure. However, no changes in levels of brain APH were observed among groups. In conclusion, our data point to a relationship between cognitive impairments and changes in AChE forms, specifically to a high inhibition of the particulate form and a modification of alternative splicing of mRNA during CPF dietary exposure. PMID:23545134

  3. Expression of key hydrolases for soy sauce fermentation in Zygosaccharomyces rouxii.

    PubMed

    Yuzuki, Masanobu; Matsushima, Kenichiro; Koyama, Yasuji

    2015-01-01

    Several key hydrolases in soy sauce fermentation such as proteases, peptidases, and glutaminases are supplied by Aspergillus sojae or Aspergillus oryzae. The genes encoding these hydrolases were successfully expressed in salt-tolerant yeast Zygosaccharomyces rouxii. These transformants are expected to supply extra hydrolases during soy sauce fermentation process. PMID:25073685

  4. Curation of characterized glycoside hydrolases of fungal origin.

    PubMed

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

    2011-01-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  6. Peptidyl-urea based inhibitors of soluble epoxide hydrolases

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed

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

    2006-12-01

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

  8. Curation of characterized glycoside hydrolases of Fungal origin

    PubMed Central

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

    2011-01-01

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

  9. ORGANOPHOSPHORUS HYDROLASE-BASED ASSAY FOR ORGANOPHOSPHATE PESTICIDES

    EPA Science Inventory

    We report a rapid and versatile Organophosphorus hydrolase (OPH)-based method for measurement of organophosphates. This assay is based on a substrate-dependent change in pH at the local vicinity of the enzyme. The pH change is monitored using fluorescein isothiocyanate (FITC), ...

  10. ENGINEERING OF PEPTIDOGLYCAN HYDROLASES FOR CONTROL OF PATHOGENIC BACTERIA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacteriophages are viruses exclusively infecting bacteria and therefore offer suitable tools for their detection and control. At the end of their multiplication cycle, most phages lyse their hosts from within by means of an endolysin (peptidoglycan hydrolase), thereby enabling release of the phage p...

  11. Bacteriophage virion-associated peptidoglycan hydrolases: potential new enzybiotics

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virion-associated peptidoglycan hydrolases (VAPGH) are phage-encoded lytic enzymes that locally degrade the peptidoglycan (PG) of the bacterial cell wall during infection. Their action usually generates a small hole through which the phage tail crosses the cell envelope to inject the phage genetic m...

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

    PubMed

    Lin, Jun; Negga, Rekek; Zeng, Ximin; Smith, Katie

    2014-01-01

    Bile salt hydrolase (BSH), a widely distributed function of the gut microbiota, has a profound impact on host lipid metabolism and energy harvest. Recent studies suggest that BSH inhibitors are promising alternatives to antibiotic growth promoters (AGP) for enhanced animal growth performance and food safety. Using a high-purity BSH from Lactobacillus salivarius strain, we have identified a panel of BSH inhibitors. However, it is still unknown if these inhibitors also effectively inhibit the function of the BSH enzymes from other bacterial species with different sequence and substrate spectrum. In this study, we performed bioinformatics analysis and determined the inhibitory effect of identified BSH inhibitors on a BSH from L. acidophilus. Although the L. acidophilus BSH is phylogenetically distant from the L. salivarius BSH, sequence analysis and structure modeling indicated the two BSH enzymes contain conserved, catalytically important amino residues and domain. His-tagged recombinant BSH from L. acidophilus was further purified and used to determine inhibitory effect of specific compounds. Previously identified BSH inhibitors also exhibited potent inhibitory effects on the L. acidophilus BSH. In conclusion, this study demonstrated that the BSH from L. salivarius is an ideal candidate for screening BSH inhibitors, the promising alternatives to AGP for enhanced feed efficiency, growth performance and profitability of food animals. PMID:25526498

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

    PubMed

    Hiraishi, Tomohiro

    2016-02-01

    Thermally synthesized poly(aspartic acid) (tPAA) is a bio-based, biocompatible, biodegradable, and water-soluble polymer that has a high proportion of β-Asp units and equivalent moles of D- and L-Asp units. Poly(aspartic acid) (PAA) hydrolase-1 and hydrolase-2 are tPAA biodegradation enzymes purified from Gram-negative bacteria. PAA hydrolase-1 selectively cleaves amide bonds between β-Asp units via an endo-type process, whereas PAA hydrolase-2 catalyzes the exo-type hydrolysis of the products of tPAA hydrolysis by PAA hydrolase-1. The novel reactivity of PAA hydrolase-1 makes it a good candidate for a biocatalyst in β-peptide synthesis. This mini-review gives an overview of PAA hydrolases with emphasis on their biochemical and functional properties, in particular, PAA hydrolase-1. Functionally related enzymes, such as poly(R-3-hydroxybutyrate) depolymerases and β-aminopeptidases, are compared to PAA hydrolases. This mini-review also provides findings that offer an insight into the catalytic mechanisms of PAA hydrolase-1 from Pedobacter sp. KP-2. PMID:26695157

  14. Potent Urea and Carbamate Inhibitors of Soluble Epoxide Hydrolases

    NASA Astrophysics Data System (ADS)

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

    1999-08-01

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

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

    PubMed

    Naumoff, D G

    2016-01-01

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

  16. Isolation and characterization of Xenopus soluble epoxide hydrolase.

    PubMed

    Purba, Endang R; Oguro, Ami; Imaoka, Susumu

    2014-07-01

    Soluble epoxide hydrolase (sEH) contributes to cell growth, but the contribution of sEH to embryonic development is not well understood. In this study, Xenopus sEH cDNA was isolated from embryos of Xenopus laevis. The Xenopus sEH was expressed in Escherichia coli and was purified. The epoxide hydrolase and phosphatase activities of purified sEH were investigated. The Xenopus sEH did not show phosphatase activity toward 4-methylumbelliferyl phosphate or several lysophosphatidic acids although it had EH activity. The amino acid sequence of Xenopus sEH was compared with that reported previously. We found amino acid substitutions of the 29th Thr to Asn and the 146th Arg to His and prepared a sEH mutant (N29T/H146R), designed as mutant 1. Neither wild-type sEH nor mutant 1 had phosphatase activity. Additional substitution of the 11th Gly with Asp was found by comparison with human sEH which has phosphatase activity, but the Xenopus sEH mutant G11D prepared as mutant 2 did not have phosphatase activity. The epoxide hydrolase activity of sEH seemed to be similar to that of human sEH, while Xenopus sEH did not have phosphatase activity toward several substrates that human sEH metabolizes. PMID:24681163

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

    SciTech Connect

    Tyler, Ludmila; Bragg, Jennifer; Wu, Jiajie; Yang, Xiaohan; Tuskan, Gerald A; Vogel, John

    2010-01-01

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

  18. The Responses of Rat Intestinal Brush Border and Cytosol Peptide Hydrolase Activities to Variation in Dietary Protein Content DIETARY REGULATION OF INTESTINAL PEPTIDE HYDROLASES

    PubMed Central

    Nicholson, J. Alex; McCarthy, Denis M.; Kim, Young S.

    1974-01-01

    The effects of variation in dietary protein content on small intestinal brush border and cytosol peptide hydrolase activities have been investigated. One group of rats was fed a high protein diet (55% casein) and another group was fed a low protein diet (10% casein). After 1 wk, brush border peptide hydrolase activity (L-leucyl-β-naphthylamide as substrate) and cytosol peptide hydrolase activity (L-prolyl-L-leucine as substrate) were determined in mucosae taken from the proximal, middle, and distal small intestine. As judged by several parameters, brush border peptide hydrolase activity was significantly greater in rats fed the high protein diet when data for corresponding segments were compared. In contrast, no significant difference was seen in cytosol peptide hydrolase activity. In a second study, brush border and cytosol peptide hydrolase activities were determined in the proximal intestine by utilizing an additional three peptide substrates: L-leucyl-L-alanine, L-phenylalanylglycine, and glycyl-L-phenylalanine. Sucrase, maltase, and alkaline phosphatase activities were also determined. As before, brush border peptide hydrolase activities were significantly greater in rats fed the high protein diet. However, activities of the nonproteolytic brush border enzymes did not vary significantly with diet. In contrast to the results obtained with L-prolyl-L-leucine as substrate for the cytosol enzymes, cytosol activity against the three additional peptide substrates was greater in rats fed the high protein diet. It is suggested that the brush border peptide hydrolase response to variation in dietary protein content represents a functional adaptation analogous to the regulation of intestinal disaccharidases by dietary carbohydrates. The implication of the differential responses of the cytosol peptide hydrolases is uncertain, since little is known of the functional role of these nonorgan-specific enzymes. PMID:4430719

  19. Active Site and Laminarin Binding in Glycoside Hydrolase Family 55*

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-05-01

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

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

    PubMed Central

    2011-01-01

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

  2. Allophanate hydrolase, not urease, functions in bacterial cyanuric acid metabolism.

    PubMed

    Cheng, Gang; Shapir, Nir; Sadowsky, Michael J; Wackett, Lawrence P

    2005-08-01

    Growth substrates containing an s-triazine ring are typically metabolized by bacteria to liberate 3 mol of ammonia via the intermediate cyanuric acid. Over a 25-year period, a number of original research papers and reviews have stated that cyanuric acid is metabolized in two steps to the 2-nitrogen intermediate urea. In the present study, allophanate, not urea, was shown to be the 2-nitrogen intermediate in cyanuric acid metabolism in all the bacteria examined. Six different experimental results supported this conclusion: (i) synthetic allophanate was shown to readily decarboxylate to form urea under acidic extraction and chromatography conditions used in previous studies; (ii) alkaline extraction methods were used to stabilize and detect allophanate in bacteria actively metabolizing cyanuric acid; (iii) the kinetic course of allophanate formation and disappearance was consistent with its being an intermediate in cyanuric acid metabolism, and no urea was observed in those experiments; (iv) protein extracts from cells grown on cyanuric acid contained allophanate hydrolase activity; (v) genes encoding the enzymes AtzE and AtzF, which produce and hydrolyze allophanate, respectively, were found in several cyanuric acid-metabolizing bacteria; and (vi) TrzF, an AtzF homolog found in Enterobacter cloacae strain 99, was cloned, expressed in Escherichia coli, and shown to have allophanate hydrolase activity. In addition, we have observed that there are a large number of genes homologous to atzF and trzF distributed in phylogenetically distinct bacteria. In total, the data indicate that s-triazine metabolism in a broad class of bacteria proceeds through allophanate via allophanate hydrolase, rather than through urea using urease. PMID:16085834

  3. Inverting hydrolases and their use in enantioconvergent biotransformations

    PubMed Central

    Schober, Markus; Faber, Kurt

    2013-01-01

    Owing to the more abundant occurrence of racemic compounds compared to prochiral or meso forms, most enantiomerically pure products are obtained via racemate resolution. This review summarizes (chemo)enzymatic enantioconvergent processes based on the use of hydrolytic enzymes, which are able to invert a stereocenter during catalysis that can overcome the 50%-yield limitation of kinetic resolution. Recent developments are presented in the fields of inverting or retaining sulfatases, epoxide hydrolases and dehalogenases, which allow the production of secondary alcohols or vicinal diols at a 100% theoretical yield from a racemate via enantioconvergent processes. PMID:23809848

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glycoside hydrolase genes have been previously cataloged for Oryza sativa (rice), the model dicotyledonous plant Arabidopsis thaliana, and the fast-growing tree Populus trichocarpa (poplar). To improve our understanding of glycoside hydrolases in plants generally and in grasses specifically, we ann...

  5. Gulosibacter molinativorax ON4T Molinate Hydrolase, a Novel Cobalt-Dependent Amidohydrolase ▿ ‡

    PubMed Central

    Duarte, Márcia; Ferreira-da-Silva, Frederico; Lünsdorf, Heinrich; Junca, Howard; Gales, Luís; Pieper, Dietmar H.; Nunes, Olga C.

    2011-01-01

    A new pathway of molinate mineralization has recently been described. Among the five members of the mixed culture able to promote such a process, Gulosibacter molinativorax ON4T has been observed to promote the initial breakdown of the herbicide into ethanethiol and azepane-1-carboxylate. In the current study, the gene encoding the enzyme responsible for molinate hydrolysis was identified and heterologously expressed, and the resultant active protein was purified and characterized. Nucleotide sequence analysis revealed that the gene encodes a 465-amino-acid protein of the metal-dependent hydrolase A subfamily of the amidohydrolase superfamily with a predicted molecular mass of 50.9 kDa. Molinate hydrolase shares the highest amino acid sequence identity (48 to 50%) with phenylurea hydrolases of Arthrobacter globiformis and Mycobacterium brisbanense. However, in contrast to previously described members of the metal-dependent hydrolase A subfamily, molinate hydrolase contains cobalt as the only active-site metal. PMID:21840982

  6. Structure of unsaturated rhamnogalacturonyl hydrolase complexed with substrate

    SciTech Connect

    Itoh, Takafumi; Ochiai, Akihito; Mikami, Bunzo; Hashimoto, Wataru; Murata, Kousaku . E-mail: kmurata@kais.kyoto-u.ac.jp

    2006-09-08

    Bacillus subtilis strain 168 YteR has been identified as a novel enzyme 'unsaturated rhamnogalacturonyl hydrolase' classified in glycoside hydrolase family 105. This enzyme acts specifically on unsaturated rhamnogalacturonan (RG) produced from plant cell wall RG type-I treated with RG lyases, releasing unsaturated galacturonic acid ({delta}GalA) from the substrate. The most likely candidate catalytic residue is Asp-143. Here, we show the structure of D143N in complex with unsaturated RG disaccharide (substrate) determined at 1.9 A resolution by X-ray crystallography. This structural feature directly contributes to the postulation of the enzyme reaction mechanism. YteR triggers the hydration of vinyl ether group in {delta}GalA, but not of glycoside bond, by using Asp-143 as a general acid and base catalyst. Asp-143 donates proton to the double bond of {delta}GalA as an acid catalyst and also deprotonates a water molecule as a base catalyst. Deprotonated water molecule attacks the C5 atom of {delta}GalA.

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

    PubMed

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

    2006-08-11

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

  8. Marine extremophiles: a source of hydrolases for biotechnological applications.

    PubMed

    Dalmaso, Gabriel Zamith Leal; Ferreira, Davis; Vermelho, Alane Beatriz

    2015-04-01

    The marine environment covers almost three quarters of the planet and is where evolution took its first steps. Extremophile microorganisms are found in several extreme marine environments, such as hydrothermal vents, hot springs, salty lakes and deep-sea floors. The ability of these microorganisms to support extremes of temperature, salinity and pressure demonstrates their great potential for biotechnological processes. Hydrolases including amylases, cellulases, peptidases and lipases from hyperthermophiles, psychrophiles, halophiles and piezophiles have been investigated for these reasons. Extremozymes are adapted to work in harsh physical-chemical conditions and their use in various industrial applications such as the biofuel, pharmaceutical, fine chemicals and food industries has increased. The understanding of the specific factors that confer the ability to withstand extreme habitats on such enzymes has become a priority for their biotechnological use. The most studied marine extremophiles are prokaryotes and in this review, we present the most studied archaea and bacteria extremophiles and their hydrolases, and discuss their use for industrial applications. PMID:25854643

  9. Mechanistic Investigations of Unsaturated Glucuronyl Hydrolase from Clostridium perfringens*

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-04-18

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

  11. Catalysis of potato epoxide hydrolase, StEH1

    PubMed Central

    Elfström, Lisa T.; Widersten, Mikael

    2005-01-01

    The kinetic mechanism of epoxide hydrolase (EC 3.3.2.3) from potato, StEH1 (Solanum tuberosum epoxide hydrolase 1), was studied by presteady-state and steady-state kinetics as well as by pH dependence of activity. The specific activities towards the different enantiomers of TSO (trans-stilbene oxide) as substrate were 43 and 3 μmol·min−1·mg−1 with the R,R- or S,S-isomers respectively. The enzyme was, however, enantioselective in favour of the S,S enantiomer due to a lower Km value. The pH dependences of kcat with R,R or S,S-TSO were also distinct and supposedly reflecting the pH dependences of the individual kinetic rates during substrate conversion. The rate-limiting step for TSO and cis- and trans-epoxystearate was shown by rapid kinetic measurements to be the hydrolysis of the alkylenzyme intermediate. Functional characterization of point mutants verified residues Asp105, Tyr154, Tyr235 and His300 as crucial for catalytic activity. All mutants displayed drastically decreased enzymatic activities during steady state. Presteady-state measurements revealed the base-deficient H300N (His300→Asn) mutant to possess greatly reduced efficiencies in catalysis of both chemical steps (alkylation and hydrolysis). PMID:15882148

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

    PubMed Central

    Dalmaso, Gabriel Zamith Leal; Ferreira, Davis; Vermelho, Alane Beatriz

    2015-01-01

    The marine environment covers almost three quarters of the planet and is where evolution took its first steps. Extremophile microorganisms are found in several extreme marine environments, such as hydrothermal vents, hot springs, salty lakes and deep-sea floors. The ability of these microorganisms to support extremes of temperature, salinity and pressure demonstrates their great potential for biotechnological processes. Hydrolases including amylases, cellulases, peptidases and lipases from hyperthermophiles, psychrophiles, halophiles and piezophiles have been investigated for these reasons. Extremozymes are adapted to work in harsh physical-chemical conditions and their use in various industrial applications such as the biofuel, pharmaceutical, fine chemicals and food industries has increased. The understanding of the specific factors that confer the ability to withstand extreme habitats on such enzymes has become a priority for their biotechnological use. The most studied marine extremophiles are prokaryotes and in this review, we present the most studied archaea and bacteria extremophiles and their hydrolases, and discuss their use for industrial applications. PMID:25854643

  13. A Proton Wire and Water Channel Revealed in the Crystal Structure of Isatin Hydrolase

    PubMed Central

    Bjerregaard-Andersen, Kaare; Sommer, Theis; Jensen, Jan K.; Jochimsen, Bjarne; Etzerodt, Michael; Morth, J. Preben

    2014-01-01

    The high resolution crystal structures of isatin hydrolase from Labrenzia aggregata in the apo and the product state are described. These are the first structures of a functionally characterized metal-dependent hydrolase of this fold. Isatin hydrolase converts isatin to isatinate and belongs to a novel family of metalloenzymes that include the bacterial kynurenine formamidase. The product state, mimicked by bound thioisatinate, reveals a water molecule that bridges the thioisatinate to a proton wire in an adjacent water channel and thus allows the proton released by the reaction to escape only when the product is formed. The functional proton wire present in isatin hydrolase isoform b represents a unique catalytic feature common to all hydrolases is here trapped and visualized for the first time. The local molecular environment required to coordinate thioisatinate allows stronger and more confident identification of orthologous genes encoding isatin hydrolases within the prokaryotic kingdom. The isatin hydrolase orthologues found in human gut bacteria raise the question as to whether the indole-3-acetic acid degradation pathway is present in human gut flora. PMID:24917679

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

    PubMed

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

    2016-01-01

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

  15. Cloning and characterization of two rhamnogalacturonan hydrolase genes from Aspergillus niger.

    PubMed Central

    Suykerbuyk, M E; Kester, H C; Schaap, P J; Stam, H; Musters, W; Visser, J

    1997-01-01

    A rhamnogalacturonan hydrolase gene of Aspergillus aculeatus was used as a probe for the cloning of two rhamnogalacturonan hydrolase genes of Aspergillus niger. The corresponding proteins, rhamnogalacturonan hydrolases A and B, are 78 and 72% identical, respectively, with the A. aculeatus enzyme. In A. niger cultures which were shifted from growth on sucrose to growth on apple pectin as a carbon source, the expression of the rhamnogalacturonan hydrolase A gene (rhgA) was transiently induced after 3 h of growth on apple pectin. The rhamnogalacturonan hydrolase B gene was not induced by apple pectin, but the rhgB gene was derepressed after 18 h of growth on either apple pectin or sucrose. Gene fusions of the A. niger rhgA and rhgB coding regions with the strong and inducible Aspergillus awamori exlA promoter were used to obtain high-producing A. awamori transformants which were then used for the purification of the two A. niger rhamnogalacturonan hydrolases. High-performance anion-exchange chromatography of oligomeric degradation products showed that optimal degradation of an isolated highly branched pectin fraction by A. niger rhamnogalacturonan hydrolases A and B occurred at pH 3.6 and 4.1, respectively. The specific activities of rhamnogalacturonan hydrolases A and B were then 0.9 and 0.4 U/mg, respectively, which is significantly lower than the specific activity of A. aculeatus rhamnogalacturonan hydrolase (2.5 U/mg at an optimal pH of 4.5). Compared to the A enzymes, the A. niger B enzyme appears to have a different substrate specificity, since additional oligomers are formed. PMID:9212401

  16. Inhibition of peptidoglycan hydrolase activity in vivo and in vitro by energy uncouplers in Escherichia coli.

    PubMed

    Rodionov, D G; Ishiguro, E E

    1996-01-01

    The effects of energy uncouplers on in vivo and in vitro peptidoglycan hydrolase activities in Escherichia coli were determined. Sodium azide, potassium cyanide, and carbonyl cyanide m-chlorophenylhydrazone all inhibited ampicillin-induced lysis of exponential phase cultures, even when they were added to lysis-committed cultures. These energy uncouplers also inhibited the solubilization of radiolabeled peptidoglycan by bacterial suspensions that had been treated with 5% trichloroacetic acid by the method of Hartmann et al.3 to activate the peptidoglycan hydrolases. Therefore, the in vivo and in vitro activities of peptidoglycan hydrolases in E. coli are dependent on membrane energization. PMID:9158735

  17. Rapid development of a potent photo-triggered inhibitor of the serine hydrolase RBBP9.

    PubMed

    Liu, Xiaodan; Dix, Melissa; Speers, Anna E; Bachovchin, Daniel A; Zuhl, Andrea M; Cravatt, Benjamin F; Kodadek, Thomas J

    2012-09-24

    The serine hydrolases constitute a large class of enzymes that play important roles in physiology. There is great interest in the development of potent and selective pharmacological inhibitors of these proteins. Traditional active-site inhibitors often have limited selectivity within this superfamily and are tedious and expensive to discover. Using the serine hydrolase RBBP9 as a model target, we designed a rapid and relatively inexpensive route to highly selective peptoid-based inhibitors that can be activated by visible light. This technology provides rapid access to photo-activated tool compounds capable of selectively blocking the function of particular serine hydrolases. PMID:22907802

  18. Rapid Development of a Potent Photo-Triggered Inhibitor of the Serine Hydrolase RBBP9

    PubMed Central

    Liu, Xiaodan; Dix, Melissa; Speers, Anna E.; Bachovchin, Daniel A.; Zuhl, Andrea M.

    2013-01-01

    The serine hydrolases constitute a large class of enzymes that play important roles in physiology. There is great interest in the development of potent and selective pharmacological inhibitors to these proteins. Traditional active site inhibitors often have limited selectivity within this superfamily and are tedious and expensive to discover. Using the serine hydrolase RBBP9 as a model target, we report here a rapid and relatively inexpensive route to highly selective peptoid-based inhibitors that can be activated with visible light. This technology provides rapid access to photo-activated tool compounds capable of selectively blocking the function of particular serine hydrolases. PMID:22907802

  19. Retinyl ester hydrolases and their roles in vitamin A homeostasis☆

    PubMed Central

    Schreiber, Renate; Taschler, Ulrike; Preiss-Landl, Karina; Wongsiriroj, Nuttaporn; Zimmermann, Robert; Lass, Achim

    2012-01-01

    In mammals, dietary vitamin A intake is essential for the maintenance of adequate retinoid (vitamin A and metabolites) supply of tissues and organs. Retinoids are taken up from animal or plant sources and subsequently stored in form of hydrophobic, biologically inactive retinyl esters (REs). Accessibility of these REs in the intestine, the circulation, and their mobilization from intracellular lipid droplets depends on the hydrolytic action of RE hydrolases (REHs). In particular, the mobilization of hepatic RE stores requires REHs to maintain steady plasma retinol levels thereby assuring constant vitamin A supply in times of food deprivation or inadequate vitamin A intake. In this review, we focus on the roles of extracellular and intracellular REHs in vitamin A metabolism. Furthermore, we will discuss the tissue-specific function of REHs and highlight major gaps in the understanding of RE catabolism. This article is part of a Special Issue entitled Retinoid and Lipid Metabolism. PMID:21586336

  20. Glycerol Ester Hydrolase Activity of Lactic Acid Bacteria

    PubMed Central

    Oterholm, Anders; Ordal, Z. John; Witter, Lloyd D.

    1968-01-01

    Seventeen strains of lactic acid bacteria were assayed for their glycerol ester hydrolase activity by using an improved agar-well technique, and eight strains by determining the activity in cell-free extracts using a pH-stat procedure. All cultures tested showed activity and hydrolyzed tributyrin more actively than they did tricaproin. The cell extract studies demonstrated that the cells contained intracellular esterases and lipases. The culture supernatant fluid was without activity. The lipase and the esterase differed in their relative activity to each other in the different extracts and in the ease by which they could be freed from the cellular debris. It is suggested that the lipase of these organisms is an endoenzyme and the esterase an ectoenzyme. PMID:5649866

  1. Sulfonyl fluoride inhibitors of fatty acid amide hydrolase.

    PubMed

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

    2012-11-26

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

  2. Soluble epoxide hydrolase: Gene structure, expression and deletion

    PubMed Central

    Harris, Todd R.; Hammock, Bruce D.

    2013-01-01

    Mammalian soluble epoxide hydrolase (sEH) converts epoxides to their corresponding diols through the addition of a water molecule. sEH readily hydrolyzes lipid signaling molecules, including the epoxyeicosatrienoic acids (EETs), epoxidized lipids produced from arachidonic acid by the action of cytochrome p450s. Through its metabolism of the EETs and other lipid mediators, sEH contributes to the regulation of vascular tone, nociception, angiogenesis and the inflammatory response. Because of its central physiological role in disease states such as cardiac hypertrophy, diabetes, hypertension, and pain sEH is being investigated as a therapeutic target. This review begins with a brief introduction to sEH protein structure and function. sEH evolution and gene structure are then discussed before human small nucleotide polymorphisms and mammalian gene expression are described in the context of several disease models. The review ends with an overview of studies that have employed the sEH knockout mouse model. PMID:23701967

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

    PubMed

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

    2016-05-01

    Bile salt hydrolase (BSH) is a gut-bacterial enzyme that negatively influences host fat digestion and energy harvesting. The BSH enzyme activity functions as a gateway reaction in the small intestine by the deconjugation of glycine-conjugated or taurine-conjugated bile acids. Extensive gut-microbiota studies have suggested that BSH is a key mechanistic microbiome target for the development of novel non-antibiotic food additives to improve animal feed production and for the design of new measures to control obesity in humans. However, research on BSH is still in its infancy, particularly in terms of the structural basis of BSH function, which has hampered the development of BSH-based strategies for improving human and animal health. As an initial step towards the structure-function analysis of BSH, C-terminally His-tagged BSH from Lactobacillus salivarius NRRL B-30514 was crystallized in this study. The 1.90 Å resolution crystal structure of L. salivarius BSH was determined by molecular replacement using the structure of Clostridium perfringens BSH as a starting model. It revealed this BSH to be a member of the N-terminal nucleophile hydrolase superfamily. Crystals of apo BSH belonged to space group P21212, with unit-cell parameters a = 90.79, b = 87.35, c = 86.76 Å (PDB entry 5hke). Two BSH molecules packed perfectly as a dimer in one asymmetric unit. Comparative structural analysis of L. salivarius BSH also identified potential residues that contribute to catalysis and substrate specificity. PMID:27139829

  4. Expanding the Catalytic Triad in Epoxide Hydrolases and Related Enzymes

    PubMed Central

    2015-01-01

    Potato epoxide hydrolase 1 exhibits rich enantio- and regioselectivity in the hydrolysis of a broad range of substrates. The enzyme can be engineered to increase the yield of optically pure products as a result of changes in both enantio- and regioselectivity. It is thus highly attractive in biocatalysis, particularly for the generation of enantiopure fine chemicals and pharmaceuticals. The present work aims to establish the principles underlying the activity and selectivity of the enzyme through a combined computational, structural, and kinetic study using the substrate trans-stilbene oxide as a model system. Extensive empirical valence bond simulations have been performed on the wild-type enzyme together with several experimentally characterized mutants. We are able to computationally reproduce the differences between the activities of different stereoisomers of the substrate and the effects of mutations of several active-site residues. In addition, our results indicate the involvement of a previously neglected residue, H104, which is electrostatically linked to the general base H300. We find that this residue, which is highly conserved in epoxide hydrolases and related hydrolytic enzymes, needs to be in its protonated form in order to provide charge balance in an otherwise negatively charged active site. Our data show that unless the active-site charge balance is correctly treated in simulations, it is not possible to generate a physically meaningful model for the enzyme that can accurately reproduce activity and selectivity trends. We also expand our understanding of other catalytic residues, demonstrating in particular the role of a noncanonical residue, E35, as a “backup base” in the absence of H300. Our results provide a detailed view of the main factors driving catalysis and regioselectivity in this enzyme and identify targets for subsequent enzyme design efforts. PMID:26527505

  5. DETOXIFICATION OF ORGANOPHOSPHATE PESTICIDES BY IMMOBILIZED ESCHERICHIA COLI EXPRESSING ORGANOPHOSPHORUS HYDROLASE ON CELL SURFACE. (R823663)

    EPA Science Inventory

    An improved whole-cell technology for detoxifying organophosphate nerve agents was recently developed based on genetically engineered Escherichia coli with organophosphorus hydrolase anchored on the surface. This article reports the immobilization of these novel biocatalys...

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

    PubMed Central

    Duong-ly, Krisna C.; Schoeffield, Andrew J.; Pizarro-Dupuy, Mario A.; Zarr, Melissa; Pineiro, Silvia A.; Amzel, L. Mario; Gabelli, Sandra B.

    2015-01-01

    Given the broad range of substrates hydrolyzed by Nudix (nucleoside diphosphate linked to X) enzymes, identification of sequence and structural elements that correctly predict a Nudix substrate or characterize a family is key to correctly annotate the myriad of Nudix enzymes. Here, we present the structure determination and characterization of Bd3179 –- a Nudix hydrolase from Bdellovibrio bacteriovorus–that we show localized in the periplasmic space of this obligate Gram-negative predator. We demonstrate that the enzyme is a nucleoside diphosphate sugar hydrolase (NDPSase) and has a high degree of sequence and structural similarity to a canonical ADP-ribose hydrolase and to a nucleoside diphosphate sugar hydrolase (1.4 and 1.3 Å Cα RMSD respectively). Examination of the structural elements conserved in both types of enzymes confirms that an aspartate-X-lysine motif on the C-terminal helix of the α-β-α NDPSase fold differentiates NDPSases from ADPRases. PMID:26524597

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

    PubMed Central

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

    2015-01-01

    The occurrence of genes encoding biotechnologically relevant α/β-hydrolases in mangrove soil microbial communities was assessed using data obtained by whole-metagenome sequencing of four mangroves areas, denoted BrMgv01 to BrMgv04, in São Paulo, Brazil. The sequences (215 Mb in total) were filtered based on local amino acid alignments against the Lipase Engineering Database. In total, 5923 unassembled sequences were affiliated with 30 different α/β-hydrolase fold superfamilies. The most abundant predicted proteins encompassed cytosolic hydrolases (abH08; ∼ 23%), microsomal hydrolases (abH09; ∼ 12%) and Moraxella lipase-like proteins (abH04 and abH01; < 5%). Detailed analysis of the genes predicted to encode proteins of the abH08 superfamily revealed a high proportion related to epoxide hydrolases and haloalkane dehalogenases in polluted mangroves BrMgv01-02-03. This suggested selection and putative involvement in local degradation/detoxification of the pollutants. Seven sequences that were annotated as genes for putative epoxide hydrolases and five for putative haloalkane dehalogenases were found in a fosmid library generated from BrMgv02 DNA. The latter enzymes were predicted to belong to Actinobacteria, Deinococcus-Thermus, Planctomycetes and Proteobacteria. Our integrated approach thus identified 12 genes (complete and/or partial) that may encode hitherto undescribed enzymes. The low amino acid identity (< 60%) with already-described genes opens perspectives for both production in an expression host and genetic screening of metagenomes. PMID:25171437

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

    PubMed

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

    2015-05-01

    The occurrence of genes encoding biotechnologically relevant α/β-hydrolases in mangrove soil microbial communities was assessed using data obtained by whole-metagenome sequencing of four mangroves areas, denoted BrMgv01 to BrMgv04, in São Paulo, Brazil. The sequences (215 Mb in total) were filtered based on local amino acid alignments against the Lipase Engineering Database. In total, 5923 unassembled sequences were affiliated with 30 different α/β-hydrolase fold superfamilies. The most abundant predicted proteins encompassed cytosolic hydrolases (abH08; ∼ 23%), microsomal hydrolases (abH09; ∼ 12%) and Moraxella lipase-like proteins (abH04 and abH01; < 5%). Detailed analysis of the genes predicted to encode proteins of the abH08 superfamily revealed a high proportion related to epoxide hydrolases and haloalkane dehalogenases in polluted mangroves BrMgv01-02-03. This suggested selection and putative involvement in local degradation/detoxification of the pollutants. Seven sequences that were annotated as genes for putative epoxide hydrolases and five for putative haloalkane dehalogenases were found in a fosmid library generated from BrMgv02 DNA. The latter enzymes were predicted to belong to Actinobacteria, Deinococcus-Thermus, Planctomycetes and Proteobacteria. Our integrated approach thus identified 12 genes (complete and/or partial) that may encode hitherto undescribed enzymes. The low amino acid identity (< 60%) with already-described genes opens perspectives for both production in an expression host and genetic screening of metagenomes. PMID:25171437

  9. SARS coronavirus protein 7a interacts with human Ap4A-hydrolase

    PubMed Central

    2010-01-01

    The SARS coronavirus (SARS-CoV) open reading frame 7a (ORF 7a) encodes a 122 amino acid accessory protein. It has no significant sequence homology with any other known proteins. The 7a protein is present in the virus particle and has been shown to interact with several host proteins; thereby implicating it as being involved in several pathogenic processes including apoptosis, inhibition of cellular protein synthesis, and activation of p38 mitogen activated protein kinase. In this study we present data demonstrating that the SARS-CoV 7a protein interacts with human Ap4A-hydrolase (asymmetrical diadenosine tetraphosphate hydrolase, EC 3.6.1.17). Ap4A-hydrolase is responsible for metabolizing the "allarmone" nucleotide Ap4A and therefore likely involved in regulation of cell proliferation, DNA replication, RNA processing, apoptosis and DNA repair. The interaction between 7a and Ap4A-hydrolase was identified using yeast two-hybrid screening. The interaction was confirmed by co-immunoprecipitation from cultured human cells transiently expressing V5-His tagged 7a and HA tagged Ap4A-hydrolase. Human tissue culture cells transiently expressing 7a and Ap4A-hydrolase tagged with EGFP and Ds-Red2 respectively show these proteins co-localize in the cytoplasm. PMID:20144233

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

    NASA Astrophysics Data System (ADS)

    Tanaka, Sayuri; Sugimoto, Manabu; Kihara, Makoto

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

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

    PubMed

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

    2015-12-01

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

  12. Nudix hydrolases degrade protein-conjugated ADP-ribose.

    PubMed

    Daniels, Casey M; Thirawatananond, Puchong; Ong, Shao-En; Gabelli, Sandra B; Leung, Anthony K L

    2015-01-01

    ADP-ribosylation refers to the transfer of the ADP-ribose group from NAD(+) to target proteins post-translationally, either attached singly as mono(ADP-ribose) (MAR) or in polymeric chains as poly(ADP-ribose) (PAR). Though ADP-ribosylation is therapeutically important, investigation of this protein modification has been limited by a lack of proteomic tools for site identification. Recent work has demonstrated the potential of a tag-based pipeline in which MAR/PAR is hydrolyzed down to phosphoribose, leaving a 212 Dalton tag at the modification site. While the pipeline has been proven effective by multiple groups, a barrier to application has become evident: the enzyme used to transform MAR/PAR into phosphoribose must be purified from the rattlesnake Crotalus adamanteus venom, which is contaminated with proteases detrimental for proteomic applications. Here, we outline the steps necessary to purify snake venom phosphodiesterase I (SVP) and describe two alternatives to SVP-the bacterial Nudix hydrolase EcRppH and human HsNudT16. Importantly, expression and purification schemes for these Nudix enzymes have already been proven, with high-quality yields easily attainable. We demonstrate their utility in identifying ADP-ribosylation sites on Poly(ADP-ribose) Polymerase 1 (PARP1) with mass spectrometry and discuss a structure-based rationale for this Nudix subclass in degrading protein-conjugated ADP-ribose, including both MAR and PAR. PMID:26669448

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

    PubMed

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

    2016-07-19

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

  14. Cyanuric acid hydrolase: evolutionary innovation by structural concatenation

    PubMed Central

    Peat, Thomas S; Balotra, Sahil; Wilding, Matthew; French, Nigel G; Briggs, Lyndall J; Panjikar, Santosh; Cowieson, Nathan; Newman, Janet; Scott, Colin

    2013-01-01

    The cyanuric acid hydrolase, AtzD, is the founding member of a newly identified family of ring-opening amidases. We report the first X-ray structure for this family, which is a novel fold (termed the ‘Toblerone’ fold) that likely evolved via the concatenation of monomers of the trimeric YjgF superfamily and the acquisition of a metal binding site. Structures of AtzD with bound substrate (cyanuric acid) and inhibitors (phosphate, barbituric acid and melamine), along with mutagenesis studies, allowed the identification of the active site. The AtzD monomer, active site and substrate all possess threefold rotational symmetry, to the extent that the active site possesses three potential Ser–Lys catalytic dyads. A single catalytic dyad (Ser85–Lys42) is hypothesized, based on biochemical evidence and crystallographic data. A plausible catalytic mechanism based on these observations is also presented. A comparison with a homology model of the related barbiturase, Bar, was used to infer the active-site residues responsible for substrate specificity, and the phylogeny of the 68 AtzD-like enzymes in the database were analysed in light of this structure–function relationship. PMID:23651355

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

    PubMed

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

    2016-05-01

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

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

    PubMed

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

    2016-08-01

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

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

    PubMed

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

    2014-10-01

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

  18. Extracellular Glycoside Hydrolase Activities in the Human Oral Cavity.

    PubMed

    Inui, Taichi; Walker, Lauren C; Dodds, Michael W J; Hanley, A Bryan

    2015-08-15

    Carbohydrate availability shifts when bacteria attach to a surface and form biofilm. When salivary planktonic bacteria form an oral biofilm, a variety of polysaccharides and glycoproteins are the primary carbon sources; however, simple sugar availabilities are limited due to low diffusion from saliva to biofilm. We hypothesized that bacterial glycoside hydrolase (GH) activities would be higher in a biofilm than in saliva in order to maintain metabolism in a low-sugar, high-glycoprotein environment. Salivary bacteria from 13 healthy individuals were used to grow in vitro biofilm using two separate media, one with sucrose and the other limiting carbon sources to a complex carbohydrate. All six GHs measured were higher in vitro when grown in the medium with complex carbohydrate as the sole carbon source. We then collected saliva and overnight dental plaque samples from the same individuals and measured ex vivo activities for the same six enzymes to determine how oral microbial utilization of glycoconjugates shifts between the planktonic phase in saliva and the biofilm phase in overnight dental plaque. Overall higher GH activities were observed in plaque samples, in agreement with in vitro observation. A similar pattern was observed in GH activity profiles between in vitro and ex vivo data. 16S rRNA gene analysis showed that plaque samples had a higher abundance of microorganisms with larger number of GH gene sequences. These results suggest differences in sugar catabolism between the oral bacteria located in the biofilm and those in saliva. PMID:26048943

  19. Nudix hydrolases degrade protein-conjugated ADP-ribose

    PubMed Central

    Daniels, Casey M.; Thirawatananond, Puchong; Ong, Shao-En; Gabelli, Sandra B.; Leung, Anthony K. L.

    2015-01-01

    ADP-ribosylation refers to the transfer of the ADP-ribose group from NAD+ to target proteins post-translationally, either attached singly as mono(ADP-ribose) (MAR) or in polymeric chains as poly(ADP-ribose) (PAR). Though ADP-ribosylation is therapeutically important, investigation of this protein modification has been limited by a lack of proteomic tools for site identification. Recent work has demonstrated the potential of a tag-based pipeline in which MAR/PAR is hydrolyzed down to phosphoribose, leaving a 212 Dalton tag at the modification site. While the pipeline has been proven effective by multiple groups, a barrier to application has become evident: the enzyme used to transform MAR/PAR into phosphoribose must be purified from the rattlesnake Crotalus adamanteus venom, which is contaminated with proteases detrimental for proteomic applications. Here, we outline the steps necessary to purify snake venom phosphodiesterase I (SVP) and describe two alternatives to SVP—the bacterial Nudix hydrolase EcRppH and human HsNudT16. Importantly, expression and purification schemes for these Nudix enzymes have already been proven, with high-quality yields easily attainable. We demonstrate their utility in identifying ADP-ribosylation sites on Poly(ADP-ribose) Polymerase 1 (PARP1) with mass spectrometry and discuss a structure-based rationale for this Nudix subclass in degrading protein-conjugated ADP-ribose, including both MAR and PAR. PMID:26669448

  20. Disrupting Dimerization Translocates Soluble Epoxide Hydrolase to Peroxisomes

    PubMed Central

    Nelson, Jonathan W.; Das, Anjali J.; Barnes, Anthony P.; Alkayed, Nabil J.

    2016-01-01

    The epoxyeicosatrienoic acid (EET) neutralizing enzyme soluble epoxide hydrolase (sEH) is a neuronal enzyme, which has been localized in both the cytosol and peroxisomes. The molecular basis for its dual localization remains unclear as sEH contains a functional peroxisomal targeting sequence (PTS). Recently, a missense polymorphism was identified in human sEH (R287Q) that enhances its peroxisomal localization. This same polymorphism has also been shown to generate weaker sEH homo-dimers. Taken together, these observations suggest that dimerization may mask the sEH PTS and prevent peroxisome translocation. In the current study, we test the hypothesis that dimerization is a key regulator of sEH subcellular localization. Specifically, we altered the dimerization state of sEH by introducing substitutions in amino acids responsible for the dimer-stabilizing salt-bridge. Green Fluorescent Protein (GFP) fusions of each of mutants were co-transfected into mouse primary cultured cortical neurons together with a PTS-linked red fluorescent protein to constitutively label peroxisomes. Labeled neurons were analyzed using confocal microscopy and co-localization of sEH with peroxisomes was quantified using Pearson’s correlation coefficient. We find that dimer-competent sEH constructs preferentially localize to the cytosol, whereas constructs with weakened or disrupted dimerization were preferentially targeted to peroxisomes. We conclude that the sEH dimerization status is a key regulator of its peroxisomal localization. PMID:27203283

  1. Thermus thermophilus Glycoside Hydrolase Family 57 Branching Enzyme

    PubMed Central

    Palomo, Marta; Pijning, Tjaard; Booiman, Thijs; Dobruchowska, Justyna M.; van der Vlist, Jeroen; Kralj, Slavko; Planas, Antoni; Loos, Katja; Kamerling, Johannis P.; Dijkstra, Bauke W.; van der Maarel, Marc J. E. C.; Dijkhuizen, Lubbert; Leemhuis, Hans

    2011-01-01

    Branching enzyme (EC 2.4.1.18; glycogen branching enzyme; GBE) catalyzes the formation of α1,6-branching points in glycogen. Until recently it was believed that all GBEs belong to glycoside hydrolase family 13 (GH13). Here we describe the cloning and expression of the Thermus thermophilus family GH57-type GBE and report its biochemical properties and crystal structure at 1.35-Å resolution. The enzyme has a central (β/α)7-fold catalytic domain A with an inserted domain B between β2 and α5 and an α-helix-rich C-terminal domain, which is shown to be essential for substrate binding and catalysis. A maltotriose was modeled in the active site of the enzyme which suggests that there is insufficient space for simultaneously binding of donor and acceptor substrates, and that the donor substrate must be cleaved before acceptor substrate can bind. The biochemical assessment showed that the GH57 GBE possesses about 4% hydrolytic activity with amylose and in vitro forms a glucan product with a novel fine structure, demonstrating that the GH57 GBE is clearly different from the GH13 GBEs characterized to date. PMID:21097495

  2. Bacterial 2,4-Dioxygenases: New Members of the α/β Hydrolase-Fold Superfamily of Enzymes Functionally Related to Serine Hydrolases

    PubMed Central

    Fischer, Frank; Künne, Stefan; Fetzner, Susanne

    1999-01-01

    1H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase (Qdo) from Pseudomonas putida 33/1 and 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) from Arthrobacter ilicis Rü61a catalyze an N-heterocyclic-ring cleavage reaction, generating N-formylanthranilate and N-acetylanthranilate, respectively, and carbon monoxide. Amino acid sequence comparisons between Qdo, Hod, and a number of proteins belonging to the α/β hydrolase-fold superfamily of enzymes and analysis of the similarity between the predicted secondary structures of the 2,4-dioxygenases and the known secondary structure of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 strongly suggested that Qdo and Hod are structurally related to the α/β hydrolase-fold enzymes. The residues S95 and H244 of Qdo were found to be arranged like the catalytic nucleophilic residue and the catalytic histidine, respectively, of the α/β hydrolase-fold enzymes. Investigation of the potential functional significance of these and other residues of Qdo through site-directed mutagenesis supported the hypothesis that Qdo is structurally as well as functionally related to serine hydrolases, with S95 being a possible catalytic nucleophile and H244 being a possible catalytic base. A hypothetical reaction mechanism for Qdo-catalyzed 2,4-dioxygenolysis, involving formation of an ester bond between the catalytic serine residue and the carbonyl carbon of the substrate and subsequent dioxygenolysis of the covalently bound anionic intermediate, is discussed. PMID:10482514

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2013-01-01

    N-acylethanolamines (NAEs) are endogenous lipid-based signaling molecules best known for their role in the endocannabinoid system in mammals, but they are also known to play roles in signaling pathways in plants. The regulation of NAEs in vivo is partly accomplished by the enzyme fatty acid amide hydrolase (FAAH), which hydrolyses NAEs to ethanolamine and their corresponding fatty acid. Inhibition of FAAH has been shown to increase the levels of NAEs in vivo and to produce desirable phenotypes. This has led to the development of pharmaceutical-based therapies for a variety of conditions targeting FAAH. Recently, our group identified a functional FAAH homolog in Dictyostelium discoideum, leading to our hypothesis that D. discoideum also possesses NAEs. In this study, we provide a further characterization of FAAH and identify NAEs in D. discoideum for the first time. We also demonstrate the ability to modulate their levels in vivo through the use of a semispecific FAAH inhibitor and confirm that these NAEs are FAAH substrates through in vitro studies. We believe the demonstration of the in vivo modulation of NAE levels suggests that D. discoideum could be a good simple model organism in which to study NAE-mediated signaling. PMID:23187822

  5. John Montgomery's legacy: carbocyclic adenosine analogues as SAH hydrolase inhibitors with broad-spectrum antiviral activity.

    PubMed

    De Clercq, Erik

    2005-01-01

    Ever since the S-adenosylhomocysteine (AdoHcy, SAH) hydrolase was recognized as a pharmacological target for antiviral agents (J. A. Montgomery et al., J. Med. Chem. 25:626-629, 1982), an increasing number of adenosine, acyclic adenosine, and carbocyclic adenosine analogues have been described as potent SAH hydrolase inhibitors endowed with broad-spectrum antiviral activity. The antiviral activity spectrum of the SAH hydrolase inhibitors include pox-, rhabdo-, filo-, arena-, paramyxo-, reo-, and retroviruses. Among the most potent SAH hydrolase inhibitors and antiviral agents rank carbocyclic 3-deazaadenosine (C-c3 Ado), neplanocin A, 3-deazaneplanocin A, the 5'-nor derivatives of carbocyclic adenosine (C-Ado, aristeromycin), and the 2-halo (i.e., 2-fluoro) and 6'-R-alkyl (i.e., 6'-R-methyl) derivatives of neplanocin A. These compounds are particularly active against poxviruses (i.e., vaccinia virus), and rhabdoviruses (i.e., vesicular stomatitis virus). The in vivo efficacy of C-c3 Ado and 3-deazaneplanocin A has been established in mouse models for vaccinia virus, vesicular stomatitis virus, and Ebola virus. SAH hydrolase inhibitors such as C-c3Ado and 3-deazaneplanocin A should in thefirst place be considered for therapeutic (or prophylactic) use against poxvirus infections, including smallpox, and hemorrhagic fever virus infections such as Ebola. PMID:16438025

  6. A Bifunctional Enzyme That Has Both Monoacylglycerol Acyltransferase and Acyl Hydrolase Activities1[W][OA

    PubMed Central

    Vijayaraj, Panneerselvam; Jashal, Charnitkaur B.; Vijayakumar, Anitha; Rani, Sapa Hima; Venkata Rao, D.K.; Rajasekharan, Ram

    2012-01-01

    Monoacylglycerol acyltransferase (MGAT) catalyzes the synthesis of diacylglycerol, the precursor of triacylglycerol biosynthesis and an important signaling molecule. Here, we describe the isolation and characterization of the peanut (Arachis hypogaea) MGAT gene. The soluble enzyme utilizes invariant histidine-62 and aspartate-67 residues of the acyltransferase motif for its MGAT activity. A sequence analysis revealed the presence of a hydrolase (GXSXG) motif, and enzyme assays revealed the presence of monoacylglycerol (MAG) and lysophosphatidylcholine (LPC) hydrolytic activities, indicating the bifunctional nature of the enzyme. The overexpression of the MGAT gene in yeast (Saccharomyces cerevisiae) caused an increase in triacylglycerol accumulation. Similar to the peanut MGAT, the Arabidopsis (Arabidopsis thaliana) homolog (At1g52760) also exhibited both acyltransferase and hydrolase activities. Interestingly, the yeast homolog lacks the conserved HX4D motif, and it is deficient in the acyltransferase function but exhibits MAG and LPC hydrolase activities. This study demonstrates the presence of a soluble MGAT/hydrolase in plants. The predicted three-dimensional homology modeling and substrate docking suggested the presence of two separate substrate (MAG and LPC)-binding sites in a single polypeptide. Our study describes a soluble bifunctional enzyme that has both MGAT and hydrolase functions. PMID:22915575

  7. Development of a differential medium for bile salt hydrolase-active Lactobacillus spp.

    PubMed Central

    Dashkevicz, M P; Feighner, S D

    1989-01-01

    An agar plate assay was developed to detect bile salt hydrolase activity in lactobacilli. On Lactobacillus-selective MRS or Rogosa SL medium supplemented with taurodeoxycholic, taurocholic, or taurochenodeoxycholic acids, bile salt hydrolysis was manifested at two intensities: (i) the formation of precipitate halos around colonies or (ii) the formation of opaque granular white colonies. Sixty-six lactobacilli were tested for bile salt hydrolase activity by both the plate assay and a sensitive radiochemical assay. No false-positive or false-negative results were detected by the plate assay. Based on results of experiments with Eubacterium lentum and Bacteroides species, the plate assay was dependent on two factors: (i) the presence of bile salt hydrolytic activity and (ii) the ability of the organism to sufficiently acidify the medium to protonate free bile acids. The availability of a differential medium for determination of bile salt hydrolase activity will provide a rapid method for determining shifts in a specific functional activity of intestinal Lactobacillus species and provide a rapid screening capability for identifying bile salt hydrolase-deficient mutants. The latter application should allow bile salt hydrolase activity to be used as a marker enzyme in genetic experiments. Images PMID:2705765

  8. Soluble epoxide hydrolase limits mechanical hyperalgesia during inflammation

    PubMed Central

    2011-01-01

    Background Cytochrome-P450 (CYP450) epoxygenases metabolise arachidonic acid (AA) into four different biologically active epoxyeicosatrienoic acid (EET) regioisomers. Three of the EETs (i.e., 8,9-, 11,12- and 14,15-EET) are rapidly hydrolysed by the enzyme soluble epoxide hydrolase (sEH). Here, we investigated the role of sEH in nociceptive processing during peripheral inflammation. Results In dorsal root ganglia (DRG), we found that sEH is expressed in medium and large diameter neurofilament 200-positive neurons. Isolated DRG-neurons from sEH-/- mice showed higher EET and lower DHET levels. Upon AA stimulation, the largest changes in EET levels occurred in culture media, indicating both that cell associated EET concentrations quickly reach saturation and EET-hydrolyzing activity mostly effects extracellular EET signaling. In vivo, DRGs from sEH-deficient mice exhibited elevated 8,9-, 11,12- and 14,15-EET-levels. Interestingly, EET levels did not increase at the site of zymosan-induced inflammation. Cellular imaging experiments revealed direct calcium flux responses to 8,9-EET in a subpopulation of nociceptors. In addition, 8,9-EET sensitized AITC-induced calcium increases in DRG neurons and AITC-induced calcitonin gene related peptide (CGRP) release from sciatic nerve axons, indicating that 8,9-EET sensitizes TRPA1-expressing neurons, which are known to contribute to mechanical hyperalgesia. Supporting this, sEH-/- mice showed increased nociceptive responses to mechanical stimulation during zymosan-induced inflammation and 8,9-EET injection reduced mechanical thresholds in naive mice. Conclusion Our results show that the sEH can regulate mechanical hyperalgesia during inflammation by inactivating 8,9-EET, which sensitizes TRPA1-expressing nociceptors. Therefore we suggest that influencing the CYP450 pathway, which is actually highly considered to treat cardiovascular diseases, may cause pain side effects. PMID:21970373

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

    PubMed Central

    2011-01-01

    Background To efficiently deconstruct recalcitrant plant biomass to fermentable sugars in industrial processes, biocatalysts of higher performance and lower cost are required. The genetic diversity found in the metagenomes of natural microbial biomass decay communities may harbor such enzymes. Our goal was to discover and characterize new glycoside hydrolases (GHases) from microbial biomass decay communities, especially those from unknown or never previously cultivated microorganisms. Results From the metagenome sequences of an anaerobic microbial community actively decaying poplar biomass, we identified approximately 4,000 GHase homologs. Based on homology to GHase families/activities of interest and the quality of the sequences, candidates were selected for full-length cloning and subsequent expression. As an alternative strategy, a metagenome expression library was constructed and screened for GHase activities. These combined efforts resulted in the cloning of four novel GHases that could be successfully expressed in Escherichia coli. Further characterization showed that two enzymes showed significant activity on p-nitrophenyl-α-L-arabinofuranoside, one enzyme had significant activity against p-nitrophenyl-β-D-glucopyranoside, and one enzyme showed significant activity against p-nitrophenyl-β-D-xylopyranoside. Enzymes were also tested in the presence of ionic liquids. Conclusions Metagenomics provides a good resource for mining novel biomass degrading enzymes and for screening of cellulolytic enzyme activities. The four GHases that were cloned may have potential application for deconstruction of biomass pretreated with ionic liquids, as they remain active in the presence of up to 20% ionic liquid (except for 1-ethyl-3-methylimidazolium diethyl phosphate). Alternatively, ionic liquids might be used to immobilize or stabilize these enzymes for minimal solvent processing of biomass. PMID:21816041

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

    PubMed Central

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

    2004-01-01

    We isolated a soybean saponin hydrolase from Neocosmospora vasinfecta var. vasinfecta PF1225, a filamentous fungus that can degrade soybean saponin and generate soyasapogenol B. This enzyme was found to be a monomer with a molecular mass of about 77 kDa and a glycoprotein. Nucleotide sequence analysis of the corresponding gene (sdn1) indicated that this enzyme consisted of 612 amino acids and had a molecular mass of 65,724 Da, in close agreement with that of the apoenzyme after the removal of carbohydrates. The sdn1 gene was successfully expressed in Trichoderma viride under the control of the cellobiohydrolase I gene promoter. The molecular mass of the recombinant enzyme, about 69 kDa, was smaller than that of the native enzyme due to fewer carbohydrate modifications. Examination of the degradation products obtained by treatment of soyasaponin I with the recombinant enzyme showed that the enzyme hydrolyzed soyasaponin I to soyasapogenol B and triose [α-l-rhamnopyranosyl (1→2)-β-d-galactopyranosyl (1→2)-d-glucuronopyranoside]. Also, when soyasaponin II and soyasaponin V, which are different from soyasaponin I only in constituent saccharides, were treated with the enzyme, the ratio of the reaction velocities for soyasaponin I, soyasaponin II, and soyasaponin V was 2,680:886:1. These results indicate that this enzyme recognizes the fine structure of the carbohydrate moiety of soyasaponin in its catalytic reaction. The amino acid sequence of this enzyme predicted from the DNA sequence shows no clear homology with those of any of the enzymes involved in the hydrolysis of carbohydrates. PMID:14766566

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

    PubMed

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

    2016-06-28

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

  12. Optical solid-state detection of organophosphates using organophosphorus hydrolase.

    PubMed

    White, Brandy J; Harmon, H James

    2005-04-15

    We have developed a sensor surface for optical detection of organophosphates based on reversible inhibition of organophosphorus hydrolase (OPH) by copper complexed meso-tri(4-sulfonato phenyl) mono(4-carboxy phenyl) porphyrin (CuC1TPP). OPH immobilized onto glass microscope slides retains catalytic activity for more than 232 days. CuC1TPP is a reversible, competitive inhibitor of OPH, binding at the active site of the immobilized enzyme. The absorbance spectrum of the porphyrin-enzyme complex is measured via planar waveguide evanescent wave absorbance spectroscopy using a blue LED as a light source and an Ocean Optics USB2000 as the spectrophotometer. The characteristics of the absorbance spectrum of CuC1TPP are specific and different when the porphyrin is bound to the enzyme or is bound non-specifically to the surface of the slide. Addition of a substrate of OPH such as one of the organophosphates paraoxon, coumaphos, diazinon, or malathion displaces the porphyrin from the enzyme resulting in reduced absorbance intensity at 412 nm. Absorbance changes at 412 nm show log-linear dependence on substrate concentration. Paraoxon concentrations between 7 parts per trillion (ppt) and 14 parts per million (ppm) were investigated and a 3:1 S/N detection limit of 7 ppt was determined. Concentrations of 700 ppt to 40 ppm were investigated for diazinon, malathion, and coumaphos with detection limits of 800 ppt, 1 part per billion, and 250 ppt, respectively. This optical technique does not require the addition of reagents or solutions other than the sample and absorbance spectra can be collected in less than 6 s. PMID:15741066

  13. Prunasin hydrolases during fruit development in sweet and bitter almonds.

    PubMed

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

    2012-04-01

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

  14. Long-acting cocaine hydrolase for addiction therapy.

    PubMed

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

    2016-01-12

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

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

    SciTech Connect

    Li L. L.; van der Lelie D.; Taghavi, S.; McCorkle, S. M.; Zhang, Y.-B.; Blewitt, M. G.; Brunecky, R.; Adney, W. S.; Himmel, M. E.; Brumm, P.; Drinkwater, C.; Mead, D. A.; Tringe, S. G.

    2011-08-01

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

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

    PubMed Central

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

    2001-01-01

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

  17. Reward and Toxicity of Cocaine Metabolites Generated by Cocaine Hydrolase.

    PubMed

    Murthy, Vishakantha; Geng, Liyi; Gao, Yang; Zhang, Bin; Miller, Jordan D; Reyes, Santiago; Brimijoin, Stephen

    2015-08-01

    Butyrylcholinesterase (BChE) gene therapy is emerging as a promising concept for treatment of cocaine addiction. BChE levels after gene transfer can rise 1000-fold above those in untreated mice, making this enzyme the second most abundant plasma protein. For months or years, gene transfer of a BChE mutated into a cocaine hydrolase (CocH) can maintain enzyme levels that destroy cocaine within seconds after appearance in the blood stream, allowing little to reach the brain. Rapid enzyme action causes a sharp rise in plasma levels of two cocaine metabolites, benzoic acid (BA) and ecgonine methyl ester (EME), a smooth muscle relaxant that is mildly hypotensive and, at best, only weakly rewarding. The present study, utilizing Balb/c mice, tested reward effects and cardiovascular effects of administering EME and BA together at molar levels equivalent to those generated by a given dose of cocaine. Reward was evaluated by conditioned place preference. In this paradigm, cocaine (20 mg/kg) induced a robust positive response but the equivalent combined dose of EME + BA failed to induce either place preference or aversion. Likewise, mice that had undergone gene transfer with mouse CocH (mCocH) showed no place preference or aversion after repeated treatments with a near-lethal 80 mg/kg cocaine dose. Furthermore, a single administration of that same high cocaine dose failed to affect blood pressure as measured using the noninvasive tail-cuff method. These observations confirm that the drug metabolites generated after CocH gene transfer therapy are safe even after a dose of cocaine that would ordinarily be lethal. PMID:25814464

  18. Alterations of intestinal glycoprotein hydrolases in congenital diabetes

    SciTech Connect

    Najjar, S.M.

    1989-01-01

    The diabetic BioBreed (BB{sub d}) rat was used for the study of the molecular structure of intestinal brush border sucrase-{alpha}-dextrinase (SD) and aminooligopeptidase (AOP) in diabetes mellitus. The specific catalytic activity of S-D and AOP in the BB{sub d} rat is normal. However, solid-phase radioimmunoassay revealed loss of some antigenic determinants in the BB{sub d} rat. S-D and AOP migrated abnormally on 6% SDS-gel electrophoresis in the BB{sub d} rat. S was larger (+5 kDa), D was either smaller (-5 kDa) or unaltered, and AOP was smaller (-5 kDa) in the BB{sub d} than in the normal Wistar. The structural abnormalities were independent of hyperglycemia or ketoacidosis and restored to normal by daily insulin treatment (NPH, 3-4 units/rat) for two to three weeks. Newly-synthesized brush border hydrolases were examined after 6 hours of intraperitoneal injection of ({sup 35}S) methionine (2 mCi) and found to be altered, suggesting that structural abnormality appeared acutely during intracellular synthesis rather than being due to slow extracellular modifications such as non-enzymatic glycosylation. Deglycosylation of brush border proteins by trifluoromethanesulfonic acid resulted in an apoprotein with normal electrophoretic migration in BB{sub d}, indicating that the alteration was due to the carbohydrates component of the glycoprotein. Pulse-chase studies with ({sup 35}S) methionine were consistent with normal protein an co-translational and initial N-linked carbohydrate assembly in association with the endoplasmic reticulum in BB{sub d}. However, the post-translational maturation of N-linked and addition of 0-linked carbohydrate chains in Golgi were prolonged, and produced a larger single-chain precursor of S-D in BB{sub d} than normal.

  19. Analyzing S-adenosylhomocysteine hydrolase gene sequences in deuterostome genomes.

    PubMed

    Zhao, Jing-Nan; Wang, Yuan; Zhao, Bo-Sheng; Chen, Ling-Ling

    2009-12-01

    S-adenosylhomocysteine hydrolase (SAHH) gene sequences of sea-urchin, two amphioxus, sea-squirt and eight vertebrates are comparatively analyzed in the current analysis. Although SAHH protein sequences are highly conserved in these species, their nucleotide sequences are much different, ranging from 5,446 bp in amphioxus to 40,174 bp in zebra fish. The length divergence is mainly caused by distinct introns in some species. SAHH genes in amphioxus (or sea-urchin), sea-squirt and vertebrates are composed of eight, nine and ten exons, respectively. Sequence alignment shows that exon 3 in amphioxus and sea-urchin is similar to exons 3 + 4 in vertebrates, exon 5 in amphioxus and sea-urchin is similar to exons 5 + 6 in sea-squirt, and the two exons are fused into exon 6 in vertebrates. Furthermore, exon 7 in sea-squirt is similar to exons 7 + 8 in vertebrates, indicating that exon-fission and exon-fusion events have been taken place during the evolution of deuterostome SAHH genes. Active sites and NAD+-binding sites are located in exons 2 7 in amphioxus, which are dispersed into much more exons along with the evolution of vertebrates. It is speculated that ten-exon organization of SAHH gene occurred after the separation of invertebrates and vertebrates. Synonymous and non-synonymous substitution analysis shows that negative selection plays a dominant role in the evolution of SAHH genes. Phylogenetic analysis shows that SAHH genes in amphioxus, sea-urchin and sea-squirt form a cluster and locate at the base of neighbor-joining tree, suggesting that they are the archetype of vertebrate SAHH genes. PMID:19795919

  20. GENETIC VARIATION IN SOLUBLE EPOXIDE HYDROLASE (EPHX2) AND RISK OF CORONARY HEART DISEASE: THE ATHEROSCLEROSIS RISK IN COMMUNITIES (ARIC) STUDY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Endothelial dysfunction contributes to the development of coronary heart disease (CHD). Soluble epoxide hydrolase metabolizes epoxyeicosatrienoic acids in the vasculature and regulates endothelial function. We sought to determine whether genetic variation in soluble epoxide hydrolase (EPHX2) was ass...

  1. Genetic variation in soluble epoxide hydrolase (EPHX2) and risk of coronary heart disease: The Atherosclerosis Risk in Communities (ARIC) study

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Endothelial dysfunction contributes to the development of coronary heart disease (CHD). Soluble epoxide hydrolase metabolizes epoxyeicosatrienoic acids in the vasculature and regulates endothelial function. We sought to determine whether genetic variation in soluble epoxide hydrolase (EPHX2) was ass...

  2. Evaluation of NHS Carbamates as a Potent and Selective Class of Endocannabinoid Hydrolase Inhibitors

    PubMed Central

    2013-01-01

    Monoacylglycerol lipase (MAGL) is a principal metabolic enzyme responsible for hydrolyzing the endogenous cannabinoid (endocannabinoid) 2-arachidonoylglycerol (2-AG). Selective inhibitors of MAGL offer valuable probes to further understand the enzyme’s function in biological systems and may lead to drugs for treating a variety of diseases, including psychiatric disorders, neuroinflammation, and pain. N-Hydroxysuccinimidyl (NHS) carbamates have recently been identified as a promising class of serine hydrolase inhibitors that shows minimal cross-reactivity with other proteins in the proteome. Here, we explore NHS carbamates more broadly and demonstrate their potential as inhibitors of endocannabinoid hydrolases and additional enzymes from the serine hydrolase class. We extensively characterize an NHS carbamate 1a (MJN110) as a potent, selective, and in-vivo-active MAGL inhibitor. Finally, we demonstrate that MJN110 alleviates mechanical allodynia in a rat model of diabetic neuropathy, marking NHS carbamates as a promising class of MAGL inhibitors. PMID:23731016

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

    PubMed

    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. PMID:25905461

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

    PubMed Central

    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. PMID:25905461

  5. Competitive Activity-Based Protein Profiling Identifies Aza-β-Lactams as a Versatile Chemotype for Serine Hydrolase Inhibition

    PubMed Central

    Zuhl, Andrea M.; Mohr, Justin T.; Bachovchin, Daniel A.; Niessen, Sherry; Hsu, Ku-Lung; Berlin, Jacob M.; Dochnahl, Maximilian; López-Alberca, María P.; Fu, Gregory C.; Cravatt, Benjamin F.

    2012-01-01

    Serine hydrolases are one of the largest and most diverse enzyme classes in Nature. Most serine hydrolases lack selective inhibitors, which are needed for assigning functions to these enzymes. We recently discovered a set of aza-β-lactams (ABLs) that act as potent and selective inhibitors of the mammalian serine hydrolase protein-phosphatase methylesterase-1 (PME-1). The ABLs inactivate PME-1 by covalent acylation of the enzyme’s serine nucleophile, suggesting that they could offer a general scaffold for serine hydrolase inhibitor discovery. Here, we have tested this hypothesis by screening ABLs more broadly against cell and tissue proteomes by competitive activity-based protein profiling (ABPP), leading to the discovery of lead inhibitors for several serine hydrolases, including the uncharacterized enzyme alpha, beta-hydrolase-10 (ABHD10). ABPP-guided medicinal chemistry yielded a compound ABL303 that potently (IC50 value ~ 30 nM) and selectively inactivated ABHD10 in vitro and in living cells. A comparison of optimized inhibitors for PME-1 and ABHD10 indicates that modest structural changes that alter steric bulk can tailor the ABL to selectively react with distinct, sequence-unrelated serine hydrolases. Our findings, taken together, designate the ABL as a versatile reactive group for creating first-in-class serine hydrolase inhibitors. PMID:22400490

  6. Competitive activity-based protein profiling identifies aza-β-lactams as a versatile chemotype for serine hydrolase inhibition.

    PubMed

    Zuhl, Andrea M; Mohr, Justin T; Bachovchin, Daniel A; Niessen, Sherry; Hsu, Ku-Lung; Berlin, Jacob M; Dochnahl, Maximilian; López-Alberca, María P; Fu, Gregory C; Cravatt, Benjamin F

    2012-03-21

    Serine hydrolases are one of the largest and most diverse enzyme classes in Nature. Most serine hydrolases lack selective inhibitors, which are valuable probes for assigning functions to these enzymes. We recently discovered a set of aza-β-lactams (ABLs) that act as potent and selective inhibitors of the mammalian serine hydrolase protein-phosphatase methylesterase-1 (PME-1). The ABLs inactivate PME-1 by covalent acylation of the enzyme's serine nucleophile, suggesting that they could offer a general scaffold for serine hydrolase inhibitor discovery. Here, we have tested this hypothesis by screening ABLs more broadly against cell and tissue proteomes by competitive activity-based protein profiling (ABPP), leading to the discovery of lead inhibitors for several serine hydrolases, including the uncharacterized enzyme α,β-hydrolase domain-containing 10 (ABHD10). ABPP-guided medicinal chemistry yielded a compound ABL303 that potently (IC(50) ≈ 30 nM) and selectively inactivated ABHD10 in vitro and in living cells. A comparison of optimized inhibitors for PME-1 and ABHD10 indicates that modest structural changes that alter steric bulk can tailor the ABL to selectively react with distinct, distantly related serine hydrolases. Our findings, taken together, designate the ABL as a versatile reactive group for creating first-in-class serine hydrolase inhibitors. PMID:22400490

  7. Polyglycine hydrolases: fungal b-lactamase-like endoproteases that cleave polyglycine regions within plant class IV chitinases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyglycine hydrolases are secreted fungal proteases that cleave glycine-glycine peptide bonds in the inter-domain linker region of specific plant defense chitinases. Previously, we reported the catalytic activity of polyglycine hydrolases from the phytopathogens Epicoccum sorghi (Es-cmp) and Cochli...

  8. Structural Determinants Allowing Transferase Activity in SENSITIVE TO FREEZING 2, Classified as a Family I Glycosyl Hydrolase*

    PubMed Central

    Roston, Rebecca L.; Wang, Kun; Kuhn, Leslie A.; Benning, Christoph

    2014-01-01

    SENSITIVE TO FREEZING 2 (SFR2) is classified as a family I glycosyl hydrolase but has recently been shown to have galactosyltransferase activity in Arabidopsis thaliana. Natural occurrences of apparent glycosyl hydrolases acting as transferases are interesting from a biocatalysis standpoint, and knowledge about the interconversion can assist in engineering SFR2 in crop plants to resist freezing. To understand how SFR2 evolved into a transferase, the relationship between its structure and function are investigated by activity assay, molecular modeling, and site-directed mutagenesis. SFR2 has no detectable hydrolase activity, although its catalytic site is highly conserved with that of family 1 glycosyl hydrolases. Three regions disparate from glycosyl hydrolases are identified as required for transferase activity as follows: a loop insertion, the C-terminal peptide, and a hydrophobic patch adjacent to the catalytic site. Rationales for the effects of these regions on the SFR2 mechanism are discussed. PMID:25100720

  9. Immunocytochemical Localization of Prunasin Hydrolase and Mandelonitrile Lyase in Stems and Leaves of Prunus serotina.

    PubMed Central

    Swain, E.; Poulton, J. E.

    1994-01-01

    In macerates of black cherry (Prunus serotina Ehrh.) leaves and stems, (R)-prunasin is catabolized to HCN, benzaldehyde, and D-glucose by the sequential action of prunasin hydrolase (EC 3.2.1.21) and (R)-(+)-mandelonitrile lyase (EC 4.1.2.10). Immuno-cytochemical techniques have shown that within these organs prunasin hydrolase occurs within the vacuoles of phloem parenchyma cells. In arborescent leaves, mandelonitrile lyase was also located in phloem parenchyma vacuoles, but comparison of serial sections revealed that these two degradative enzymes are usually localized within different cells. PMID:12232409

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

    PubMed

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

    2016-09-01

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

  11. Molecular Models to Emulate Confinement Effects on the Internal Dynamics of Organophosphorous Hydrolase

    SciTech Connect

    Gomes, Diego Enry B.; Lins, Roberto D.; Pascutti, Pedro G.; Straatsma, TP; Soares, Thereza A.

    2008-09-28

    The confinement of the metalloenzyme organophosphorous hydrolase in functionalized mesoporous silica (FMS) enhances the stability and increases catalytic specific activity by 200% compared to the enzyme in solution. The mechanism by which these processes take place is not well understood. We have developed two coarse-grain models of confinement to provide insights into how the nanocage environment steers enzyme conformational dynamics towards enhanced stability and enzymatic activity. The structural dynamics of organophosphorous hydrolase under the two confinement models are very distinct from each other. Comparisons of the present simulations show that only one model leads to an accurate depiction of the internal dynamics of the enzyme.

  12. Diversity of glycosyl hydrolase enzymes from metagenome and their application in food industry.

    PubMed

    Sathya, T A; Khan, Mahejibin

    2014-11-01

    Traditional use of enzymes for food processing and production of food ingredients resulted in fast-growing enzyme industries world over. The advances in technologies gave rise to exploring newer enzymes and/or modified enzymes for specific application. Search for novel enzymes that can augment catalytic efficiency and advances in molecular biology techniques including sequencing has targeted microbial diversity through metagenomic approaches for sourcing enzymes from difficult to culture organisms. Such mining studies have received more attention in characterizing hydrolases, their prevalence, broad substrate specificities, stability, and independence of cofactors. The focus on glycosyl hydrolases from metagenome for their application in food sector is reviewed. PMID:25311940

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

    SciTech Connect

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

    2011-12-31

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  17. Oxidoreductive cellulose depolymerization by the enzymes cellobiose dehydrogenase and glycoside hydrolase 61.

    PubMed

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

    2011-10-01

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

  18. Inhibitory activity of S-adenosylhomocysteine hydrolase inhibitors against human cytomegalovirus replication.

    PubMed

    Snoeck, R; Andrei, G; Neyts, J; Schols, D; Cools, M; Balzarini, J; De Clercq, E

    1993-07-01

    Various acyclic and carbocyclic adenosine analogues, which are apparently targeted at the S-adenosylhomocysteine (AdoHcy) hydrolase have been reported to inhibit the replication of a number of pox-, rhabdo-, paramyxo-, arena-, and reoviruses. Here we show that this activity spectrum extends to human cytomegalovirus (HCMV). Of the compounds tested, neplanocin A, 3-deazaneplanocin A, 6'-C-methylneplanocin A and 5'-noraristeromycin were found to be the most potent inhibitors of HCMV replication in vitro. Their 50% inhibitory concentration ranged from 0.05 to 1.35 micrograms/ml. In general, the anti-HCMV activity of the adenosine analogues correlated well with their affinity (Ki) for AdoHcy hydrolase, suggesting that AdoHcy hydrolase may be considered as a target enzyme for anti-HCMV agents. For four compounds (3-deazaneplanocin A, 6'-C-methylneplanocin A (isomers I and II) and 3-deazaadenosine), anti-HCMV potency was greater than could be expected solely from their interaction with AdoHcy hydrolase, suggesting that these compounds may be functioning by an additional mechanism. PMID:8215298

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

    PubMed Central

    Ha, Christina Hung Hung; Fatima, Ayesha; Gaurav, Anand

    2015-01-01

    Human African Trypanosomiasis is endemic to 37 countries of sub-Saharan Africa. It is caused by two related species of Trypanosoma brucei. Current therapies suffer from resistance and public accessibility of expensive medicines. Finding safer and effective therapies of natural origin is being extensively explored worldwide. Pentamidine is the only available therapy for inhibiting the P2 adenosine transporter involved in the purine salvage pathway of the trypanosomatids. The objective of the present study is to use computational studies for the investigation of the probable trypanocidal mechanism of flavonoids. Docking experiments were carried out on eight flavonoids of varying level of hydroxylation, namely, flavone, 5-hydroxyflavone, 7-hydroxyflavone, chrysin, apigenin, kaempferol, fisetin, and quercetin. Using AutoDock 4.2, these compounds were tested for their affinity towards inosine-adenosine-guanosine nucleoside hydrolase and the inosine-guanosine nucleoside hydrolase, the major enzymes of the purine salvage pathway. Our results showed that all of the eight tested flavonoids showed high affinities for both hydrolases (lowest free binding energy ranging from −10.23 to −7.14 kcal/mol). These compounds, especially the hydroxylated derivatives, could be further studied as potential inhibitors of the nucleoside hydrolases. PMID:26640486

  20. A novel meta-cleavage product hydrolase from Flavobacterium sp. ATCC27551

    SciTech Connect

    Khajamohiddin, Syed; Babu, Pakala Suresh; Chakka, Deviprasanna; Merrick, Mike; Bhaduri, Anirban; Sowdhamini, Ramanathan; Siddavattam, Dayananda . E-mail: sdsl@uohyd.ernet.in

    2006-12-22

    The organophosphate degrading (opd) gene cluster of plasmid pPDL2 of Flavobacterium sp. ATCC27551 contains a novel open-reading frame, orf243. This was predicted to encode an {alpha}/{beta} hydrolase distantly related to the meta-fission product (MFP) hydrolases such as XylF, PhnD, and CumD. By homology modeling Orf243 has most of the structural features of MFP hydrolases including the characteristic active site catalytic triad. The purified protein (designated MfhA) is a homotetramer and shows similar affinity for 2-hydroxy-6-oxohepta-2,4-dienoate (HOHD), 2-hydroxymuconic semialdehyde (HMSA), and 2-hydroxy-5-methylmuconic semialdehyde (HMMSA), the meta-fission products of 3-methyl catechol, catechol, and 4-methyl catechol. The unique catalytic properties of MfhA and the presence near its structural gene of cis-elements required for transposition suggest that mfhA has evolved towards encoding a common hydrolase that can act on meta-fission products containing either aldehyde or ketone groups.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several atoxigenic Aspergillus flavus isolates, including some biocontrol agents, and one toxigenic isolate were surveyed for the ability to produce extracellular xylanolytic and pectinolytic hydrolases. All of the tested isolates displayed good production of endoxylanases when grown on a medium ut...

  2. Proteomic Analysis of a Novel Bacillus Jumbo Phage Revealing Glycoside Hydrolase As Structural Component

    PubMed Central

    Yuan, Yihui; Gao, Meiying

    2016-01-01

    Tailed phages with genomes of larger than 200 kbp are classified as Jumbo phages and exhibited extremely high uncharted diversity. The genomic annotation of Jumbo phage is often disappointing because most of the predicted proteins, including structural proteins, failed to make good hits to the sequences in the databases. In this study, 23 proteins of a novel Bacillus Jumbo phage, vB_BpuM_BpSp, were identified as phage structural proteins by the structural proteome analysis, including 14 proteins of unknown function, 5 proteins with predicted function as structural proteins, a glycoside hydrolase, a Holliday junction resolvase, a RNA-polymerase β-subunit, and a host-coding portal protein, which might be hijacked from the host strain during phage virion assembly. The glycoside hydrolase (Gp255) was identified as phage virion component and was found to interact with the phage baseplate protein. Gp255 shows specific lytic activity against the phage host strain GR8 and has high temperature tolerance. In situ peptidoglycan-hydrolyzing activities analysis revealed that the expressed Gp255 and phage structural proteome exhibited glycoside hydrolysis activity against the tested GR8 cell extracts. This study identified the first functional individual structural glycoside hydrolase in phage virion. The presence of activated glycoside hydrolase in phage virions might facilitate the injection of the phage genome during infection by forming pores on the bacterial cell wall. PMID:27242758

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

    EPA Science Inventory

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

  4. Rehabilitation of faulty kinetic determinations and misassigned glycoside hydrolase family of retaining mechanism ß-xylosidases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We obtained Cx1 from a commercial supplier, whose catalog listed it as a ß-xylosidase of glycoside hydrolase family 43. NMR experiments indicate retention of anomeric configuration in its reaction stereochemistry, opposing the assignment of GH43, which follows an inverting mechanism. Partial protein...

  5. Highly active ß-xylosidases of glycoside hydrolase family 43 operating on natural and artificial substrates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The hemicellulose xylan constitutes a major portion of plant biomass, a renewable feedstock available for conversion to biofuels and other bioproducts. ß-xylosidase operates in the deconstruction of the polysaccharide to fermentable sugars. Glycoside hydrolase family 43 has been identified as a so...

  6. Expression of recombinant organophosphorus hydrolase in the original producer of the enzyme, Sphingobium fuliginis ATCC 27551.

    PubMed

    Nakayama, Kosuke; Ohmori, Takeshi; Ishikawa, Satoshi; Iwata, Natsumi; Seto, Yasuo; Kawahara, Kazuyoshi

    2016-05-01

    The plasmid encoding His-tagged organophosphorus hydrolase (OPH) cloned from Sphingobium fuliginis was modified to be transferred back to this bacterium. The replication function of S. amiense plasmid was inserted at downstream of OPH gene, and S. fuliginis was transformed with this plasmid. The transformant produced larger amount of active OPH with His-tag than E. coli. PMID:26784883

  7. Purification and Characterization of TrzF: Biuret Hydrolysis by Allophanate Hydrolase Supports Growth

    PubMed Central

    Shapir, Nir; Cheng, Gang; Sadowsky, Michael J.; Wackett, Lawrence P.

    2006-01-01

    TrzF, the allophanate hydrolase from Enterobacter cloacae strain 99, was cloned, overexpressed in the presence of a chaperone protein, and purified to homogeneity. Native TrzF had a subunit molecular weight of 65,401 and a subunit stoichiometry of α2 and did not contain significant levels of metals. TrzF showed time-dependent inhibition by phenyl phosphorodiamidate and is a member of the amidase signature protein family. TrzF was highly active in the hydrolysis of allophanate but was not active with urea, despite having been previously considered a urea amidolyase. TrzF showed lower activity with malonamate, malonamide, and biuret. The allophanate hydrolase from Pseudomonas sp. strain ADP, AtzF, was also shown to hydrolyze biuret slowly. Since biuret and allophanate are consecutive metabolites in cyanuric acid metabolism, the low level of biuret hydrolase activity can have physiological significance. A recombinant Escherichia coli strain containing atzD, encoding cyanuric acid hydrolase that produces biuret, and atzF grew slowly on cyanuric acid as a source of nitrogen. The amount of growth produced was consistent with the liberation of 3 mol of ammonia from cyanuric acid. In vitro, TrzF was shown to hydrolyze biuret to liberate 3 mol of ammonia. The biuret hydrolyzing activity of TrzF might also be physiologically relevant in native strains. E. cloacae strain 99 grows on cyanuric acid with a significant accumulation of biuret. PMID:16597948

  8. High-Throughput In Vitro Glycoside Hydrolase (HIGH) Screening for Enzyme Discovery

    SciTech Connect

    Kim, Tae-Wan; Chokhawala, Harshal A.; Hess, Matthias; Dana, Craig M.; Baer, Zachary; Sczyrba, Alexander; Rubin, Edward M.; Blanch, Harvey W.; Clark, Douglas S.

    2011-09-16

    A high-throughput protein-expression and screening method (HIGH method, see picture) provides a rapid approach to the discovery of active glycoside hydrolases in environmental samples. Finally, HIGH screening combines cloning, protein expression, and enzyme hydrolysis in one pot; thus, the entire process from gene expression to activity detection requires only three hours.

  9. Cloning, Expression and Characterization of a Glycoside Hydrolase Family 39 Xylosidase from Bacillus Halodurans C-125

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The gene encoding a glycoside hydrolase family 39 xylosidase (BH1068) from the alkaliphile Bacillus halodurans strain C-125 was cloned with a C-terminal His-tag and the recombinant gene product termed XylBH1068 was expressed in E. coli. Of the artificial substrates tested, XylBH1068 hydrolyzed nitro...

  10. BIODEGRADATION OF ORGANOPHOSPHORUS PESTICIDES BY SURFACE-EXPRESSED ORGANOPHOSPHORUS HYDROLASE. (R823663)

    EPA Science Inventory

    Organophosphorus hydrolase (OPH) was displayed and anchored onto the surface of
    Escherichia coli using an Lpp-OmpA fusion system. Production of the fusion proteins in membrane
    fractions was verified by immunoblotting with OmpA antisera. inclusion of the organophosphorus
    ...

  11. ORGANOPHOSPHORUS HYDROLASE-BASED AMPEROMETRIC SENSOR: MODULATION OF SENSITIVITY AND SUBSTRATE SELECTIVITY

    EPA Science Inventory

    The detection of organophosphate (OP) insecticides with nitrophenyl substituents is reported using an enzyme electrode composed of Organophosphorus Hydrolase (OPH) and albumin co-immobilized to a nylon net and attached to a carbon paste electrode. The mechanism for this biosen...

  12. Epoxide hydrolase activities and epoxy fatty acids in the mosquito Culex quinquefasciatus

    PubMed Central

    Xu, Jiawen; Morisseau, Christophe; Yang, Jun; Mamatha, Dadala M.

    2015-01-01

    Culex mosquitoes have emerged as important model organisms for mosquito biology, and are disease vectors for multiple mosquito-borne pathogens, including West Nile virus. We characterized epoxide hydrolase activities in the mosquito Culex quinquefasciatus, which suggested multiple forms of epoxide hydrolases were present. We found EH activities on epoxy eicosatrienoic acids (EETs). EETs and other eicosanoids are well-established lipid signaling molecules in vertebrates. We showed EETs can be synthesized in vitro from arachidonic acids by mosquito lysate, and EETs were also detected in vivo both in larvae and adult mosquitoes by LC-MS/MS. The EH activities on EETs can be induced by blood feeding, and the highest activity was observed in the midgut of female mosquitoes. The enzyme activities on EETs can be inhibited by urea-based inhibitors designed for mammalian soluble epoxide hydrolases (sEH). The sEH inhibitors have been shown to play diverse biological roles in mammalian systems, and they can be useful tools to study the function of EETs in mosquitoes. Besides juvenile hormone metabolism and detoxification, insect epoxide hydrolases may also play a role in regulating lipid signaling molecules, such as EETs and other epoxy fatty acids, synthesized in vivo or obtained from blood feeding by female mosquitoes. PMID:25686802

  13. Proteomic Analysis of a Novel Bacillus Jumbo Phage Revealing Glycoside Hydrolase As Structural Component.

    PubMed

    Yuan, Yihui; Gao, Meiying

    2016-01-01

    Tailed phages with genomes of larger than 200 kbp are classified as Jumbo phages and exhibited extremely high uncharted diversity. The genomic annotation of Jumbo phage is often disappointing because most of the predicted proteins, including structural proteins, failed to make good hits to the sequences in the databases. In this study, 23 proteins of a novel Bacillus Jumbo phage, vB_BpuM_BpSp, were identified as phage structural proteins by the structural proteome analysis, including 14 proteins of unknown function, 5 proteins with predicted function as structural proteins, a glycoside hydrolase, a Holliday junction resolvase, a RNA-polymerase β-subunit, and a host-coding portal protein, which might be hijacked from the host strain during phage virion assembly. The glycoside hydrolase (Gp255) was identified as phage virion component and was found to interact with the phage baseplate protein. Gp255 shows specific lytic activity against the phage host strain GR8 and has high temperature tolerance. In situ peptidoglycan-hydrolyzing activities analysis revealed that the expressed Gp255 and phage structural proteome exhibited glycoside hydrolysis activity against the tested GR8 cell extracts. This study identified the first functional individual structural glycoside hydrolase in phage virion. The presence of activated glycoside hydrolase in phage virions might facilitate the injection of the phage genome during infection by forming pores on the bacterial cell wall. PMID:27242758

  14. Channel Catfish, Ictalurus punctatus, ubiquitin carboxy-terminal hydrolase L5 cDNA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ubiquitin-proteasome cycle is a complex, non-lysosomal biochemical process for intracellular protein degradation. This process involves many enzymes. One of them is ubiquitin carboxy-terminal hydrolase (UCT). In this report, we cloned, sequenced and characterized the channel catfish UCT L5 cDNA....

  15. Functional characterization and structural modeling of synthetic polyester-degrading hydrolases from Thermomonospora curvata

    PubMed Central

    2014-01-01

    Thermomonospora curvata is a thermophilic actinomycete phylogenetically related to Thermobifida fusca that produces extracellular hydrolases capable of degrading synthetic polyesters. Analysis of the genome of T. curvata DSM43183 revealed two genes coding for putative polyester hydrolases Tcur1278 and Tcur0390 sharing 61% sequence identity with the T. fusca enzymes. Mature proteins of Tcur1278 and Tcur0390 were cloned and expressed in Escherichia coli TOP10. Tcur1278 and Tcur0390 exhibited an optimal reaction temperature against p-nitrophenyl butyrate at 60°C and 55°C, respectively. The optimal pH for both enzymes was determined at pH 8.5. Tcur1278 retained more than 80% and Tcur0390 less than 10% of their initial activity following incubation for 60 min at 55°C. Tcur0390 showed a higher hydrolytic activity against poly(ε-caprolactone) and polyethylene terephthalate (PET) nanoparticles compared to Tcur1278 at reaction temperatures up to 50°C. At 55°C and 60°C, hydrolytic activity against PET nanoparticles was only detected with Tcur1278. In silico modeling of the polyester hydrolases and docking with a model substrate composed of two repeating units of PET revealed the typical fold of α/β serine hydrolases with an exposed catalytic triad. Molecular dynamics simulations confirmed the superior thermal stability of Tcur1278 considered as the main reason for its higher hydrolytic activity on PET. PMID:25405080

  16. A novel activity of microsomal epoxide hydrolase: metabolism of the endocannabinoid 2-arachidonoylglycerol

    PubMed Central

    Nithipatikom, Kasem; Endsley, Michael P.; Pfeiffer, Adam W.; Falck, John R.; Campbell, William B.

    2014-01-01

    Microsomal epoxide hydrolase (EPHX1, EC 3.3.2.9) is a highly abundant α/β-hydrolase enzyme that is known for its catalytical epoxide hydrolase activity. A wide range of EPHX1 functions have been demonstrated including xenobiotic metabolism; however, characterization of its endogenous substrates is limited. In this study, we present evidence that EPHX1 metabolizes the abundant endocannabinoid 2-arachidonoylglycerol (2-AG) to free arachidonic acid (AA) and glycerol. The EPHX1 metabolism of 2-AG was demonstrated using commercially available EPHX1 microsomes as well as PC-3 cells overexpressing EPHX1. Conversely, EPHX1 siRNA markedly reduced the EPHX1 expression and 2-AG metabolism in HepG2 cells and LNCaP cells. A selective EPHX1 inhibitor, 10-hydroxystearamide, inhibited 2-AG metabolism and hydrolysis of a well-known EPHX1 substrate, cis-stilbene oxide. Among the inhibitors studied, a serine hydrolase inhibitor, methoxy-arachidonyl fluorophosphate, was the most potent inhibitor of 2-AG metabolism by EPHX1 microsomes. These results demonstrate that 2-AG is an endogenous substrate for EPHX1, a potential role of EPHX1 in the endocannabinoid signaling and a new AA biosynthetic pathway. PMID:24958911

  17. γ-PGA Hydrolases of Phage Origin in Bacillus subtilis and Other Microbial Genomes.

    PubMed

    Mamberti, Stefania; Prati, Paola; Cremaschi, Paolo; Seppi, Claudio; Morelli, Carlo F; Galizzi, Alessandro; Fabbi, Massimo; Calvio, Cinzia

    2015-01-01

    Poly-γ-glutamate (γ-PGA) is an industrially interesting polymer secreted mainly by members of the class Bacilli which forms a shield able to protect bacteria from phagocytosis and phages. Few enzymes are known to degrade γ-PGA; among them is a phage-encoded γ-PGA hydrolase, PghP. The supposed role of PghP in phages is to ensure access to the surface of bacterial cells by dismantling the γ-PGA barrier. We identified four unannotated B. subtilis genes through similarity of their encoded products to PghP; in fact these genes reside in prophage elements of B. subtilis genome. The recombinant products of two of them demonstrate efficient polymer degradation, confirming that sequence similarity reflects functional homology. Genes encoding similar γ-PGA hydrolases were identified in phages specific for the order Bacillales and in numerous microbial genomes, not only belonging to that order. The distribution of the γ-PGA biosynthesis operon was also investigated with a bioinformatics approach; it was found that the list of organisms endowed with γ-PGA biosynthetic functions is larger than expected and includes several pathogenic species. Moreover in non-Bacillales bacteria the predicted γ-PGA hydrolase genes are preferentially found in species that do not have the genetic asset for polymer production. Our findings suggest that γ-PGA hydrolase genes might have spread across microbial genomes via horizontal exchanges rather than via phage infection. We hypothesize that, in natural habitats rich in γ-PGA supplied by producer organisms, the availability of hydrolases that release glutamate oligomers from γ-PGA might be a beneficial trait under positive selection. PMID:26158264

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

    PubMed Central

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

    2011-01-01

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

  19. Different types of dienelactone hydrolase in 4-fluorobenzoate-utilizing bacteria.

    PubMed Central

    Schlömann, M; Schmidt, E; Knackmuss, H J

    1990-01-01

    Of various benzoate-utilizing bacteria tested, Alcaligenes eutrophus 335, A. eutrophus H16, A. eutrophus JMP222, A. eutrophus JMP134, Alcaligenes strain A7, and Pseudomonas cepacia were able to grow with 4-fluorobenzoate as the sole source of carbon and energy. P. cepacia also utilizes 3-fluorobenzoate. Except for A. eutrophus JMP134, which is known to grow with 2,4-dichlorophenoxyacetate and 3-chlorobenzoate (R. H. Don and J. M. Pemberton, J. Bacteriol. 145:681-686, 1981), the strains were unable to grow at the expense of these compounds or 4-chlorobenzoate. Assays of cell extracts revealed that all strains express dienelactone hydrolase and maleylacetate reductase activities in addition to enzymes of the catechol branch of the 3-oxoadipate pathway when growing with 4-fluorobenzoate. Induction of dienelactone hydrolase and maleylacetate reductase apparently is not necessarily connected to synthesis of catechol 1,2-dioxygenase type II and chloromuconate cycloisomerase activities, which are indispensable for the degradation of chlorocatechols. Substrate specificities of the dienelactone hydrolases provisionally differentiate among three types of this activity. (i) Extracts of A. eutrophus 335, A. eutrophus H16, A. eutrophus JMP222, and Alcaligenes strain A7 convert trans-4-carboxymethylenebut-2-en-4-olide (trans-dienelactone) much faster than the cis-isomer (type I). (ii) The enzyme present in P. cepacia shows the opposite preference for the isomeric substrates (type II). (iii) Cell extracts of A. eutrophus JMP134, as well as purified dienelactone hydrolase from Pseudomonas strain B13 (E. Schmidt and H.-J. Knackmuss, Biochem. J. 192:339-347, 1980), hydrolyze both dienelactones at rates that are of the same order of magnitude (type III). This classification implies that A. eutrophus JMP134 possesses at least two different dienelactone hydrolases, one of type III encoded by the plasmid pJP4 and one of type I, which is also present in the cured strain JMP222. PMID

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

    PubMed

    Nisha, M; Satyanarayana, T

    2016-07-01

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

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

    PubMed

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

    2011-11-01

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

  2. γ-PGA Hydrolases of Phage Origin in Bacillus subtilis and Other Microbial Genomes

    PubMed Central

    Mamberti, Stefania; Prati, Paola; Cremaschi, Paolo; Seppi, Claudio; Morelli, Carlo F.; Galizzi, Alessandro; Fabbi, Massimo; Calvio, Cinzia

    2015-01-01

    Poly-γ-glutamate (γ-PGA) is an industrially interesting polymer secreted mainly by members of the class Bacilli which forms a shield able to protect bacteria from phagocytosis and phages. Few enzymes are known to degrade γ-PGA; among them is a phage-encoded γ-PGA hydrolase, PghP. The supposed role of PghP in phages is to ensure access to the surface of bacterial cells by dismantling the γ-PGA barrier. We identified four unannotated B. subtilis genes through similarity of their encoded products to PghP; in fact these genes reside in prophage elements of B. subtilis genome. The recombinant products of two of them demonstrate efficient polymer degradation, confirming that sequence similarity reflects functional homology. Genes encoding similar γ-PGA hydrolases were identified in phages specific for the order Bacillales and in numerous microbial genomes, not only belonging to that order. The distribution of the γ-PGA biosynthesis operon was also investigated with a bioinformatics approach; it was found that the list of organisms endowed with γ-PGA biosynthetic functions is larger than expected and includes several pathogenic species. Moreover in non-Bacillales bacteria the predicted γ-PGA hydrolase genes are preferentially found in species that do not have the genetic asset for polymer production. Our findings suggest that γ-PGA hydrolase genes might have spread across microbial genomes via horizontal exchanges rather than via phage infection. We hypothesize that, in natural habitats rich in γ-PGA supplied by producer organisms, the availability of hydrolases that release glutamate oligomers from γ-PGA might be a beneficial trait under positive selection. PMID:26158264

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

    SciTech Connect

    Einstein, R.; Gabel, C.A. )

    1991-01-01

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

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

    SciTech Connect

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

    2015-07-29

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

  5. Subtherapeutic levels of antibiotics in poultry feeds and their effects on weight gain, feed efficiency, and bacterial cholyltaurine hydrolase activity.

    PubMed Central

    Feighner, S D; Dashkevicz, M P

    1987-01-01

    A radiochemical method was developed to estimate cholyltaurine hydrolase potentials and rates of cholyltaurine hydrolysis in chicken intestinal homogenates. This method was used to monitor the effects of antibiotic feed additives on cholyltaurine hydrolase activity. Avoparcin, bacitracin methylenedisalisylic acid, efrotomycin, lincomycin, penicillin G procaine, and virginiamycin improved rate of weight gain and feed conversion of chicks and decreased cholyltaurine hydrolase activity in ileal homogenates relative to those of nonmedicated control birds. The results provided the first evidence that feeding selected antibiotics at subtherapeutic levels can affect bile acid-transforming enzymes in small-intestinal homogenates. The inverse relationship between growth performance and cholyltaurine hydrolase activity raises the possibility that specific inhibitors of this enzyme may promote weight gain and feed conversion in livestock and thereby reduce or eliminate the need for antibiotic feed additives. PMID:3566269

  6. The cytotoxic activity of Bacillus anthracis lethal factor is inhibited by leukotriene A4 hydrolase and metallopeptidase inhibitors.

    PubMed Central

    Menard, A; Papini, E; Mock, M; Montecucco, C

    1996-01-01

    The lethal factor of Bacillus anthracis is central to the pathogenesis of anthrax. Its mechanism of action is still unknown. Recently, on the basis of sequence similarities, we suggested that lethal factor might act similarly to leukotriene A4 hydrolase (LTA4), a bifunctional enzyme also endowed with a metallopeptidase activity. Here we show that some inhibitors of the LTA4 hydrolase and metallopeptidase activities of LTA4 hydrolase also affect the cytotoxicity of the anthrax lethal factor on macrophage cell lines, without interfering with the ability of the lethal factor to enter cells. These results support the proposal that anthrax lethal factor might display in the cytosol of intoxicated cells a peptidase activity similar to that of LTA4 hydrolase. PMID:8973585

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

    SciTech Connect

    McCulloch, Kathryn M.; Mukherjee, Tathagata; Begley, Tadhg P.; Ealick, Steven E.

    2010-06-22

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

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

    SciTech Connect

    Hu, Wenxin; Wang, Qihai; Bi, Ruchang

    2005-12-01

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

  9. A New Insight into the Physiological Role of Bile Salt Hydrolase among Intestinal Bacteria from the Genus Bifidobacterium

    PubMed Central

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

    2014-01-01

    This study analyzes the occurrence of bile salt hydrolase in fourteen strains belonging to the genus Bifidobacterium. Deconjugation activity was detected using a plate test, two-step enzymatic reaction and activity staining on a native polyacrylamide gel. Subsequently, bile salt hydrolases from B. pseudocatenulatum and B. longum subsp. suis were purified using a two-step chromatographic procedure. Biochemical characterization of the bile salt hydrolases showed that the purified enzymes hydrolyzed all of the six major human bile salts under the pH and temperature conditions commonly found in the human gastrointestinal tract. Next, the dynamic rheometry was applied to monitor the gelation process of deoxycholic acid under different conditions. The results showed that bile acids displayed aqueous media gelating properties. Finally, gel-forming abilities of bifidobacteria exhibiting bile salt hydrolase activity were analyzed. Our investigations have demonstrated that the release of deconjugated bile acids led to the gelation phenomenon of the enzymatic reaction solution containing purified BSH. The presented results suggest that bile salt hydrolase activity commonly found among intestinal microbiota increases hydrogel-forming abilities of certain bile salts. To our knowledge, this is the first report showing that bile salt hydrolase activity among Bifidobacterium is directly connected with the gelation process of bile salts. In our opinion, if such a phenomenon occurs in physiological conditions of human gut, it may improve bacterial ability to colonize the gastrointestinal tract and their survival in this specific ecological niche. PMID:25470405

  10. Plant Microsomal Phospholipid Acyl Hydrolases Have Selectivities for Uncommon Fatty Acids.

    PubMed Central

    Stahl, U.; Banas, A.; Stymne, S.

    1995-01-01

    Developing endosperms and embryos accumulating triacylglycerols rich in caproyl (decanoyl) groups (i.e. developing embryos of Cuphea procumbens and Ulmus glabra) had microsomal acyl hydrolases with high selectivities toward phosphatidylcholine with this acyl group. Similarly, membranes from Euphorbia lagascae and Ricinus communis endosperms, which accumulate triacylglycerols with vernoleate (12-epoxy-octadeca-9-enoate) and ricinoleate (12-hydroxy-octadeca-9-enoate), respectively, had acyl hydrolases that selectively removed their respective oxygenated acyl group from the phospholipids. The activities toward phospholipid substrates with epoxy, hydroxy, and medium-chain acyl groups varied greatly between microsomal preparations from different plant species. Epoxidated and hydroxylated acyl groups in sn-1 and sn-2 positions of phosphatidylcholine and in sn-1-lysophosphatidylcholine were hydrolyzed to a similar extent, whereas the hydrolysis of caproyl groups was highly dependent on the positional localization. PMID:12228415

  11. Carbocyclic adenosine analogues as S-adenosylhomocysteine hydrolase inhibitors and antiviral agents: recent advances.

    PubMed

    De Clercq, E

    1998-01-01

    Various carbocyclic analogues of adenosine, including aristeromycin (carbocyclic adenosine), carbocyclic 3-deazaadenosine, neplanocin A, 3-deazaneplanocin A, the 5'-nor derivatives of aristeromycin, carbocylic 3-deazaadenosine, neplanocin A and 3-deazaneplanocin A, and the 2-halo (i.e., 2-fluoro) and 6'-R-alkyl (i.e., 6'-R-methyl) derivatives of neplanocin A have been recognized as potent inhibitors of S-adenosylhomocysteine (AdoHcy) hydrolase. This enzyme plays a key role in methylation reactions depending on S-adenosylmethionine (AdoMet) as methyl donor. AdoHcy hydrolase inhibitors have been shown to exert broad-spectrum antiviral activity against pox-, paramyxo-, rhabdo-, filo-, bunya-, arena-, and reoviruses. They also interfere with the replication of human immunodeficiency virus through inhibition of the Tat transactivation process. PMID:9708366

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

    PubMed

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

    2016-01-01

    This study reports thermophilic fungus Malbranchea cinnamomea as an important source of lignocellulolytic enzymes. The secretome analysis using LC-MS/MS orbitrap showed that fungus produced a spectrum of glycosyl hydrolases (cellulase/hemicellulase), polysaccharide lyases (PL) and carbohydrate esterases (CE) in addition to cellobiose dehydrogenase (CDH) indicating the presence of functional classical and oxidative cellulolytic mechanisms. The protein fractions in the secretome resolved by ion exchange chromatography were analyzed for ability to hydrolyze alkali treated carrot grass (ATCG) in the presence of Mn(2+)/Cu(2+). This strategy in tandem with peptide mass fingerprinting led to identification of metal dependent protein hydrolases with no apparent hydrolytic activity, however, showed 5.7 folds higher saccharification in presence of Mn(2+). Furthermore, adding different protein fractions to commercial cellulase (Novozymes: Cellic CTec2) resulted in enhanced hydrolysis of ATCG ranging between 1.57 and 3.43 folds indicating the enzymes from M. cinnamomea as catalytically efficient. PMID:26476165

  13. [Effect of chitosan on the cell ultrastructure and activity of hydrolases in tobacco leaves].

    PubMed

    Nagorskaia, V P; Reunov, A V; Lapshina, L A; Davydova, V N; Ermak, I M

    2012-01-01

    Effect of chitosan on the mesophyll cell ultrastucture and activity of hydrolases in leaves of tobacco cv. Samsun was studied. It was shown that, in many cells, chitosan treatment stimulated the protein-synthesizing apparatus (nucleolus dimension and amount of both mitochondria and rough endoplasmic reticulum membranes increased) and, at the same time, caused some activation of lytic compartment expressed in the stimulation of the formation of dictyosomes, smooth ER elements and cytoplasmic vacuoles, which are all prominent constituents of this compartment. In biochemical experiments, it was established that chitosan substantially enhanced activity of hydrolases (acid phosphatase, RNase, proteases) in the leaves as compared to untreated leaves. In some cells chitosan treatment caused considerable destructive changes (condensation of nuclear chromatin, collapse of cytoplasm and so on) that can be classified as a result of programmed cell death development. PMID:23461036

  14. Crystallization and preliminary X-ray analysis of neoagarobiose hydrolase from Saccharophagus degradans 2-40

    PubMed Central

    Lee, Saeyoung; Lee, Jonas Yun; Ha, Sung Chul; Jung, Jina; Shin, Dong Hae; Kim, Kyoung Heon; Choi, In-Geol

    2009-01-01

    Many agarolytic bacteria degrade agar polysaccharide into the disaccharide unit neoagarobiose [O-3,6-anhydro-α-l-galactopyranosyl-(1→3)-d-galactose] using various β-agarases. Neoagarobiose hydrolase is an enzyme that acts on the α-­1,3 linkage in neoagarobiose to yield d-galactose and 3,6-anhydro-l-galactose. This activity is essential in both the metabolism of agar by agarolytic bacteria and the production of fermentable sugars from agar biomass for bioenergy production. Neoagarobiose hydrolase from the marine bacterium Saccharophagus degradans 2-40 was overexpressed in Escherichia coli and crystallized in the monoclinic space group C2, with unit-cell parameters a = 129.83, b = 76.81, c = 90.11 Å, β = 101.86°. The crystals diffracted to 1.98 Å resolution and possibly contains two molecules in the asymmetric unit. PMID:20054134

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

    PubMed Central

    Henrissat, B; Bairoch, A

    1993-01-01

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

  16. Involvement of the Saccharomyces cerevisiae hydrolase Ldh1p in lipid homeostasis.

    PubMed

    Debelyy, Mykhaylo O; Thoms, Sven; Connerth, Melanie; Daum, Günther; Erdmann, Ralf

    2011-06-01

    Here, we report the functional characterization of the newly identified lipid droplet hydrolase Ldh1p. Recombinant Ldh1p exhibits esterase and triacylglycerol lipase activities. Mutation of the serine in the hydrolase/lipase motif GXSXG completely abolished esterase activity. Ldh1p is required for the maintenance of a steady-state level of the nonpolar and polar lipids of lipid droplets. A characteristic feature of the Saccharomyces cerevisiae Δldh1 strain is the appearance of giant lipid droplets and an excessive accumulation of nonpolar lipids and phospholipids upon growth on medium containing oleic acid as a sole carbon source. Ldh1p is thought to play a role in maintaining the lipid homeostasis in yeast by regulating both phospholipid and nonpolar lipid levels. PMID:21478434

  17. Investigation of the Microheterogeneity and Aglycone Specificity-Conferring Residues of Black Cherry Prunasin Hydrolases1

    PubMed Central

    Zhou, Jiming; Hartmann, Stefanie; Shepherd, Brianne K.; Poulton, Jonathan E.

    2002-01-01

    In black cherry (Prunus serotina Ehrh.) seed homogenates, (R)-amygdalin is degraded to HCN, benzaldehyde, and glucose by the sequential action of amygdalin hydrolase (AH), prunasin hydrolase (PH), and mandelonitrile lyase. Leaves are also highly cyanogenic because they possess (R)-prunasin, PH, and mandelonitrile lyase. Taking both enzymological and molecular approaches, we demonstrate here that black cherry PH is encoded by a putative multigene family of at least five members. Their respective cDNAs (designated Ph1, Ph2, Ph3, Ph4, and Ph5) predict isoforms that share 49% to 92% amino acid identity with members of glycoside hydrolase family 1, including their catalytic asparagine-glutamate-proline and isoleucine-threonine-glutamate-asparagine-glycine motifs. Furthermore, consistent with the vacuolar/protein body location and glycoprotein character of these hydrolases, their open reading frames predict N-terminal signal sequences and multiple potential N-glycosylation sites. Genomic sequences corresponding to the open reading frames of these PHs and of the previously isolated AH1 isoform are interrupted at identical positions by 12 introns. Earlier studies established that native AH and PH display strict specificities toward their respective glucosidic substrates. Such behavior was also shown by recombinant AH1, PH2, and PH4 proteins after expression in Pichia pastoris. Three amino acid moieties that may play a role in conferring such aglycone specificities were predicted by structural modeling and comparative sequence analysis and tested by introducing single and multiple mutations into isoform AH1 by site-directed mutagenesis. The double mutant AH ID (Y200I and G394D) hydrolyzed prunasin at approximately 150% of the rate of amygdalin hydrolysis, whereas the other mutations failed to engender PH activity. PMID:12114579

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

    PubMed

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

    2015-08-01

    Clostridium acetobutylicum ATCC 824 gene CA_C0359 encodes a putative unsaturated rhamnogalacturonyl hydrolase (URH) with distant amino-acid sequence homology to YteR of Bacillus subtilis strain 168. YteR, like other URHs, has core structural homology to unsaturated glucuronyl hydrolases, but hydrolyzes the unsaturated disaccharide derivative of rhamnogalacturonan I. The crystal structure of the recombinant CA_C0359 protein was solved to 1.6 Å resolution by molecular replacement using the phase information of the previously reported structure of YteR (PDB entry 1nc5) from Bacillus subtilis strain 168. The YteR-like protein is a six-α-hairpin barrel with two β-sheet strands and a small helix overlaying the end of the hairpins next to the active site. The protein has low primary protein sequence identity to YteR but is structurally similar. The two tertiary structures align with a root-mean-square deviation of 1.4 Å and contain a highly conserved active pocket. There is a conserved aspartic acid residue in both structures, which has been shown to be important for hydration of the C=C bond during the release of unsaturated galacturonic acid by YteR. A surface electrostatic potential comparison of CA_C0359 and proteins from CAZy families GH88 and GH105 reveals the make-up of the active site to be a combination of the unsaturated rhamnogalacturonyl hydrolase and the unsaturated glucuronyl hydrolase from Bacillus subtilis strain 168. Structural and electrostatic comparisons suggests that the protein may have a slightly different substrate specificity from that of YteR. PMID:26249707

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

    PubMed

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

    2016-08-01

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

  20. Biosynthesis, glycosylation, and intracellular transport of intestinal lactase-phlorizin hydrolase in rat.

    PubMed

    Büller, H A; Montgomery, R K; Sasak, W V; Grand, R J

    1987-12-15

    The biosynthesis of rat intestinal lactase-phlorizin hydrolase was studied by pulse-labeling of jejunal explants from 5-day-old suckling rats in organ culture. Explants were either continuously labeled with [35S] methionine for 15, 30, and 60 min or pulse-labeled for 30 min and chased for various periods of time up to 6 h in the presence or absence of protease inhibitors (PI), leupeptin, phenylmethylsulfonyl fluoride, and soybean trypsin inhibitor. Lactase-phlorizin hydrolase was immunoprecipitated from microvillus membrane (MVM) and ER-Golgi fractions with monoclonal antibodies. After pulse-labeling, lactase-phlorizin hydrolase from the ER-Golgi fraction appeared on SDS-PAGE as one band of approximately 220 kDa, regardless of the presence or absence of PI in the culture media. The 220-kDa protein band could also be labeled after incubation with [2-3H]mannose. In the absence of PI, the 220-kDa band appeared in the MVM by 30 min chase, simultaneously with a 180-kDa band, and by 60 min of chase an additional band of 130 kDa was seen. With increasing time of chase, the relative intensity of the 130-kDa band increased, whereas that of the 220-kDa band decreased, suggesting a precursor-product relationship. When PI were added to the medium, the formation of the 180-kDa band was not affected, but the conversion of the 180-kDa protein to the 130-kDa protein was virtually blocked. These findings suggest that lactase-phlorizin hydrolase is initially synthesized as a glycosylated precursor of 220 kDa, which is transported to the MVM. There it undergoes the following two cleavages: first, to the 180-kDa form, which is not prevented by PI used in these experiments, and second, to the 130-kDa form inhibited by PI. PMID:3119597

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

    SciTech Connect

    Nomura, Daniel K.; Fujioka, Kazutoshi; Issa, Roger S.; Ward, Anna M.; Cravatt, Benjamin F.; Casida, John E.

    2008-04-01

    Serine hydrolase KIAA1363 is an acetyl monoalkylglycerol ether (AcMAGE) hydrolase involved in tumor cell invasiveness. It is also an organophosphate (OP) insecticide-detoxifying enzyme. The key to understanding these dual properties was the use of KIAA1363 +/+ (wildtype) and -/- (gene deficient) mice to define the role of this enzyme in brain and other tissues and its effectiveness in vivo in reducing OP toxicity. KIAA1363 was the primary AcMAGE hydrolase in brain, lung, heart and kidney and was highly sensitive to inactivation by chlorpyrifos oxon (CPO) (IC{sub 50} 2 nM) [the bioactivated metabolite of the major insecticide chlorpyrifos (CPF)]. Although there was no difference in hydrolysis product monoalkylglycerol ether (MAGE) levels in +/+ and -/- mouse brains in vivo, isopropyl dodecylfluorophosphonate (30 mg/kg) and CPF (100 mg/kg) resulted in 23-51% decrease in brain MAGE levels consistent with inhibition of AcMAGE hydrolase activity. On incubating +/+ and -/- brain membranes with AcMAGE and cytidine-5'-diphosphocholine, the absence of KIAA1363 activity dramatically increased de novo formation of platelet-activating factor (PAF) and lyso-PAF, signifying that metabolically-stabilized AcMAGE can be converted to this bioactive lipid in brain. On considering detoxification, KIAA1363 -/- mice were significantly more sensitive than +/+ mice to ip-administered CPF (100 mg/kg) and parathion (10 mg/kg) with increased tremoring and mortality that correlated for CPF with greater brain acetylcholinesterase inhibition. Docking AcMAGE and CPO in a KIAA1363 active site model showed similar positioning of their acetyl and trichloropyridinyl moieties, respectively. This study establishes the relevance of KIAA1363 in ether lipid metabolism and OP detoxification.

  2. Hydrolysis of VX and related compounds by organophosphorus hydrolase. Final report, Februray-December 1993

    SciTech Connect

    Kolakowski, J.E.; DeFrank, J.J.; Lai, K.; Wild, J.R.

    1995-11-01

    Organophosphorus Hydrolase (OPH) is a fully characterized and cloned enzyme, derived from Pseudomonas diminuta, consisting of 365 amino acids with a total molecular weight of 38,0(X). The enzyme has a leader sequence of 29 amino acids which has been removed in the construction used in this study. OPH was evaluated for its effectiveness in catalyzing the S-(2-diisopwpylaminoethyl) methylphosphonothioate (VX) and its analogs.

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

    PubMed Central

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

    2015-01-01

    Clostridium acetobutylicum ATCC 824 gene CA_C0359 encodes a putative unsaturated rhamnogalacturonyl hydrolase (URH) with distant amino-acid sequence homology to YteR of Bacillus subtilis strain 168. YteR, like other URHs, has core structural homology to unsaturated glucuronyl hydrolases, but hydrolyzes the unsaturated disaccharide derivative of rhamnogalacturonan I. The crystal structure of the recombinant CA_C0359 protein was solved to 1.6 Å resolution by molecular replacement using the phase information of the previously reported structure of YteR (PDB entry 1nc5) from Bacillus subtilis strain 168. The YteR-like protein is a six-α-hairpin barrel with two β-sheet strands and a small helix overlaying the end of the hairpins next to the active site. The protein has low primary protein sequence identity to YteR but is structurally similar. The two tertiary structures align with a root-mean-square deviation of 1.4 Å and contain a highly conserved active pocket. There is a conserved aspartic acid residue in both structures, which has been shown to be important for hydration of the C=C bond during the release of unsaturated galacturonic acid by YteR. A surface electrostatic potential comparison of CA_C0359 and proteins from CAZy families GH88 and GH105 reveals the make-up of the active site to be a combination of the unsaturated rhamnogalacturonyl hydrolase and the unsaturated glucuronyl hydrolase from Bacillus subtilis strain 168. Structural and electrostatic comparisons suggests that the protein may have a slightly different substrate specificity from that of YteR. PMID:26249707

  4. Phosphonoacetate hydrolase from Penicillium oxalicum: purification and properties, phosphate starvation-independent expression, and partial sequencing.

    PubMed

    Klimek-Ochab, Magdalena; Raucci, Giuseppe; Lejczak, Barbara; Forlani, Giuseppe

    2006-03-01

    The enzyme responsible for the hydrolysis of phosphonoacetic acid, a non-biogenic C-P compound, was purified to electrophoretic homogeneity from a wild-type strain of Penicillium oxalicum. A 50-fold enrichment was obtained by a combination of anion exchange, hydrophobic interaction and MonoQ-fast protein liquid chromatography, with a yield of one-third of the initial activity. A characterization of the protein showed both similarities and differences with respect to the well-characterized bacterial counterpart. The fungal phosphonoacetate hydrolase is a 43-kDa monomeric protein showing low affinity toward its substrate and high sensitivity to even mildly acidic pH values. Enzyme activity neither required nor was stimulated by the presence of divalent cations. Polyclonal antibodies were raised in mouse against the purified protein, allowing the study of enzyme induction as a function of the phosphate status of the cell. Peptide mass mapping led to the determination of about 20% of the primary structure. Despite the biochemical differences, amino acid alignment showed a high degree of similarity of the fungal hydrolase with the few sequences available to date for the bacterial enzyme. The possible physiological role of a phosphonoacetate hydrolase is discussed. PMID:16129582

  5. Characterization of tunable piperidine and piperazine carbamates as inhibitors of endocannabinoid hydrolases

    PubMed Central

    Long, Jonathan Z.; Jin, Xin; Adibekian, Alexander; Li, Weiwei; Cravatt, Benjamin F.

    2010-01-01

    Monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) are two enzymes from the serine hydrolase superfamily that degrade the endocannabinoids 2-arachidonoylglycerol and anandamide, respectively. We have recently discovered that MAGL and FAAH are both inhibited by carbamates bearing an N-piperidine/piperazine group. Piperidine/piperazine carbamates show excellent in vivo activity, raising brain endocannabinoid levels and producing CB1-dependent behavioral effects in mice, suggesting that they represent a promising class of inhibitors for studying the endogenous functions of MAGL and FAAH. Herein, we disclose a full account of the syntheses, structure-activity relationships, and inhibitory activities of piperidine/piperazine carbamates against members of the serine hydrolase family. These scaffolds can be tuned for MAGL-selective or dual MAGL-FAAH inhibition by the attachment of an appropriately substituted bisarylcarbinol or aryloxybenzyl moiety, respectively, on the piperidine/piperazine ring. Modifications to the piperidine/piperazine ring ablated inhibitory activity, suggesting a strict requirement for a six-member ring to maintain potency. PMID:20099888

  6. Strategies to reduce end-product inhibition in family 48 glycoside hydrolases

    DOE PAGESBeta

    Chen, Mo; Bu, Lintao; Alahuhta, Markus; Brunecky, Roman; Xu, Qi; Lunin, Vladimir V.; Brady, John W.; Crowley, Michael F.; Himmel, Michael E.; Bomble, Yannick J.

    2016-02-01

    Family 48 cellobiohydrolases are some of the most abundant glycoside hydrolases in nature. They are able to degrade cellulosic biomass and therefore serve as good enzyme candidates for biofuel production. Family 48 cellulases hydrolyze cellulose chains via a processive mechanism, and produce end products composed primarily of cellobiose as well as other cellooligomers (dp ≤ 4). The challenge of utilizing cellulases in biofuel production lies in their extremely slow turnover rate. A factor contributing to the low enzyme activity is suggested to be product binding to enzyme and the resulting performance inhibition. In this study, we quantitatively evaluated the productmore » inhibitory effect of four family 48 glycoside hydrolases using molecular dynamics simulations and product expulsion free-energy calculations. We also suggested a series of single mutants of the four family 48 glycoside hydrolases with theoretically reduced level of product inhibition. As a result, the theoretical calculations provide a guide for future experimental studies designed to produce mutant cellulases with enhanced activity.« less

  7. Identification of Neutral Cholesterol Ester Hydrolase, a Key Enzyme Removing Cholesterol from Macrophages*S⃞

    PubMed Central

    Okazaki, Hiroaki; Igarashi, Masaki; Nishi, Makiko; Sekiya, Motohiro; Tajima, Makiko; Takase, Satoru; Takanashi, Mikio; Ohta, Keisuke; Tamura, Yoshiaki; Okazaki, Sachiko; Yahagi, Naoya; Ohashi, Ken; Amemiya-Kudo, Michiyo; Nakagawa, Yoshimi; Nagai, Ryozo; Kadowaki, Takashi; Osuga, Jun-ichi; Ishibashi, Shun

    2008-01-01

    Unstable lipid-rich plaques in atherosclerosis are characterized by the accumulation of macrophage foam cells loaded with cholesterol ester (CE). Although hormone-sensitive lipase and cholesteryl ester hydrolase (CEH) have been proposed to mediate the hydrolysis of CE in macrophages, circumstantial evidence suggests the presence of other enzymes with neutral cholesterol ester hydrolase (nCEH) activity. Here we show that the murine orthologue of KIAA1363, designated as neutral cholesterol ester hydrolase (NCEH), is a microsomal nCEH with high expression in murine and human macrophages. The effect of various concentrations of NaCl on its nCEH activity resembles that on endogenous nCEH activity of macrophages. RNA silencing of NCEH decreases nCEH activity at least by 50%; conversely, its overexpression inhibits the CE formation in macrophages. Immunohistochemistry reveals that NCEH is expressed in macrophage foam cells in atherosclerotic lesions. These data indicate that NCEH is responsible for a major part of nCEH activity in macrophages and may be a potential therapeutic target for the prevention of atherosclerosis. PMID:18782767

  8. Brucella abortus Choloylglycine Hydrolase Affects Cell Envelope Composition and Host Cell Internalization

    PubMed Central

    Marchesini, María Inés; Connolly, Joseph; Delpino, María Victoria; Baldi, Pablo C.; Mujer, Cesar V.; DelVecchio, Vito G.; Comerci, Diego J.

    2011-01-01

    Choloylglycine hydrolase (CGH, E.C. 3.5.1.24) is a conjugated bile salt hydrolase that catalyses the hydrolysis of the amide bond in conjugated bile acids. Bile salt hydrolases are expressed by gastrointestinal bacteria, and they presumably decrease the toxicity of host's conjugated bile salts. Brucella species are the causative agents of brucellosis, a disease affecting livestock and humans. CGH confers Brucella the ability to deconjugate and resist the antimicrobial action of bile salts, contributing to the establishment of a successful infection through the oral route in mice. Additionally, cgh-deletion mutant was also attenuated in intraperitoneally inoculated mice, which suggests that CGH may play a role during systemic infection other than hydrolyzing conjugated bile acids. To understand the role CGH plays in B. abortus virulence, we infected phagocytic and epithelial cells with a cgh-deletion mutant (Δcgh) and found that it is defective in the internalization process. This defect along with the increased resistance of Δcgh to the antimicrobial action of polymyxin B, prompted an analysis of the cell envelope of this mutant. Two-dimensional electrophoretic profiles of Δcgh cell envelope-associated proteins showed an altered expression of Omp2b and different members of the Omp25/31 family. These results were confirmed by Western blot analysis with monoclonal antibodies. Altogether, the results indicate that Brucella CGH not only participates in deconjugation of bile salts but also affects overall membrane composition and host cell internalization. PMID:22174816

  9. Conserved catalytic machinery and the prediction of a common fold for several families of glycosyl hydrolases.

    PubMed Central

    Henrissat, B; Callebaut, I; Fabrega, S; Lehn, P; Mornon, J P; Davies, G

    1995-01-01

    The regions surrounding the catalytic amino acids previously identified in a few "retaining" O-glycosyl hydrolases (EC 3.2.1) have been analyzed by hydrophobic cluster analysis and have been used to define sequence motifs. These motifs have been found in more than 150 glycosyl hydrolase sequences representing at least eight established protein families that act on a large variety of substrates. This allows the localization and the precise role of the catalytic residues (nucleophile and acid catalyst) to be predicted for each of these enzymes, including several lysosomal glycosidases. An identical arrangement of the catalytic nucleophile was also found for S-glycosyl hydrolases (myrosinases; EC 3.2.3.1) for which the acid catalyst is lacking. A (beta/alpha)8 barrel structure has been reported for two of the eight families of proteins that have been grouped. It is suggested that the six other families also share this fold at their catalytic domain. These enzymes illustrate how evolutionary events led to a wide diversification of substrate specificity with a similar disposition of identical catalytic residues onto the same ancestral (beta/alpha)8 barrel structure. Images Fig. 1 PMID:7624375

  10. Properties of a β-(1→4)-glucan hydrolase from Aspergillus niger

    PubMed Central

    Clarke, A. E.; Stone, B. A.

    1965-01-01

    1. A β-(1→4)-glucan hydrolase prepared from Aspergillus niger, as described by Clarke & Stone (1965a), showed a pH optimum in the range 4·5–6 and Km 0·25% when acting on a cellulose dextrin sulphate substrate. 2. The hydrolase rapidly decreased the specific viscosity of carboxymethylcellulose with a small increase in the production of reducing sugars. The identity of the products of hydrolysis of cellotetraose, cellopentaose and their reduced analogues indicate a preferential cleavage of non-terminal glucosidic linkages. The enzyme may be described as β-(1→4)-glucan 4-glucanohydrolase (EC 3.2.1.4). 3. In addition to carboxymethylcellulose, cellulose dextrins, cellopentaose and cellotetraose the enzyme fraction hydrolysed lichenin, oat and barley glucans, ivory-nut mannan and a glucomannan from Konjak flour. No hydrolysis of wheat-straw β-(1→4)-xylan, Lupinus albus β-(1→4)-galactan, pneumococcal type III polysaccharide, chitin, hyaluronic acid, laminarin, pachydextrins, carboxymethylpachyman or β-(1→3)-oligoglucosides was detected. 4. The hydrolase showed no transglycosylase activity from cellodextrin or cellopentaose substrates to glucose or methanol acceptors. 5. The hydrolysis of cellodextrins was inhibited completely by 1·0mm-Hg2+, 0·7mm-phenylmercuric nitrate and 1·0mm-iodine. PMID:5862418

  11. Properties of a beta-(1-4)-glucan hydrolase from Aspergillus niger.

    PubMed

    Clarke, A E; Stone, B A

    1965-09-01

    1. A beta-(1-->4)-glucan hydrolase prepared from Aspergillus niger, as described by Clarke & Stone (1965a), showed a pH optimum in the range 4.5-6 and K(m) 0.25% when acting on a cellulose dextrin sulphate substrate. 2. The hydrolase rapidly decreased the specific viscosity of carboxymethylcellulose with a small increase in the production of reducing sugars. The identity of the products of hydrolysis of cellotetraose, cellopentaose and their reduced analogues indicate a preferential cleavage of non-terminal glucosidic linkages. The enzyme may be described as beta-(1-->4)-glucan 4-glucanohydrolase (EC 3.2.1.4). 3. In addition to carboxymethylcellulose, cellulose dextrins, cellopentaose and cellotetraose the enzyme fraction hydrolysed lichenin, oat and barley glucans, ivory-nut mannan and a glucomannan from Konjak flour. No hydrolysis of wheat-straw beta-(1-->4)-xylan, Lupinus albus beta-(1-->4)-galactan, pneumococcal type III polysaccharide, chitin, hyaluronic acid, laminarin, pachydextrins, carboxymethylpachyman or beta-(1-->3)-oligoglucosides was detected. 4. The hydrolase showed no transglycosylase activity from cellodextrin or cellopentaose substrates to glucose or methanol acceptors. 5. The hydrolysis of cellodextrins was inhibited completely by 1.0mm-Hg(2+), 0.7mm-phenylmercuric nitrate and 1.0mm-iodine. PMID:5862418

  12. Inhibition of Diabrotica Larval Growth by Patatin, the Lipid Acyl Hydrolase from Potato Tubers.

    PubMed Central

    Strickland, J. A.; Orr, G. L.; Walsh, T. A.

    1995-01-01

    Patatin, the nonspecific lipid acyl hydrolase from potato (Solanum tuberosum L.) tubers, dose-dependently inhibits the growth of southern corn rootworm (SCR) and western corn rootworm when fed to them on artificial diet. The 50% growth reduction levels are somewhat cultivar dependent, ranging from 60 to 150 [mu]g/g diet for neonate SCR larvae. A single patatin isoform also inhibits larval growth. Neonate SCR continuously exposed to patatin are halted in larval development. Treatment with di-isopropylfluorophosphate essentially eliminates patatin's phospholipase, galactolipase, and acyl hydrolase activities. SCR growth inhibition is eliminated also, indicating that patatin's serine hydrolase activity is responsible for the observed activities. Patatin-mediated phospholipolysis is highly pH and cultivar dependent, with specific activities up to 300-fold less at pH 5.5 than at pH 8.5. Esterase or phospholipase activities do not correlate with insect growth inhibition. Galactolipase activity, being cultivar and pH independent, correlates significantly with SCR growth inhibition. Insect-growth inhibition of patatin is significantly reduced with increased dietary cholesterol levels. In conclusion, patatin represents a new class of insect-control proteins with a novel mode of action possibly involving lipid metabolism. PMID:12228621

  13. Inhibition of Diabrotica Larval Growth by Patatin, the Lipid Acyl Hydrolase from Potato Tubers.

    PubMed

    Strickland, J. A.; Orr, G. L.; Walsh, T. A.

    1995-10-01

    Patatin, the nonspecific lipid acyl hydrolase from potato (Solanum tuberosum L.) tubers, dose-dependently inhibits the growth of southern corn rootworm (SCR) and western corn rootworm when fed to them on artificial diet. The 50% growth reduction levels are somewhat cultivar dependent, ranging from 60 to 150 [mu]g/g diet for neonate SCR larvae. A single patatin isoform also inhibits larval growth. Neonate SCR continuously exposed to patatin are halted in larval development. Treatment with di-isopropylfluorophosphate essentially eliminates patatin's phospholipase, galactolipase, and acyl hydrolase activities. SCR growth inhibition is eliminated also, indicating that patatin's serine hydrolase activity is responsible for the observed activities. Patatin-mediated phospholipolysis is highly pH and cultivar dependent, with specific activities up to 300-fold less at pH 5.5 than at pH 8.5. Esterase or phospholipase activities do not correlate with insect growth inhibition. Galactolipase activity, being cultivar and pH independent, correlates significantly with SCR growth inhibition. Insect-growth inhibition of patatin is significantly reduced with increased dietary cholesterol levels. In conclusion, patatin represents a new class of insect-control proteins with a novel mode of action possibly involving lipid metabolism. PMID:12228621

  14. Probiotic characterization of potential hydrolases producing Lactococcus lactis subsp. lactis isolated from pickled yam.

    PubMed

    Bhanwar, Seema; Singh, Arashdeep; Ganguli, Abhijit

    2014-02-01

    The aim of this study was to characterize potential probiotic strain co-producing α-amylase and β-galactosidase. Sixty-three strains, isolated from pickle samples were screened for their hydrolase producing capacity by utilizing different starches as carbon source. One out of 63 strains, isolated from traditionally fermented pickled yam showing maximum hydrolase activity (α-amylase (36.9 U/ml) and β-galactosidase (42.6 U/ml)) within a period of 48 hours was identified as Lactococcus lactis subsp. lactis. Further, it was assessed for the probiotic characteristics under gastrointestinal conditions like acidic, alkaline, proteolytic enzymes, bile stress and found to exhibit tolerance to these stresses. The therapeutic potential of the isolate is implicated because of its antagonistic effect against enteric foodborne pathogens (Salmonella typhimurium, Escherichia coli 0157:H7, Staphylococcus aureus, Yersinia enterocolitica and Aeromonas hydrophila). The results of this study entail a potential applicability of the isolate in developing future probiotic foods besides the production of industrially significant hydrolases. PMID:24020495

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

    SciTech Connect

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

    2008-01-01

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

  16. Functions, structures, and applications of cellobiose 2-epimerase and glycoside hydrolase family 130 mannoside phosphorylases.

    PubMed

    Saburi, Wataru

    2016-07-01

    Carbohydrate isomerases/epimerases are essential in carbohydrate metabolism, and have great potential in industrial carbohydrate conversion. Cellobiose 2-epimerase (CE) reversibly epimerizes the reducing end d-glucose residue of β-(1→4)-linked disaccharides to d-mannose residue. CE shares catalytic machinery with monosaccharide isomerases and epimerases having an (α/α)6-barrel catalytic domain. Two histidine residues act as general acid and base catalysts in the proton abstraction and addition mechanism. β-Mannoside hydrolase and 4-O-β-d-mannosyl-d-glucose phosphorylase (MGP) were found as neighboring genes of CE, meaning that CE is involved in β-mannan metabolism, where it epimerizes β-d-mannopyranosyl-(1→4)-d-mannose to β-d-mannopyranosyl-(1→4)-d-glucose for further phosphorolysis. MGPs form glycoside hydrolase family 130 (GH130) together with other β-mannoside phosphorylases and hydrolases. Structural analysis of GH130 enzymes revealed an unusual catalytic mechanism involving a proton relay and the molecular basis for substrate and reaction specificities. Epilactose, efficiently produced from lactose using CE, has superior physiological functions as a prebiotic oligosaccharide. PMID:27031293

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

    SciTech Connect

    Brzezinski, Krzysztof; Bujacz, Grzegorz; Jaskolski, Mariusz

    2008-07-01

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

  18. Synergistic action modes of arabinan degradation by exo- and endo-arabinosyl hydrolases.

    PubMed

    Park, Jung-Mi; Jang, Myoung-Uoon; Oh, Gyo Won; Lee, Eun-Hee; Kang, Jung-Hyun; Song, Yeong-Bok; Han, Nam Soo; Kim, Tae-Jip

    2015-02-01

    Two recombinant arabinosyl hydrolases, α-L-arabinofuranosidase from Geobacillus sp. KCTC 3012 (GAFase) and endo-(1,5)-α-L-arabinanase from Bacillus licheniformis DSM13 (BlABNase), were overexpressed in Escherichia coli, and their synergistic modes of action against sugar beet (branched) arabinan were investigated. Whereas GAFase hydrolyzed 35.9% of L-arabinose residues from sugar beet (branched) arabinan, endo-action of BlABNase released only 0.5% of L-arabinose owing to its extremely low accessibility towards branched arabinan. Interestingly, the simultaneous treatment of GAFase and BlABNase could liberate approximately 91.2% of L-arabinose from arabinan, which was significantly higher than any single exo-enzyme treatment (35.9%) or even stepwise exo- after endo-enzyme treatment (75.5%). Based on their unique modes of action, both exo- and endo-arabinosyl hydrolases can work in concert to catalyze the hydrolysis of arabinan to L-arabinose. At the early stage in arabinan degradation, exo-acting GAFase could remove the terminal arabinose branches to generate debranched arabinan, which could be successively hydrolyzed into arabinooligosaccharides via the endoaction of BlABNase. At the final stage, the simultaneous actions of exo- and endo-hydrolases could synergistically accelerate the L-arabinose production with high conversion yield. PMID:25433551

  19. A novel nucleoside hydrolase from Lactobacillus buchneri LBK78 catalyzing hydrolysis of 2'-O-methylribonucleosides.

    PubMed

    Mitsukawa, Yuuki; Hibi, Makoto; Matsutani, Narihiro; Horinouchi, Nobuyuki; Takahashi, Satomi; Ogawa, Jun

    2016-08-01

    2'-O-Methylribonucleosides (2'-OMe-NRs) are promising raw materials for nucleic acid drugs because of their high thermal stability and nuclease tolerance. In the course of microbial screening for metabolic activity toward 2'-OMe-NRs, Lactobacillus buchneri LBK78 was found to decompose 2'-O-methyluridine (2'-OMe-UR). The enzyme responsible was partially purified from L. buchneri LBK78 cells by a four-step purification procedure, and identified as a novel nucleoside hydrolase. This enzyme, LbNH, belongs to the nucleoside hydrolase superfamily, and formed a homotetrameric structure composed of subunits with a molecular mass around 34 kDa. LbNH hydrolyzed 2'-OMe-UR to 2'-O-methylribose and uracil, and the kinetic constants were Km of 0.040 mM, kcat of 0.49 s(-1), and kcat/Km of 12 mM(-1) s(-1). In a substrate specificity analysis, LbNH preferred ribonucleosides and 2'-OMe-NRs as its hydrolytic substrates, but reacted weakly with 2'-deoxyribonucleosides. In a phylogenetic analysis, LbNH showed a close relationship with purine-specific nucleoside hydrolases from trypanosomes. PMID:27180876

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

    PubMed

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

    2016-02-01

    Epoxide hydrolases (EH, EC 3.3.2.3) have been proposed to be key enzymes in the biosynthesis of polyether (PE) ladder compounds such as the brevetoxins which are produced by the dinoflagellate Karenia brevis. These enzymes have the potential to catalyze kinetically disfavored endo-tet cyclization reactions. Data mining of K. brevis transcriptome libraries revealed two classes of epoxide hydrolases: microsomal and leukotriene A4 (LTA4) hydrolases. A microsomal EH was cloned and expressed for characterization. The enzyme is a monomeric protein with molecular weight 44kDa. Kinetic parameters were evaluated using a variety of epoxide substrates to assess substrate selectivity and enantioselectivity, as well as its potential to catalyze the critical endo-tet cyclization of epoxy alcohols. Monitoring of EH activity in high and low toxin producing cultures of K. brevis over a three week period showed consistently higher activity in the high toxin producing culture implicating the involvement of one or more EH in brevetoxin biosynthesis. PMID:26626160

  1. Esterase SeE of Streptococcus equi ssp. equi is a Novel Non-specific Carboxylic Ester Hydrolase

    PubMed Central

    Xie, Gang; Liu, Mengyao; Zhu, Hui; Lei, Benfang

    2009-01-01

    Extracellular carboxylic ester hydrolases are produced by many bacterial pathogens and have been shown recently to be important for virulence of some pathogens. However, these hydrolases are poorly characterized in enzymatic activity. This study prepared and characterized the secreted ester hydrolase of Streptococcus equi ssp. equi (designated SeE for S. equi esterase). SeE hydrolyzes ethyl acetate, acetylsalicylic acid, and tributyrin but not ethyl butyrate. This substrate specificity pattern does not match those of the three conventional types of non-specific carboxylic ester hydrolases (carboxylesterases, arylesterases, and acetylesterases). To determine whether SeE has lipase activity, a number of triglycerides and vinyl esters were tested in SeE-catalyzed hydrolysis. SeE does not hydrolyze triglycerides and vinyl esters of long chain carboxylic acids nor display interfacial activation, indicating that SeE is not a lipase. Like the conventional carboxylesterases, SeE is inhibited by diisopropylfluorophosphate. These findings indicate that SeE is a novel non-specific carboxylic ester hydrolase that has broader substrate specificity than the conventional carboxylesterases. PMID:19054107

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

    NASA Astrophysics Data System (ADS)

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

    2014-07-01

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

  3. Haloalkylphosphorus Hydrolases Purified from Sphingomonas sp. Strain TDK1 and Sphingobium sp. Strain TCM1

    PubMed Central

    Yoshida, Satoshi; Suzuki, Yuto; Mori, Junichi; Doi, Yuka; Takahashi, Shouji; Kera, Yoshio

    2014-01-01

    Phosphotriesterases catalyze the first step of organophosphorus triester degradation. The bacterial phosphotriesterases purified and characterized to date hydrolyze mainly aryl dialkyl phosphates, such as parathion, paraoxon, and chlorpyrifos. In this study, we purified and cloned two novel phosphotriesterases from Sphingomonas sp. strain TDK1 and Sphingobium sp. strain TCM1 that hydrolyze tri(haloalkyl)phosphates, and we named these enzymes haloalkylphosphorus hydrolases (TDK-HAD and TCM-HAD, respectively). Both HADs are monomeric proteins with molecular masses of 59.6 (TDK-HAD) and 58.4 kDa (TCM-HAD). The enzyme activities were affected by the addition of divalent cations, and inductively coupled plasma mass spectrometry analysis suggested that zinc is a native cofactor for HADs. These enzymes hydrolyzed not only chlorinated organophosphates but also a brominated organophosphate [tris(2,3-dibromopropyl) phosphate], as well as triaryl phosphates (tricresyl and triphenyl phosphates). Paraoxon-methyl and paraoxon were efficiently degraded by TCM-HAD, whereas TDK-HAD showed weak activity toward these substrates. Dichlorvos was degraded only by TCM-HAD. The enzymes displayed weak or no activity against trialkyl phosphates and organophosphorothioates. The TCM-HAD and TDK-HAD genes were cloned and found to encode proteins of 583 and 574 amino acid residues, respectively. The primary structures of TCM-HAD and TDK-HAD were very similar, and the enzymes also shared sequence similarity with fenitrothion hydrolase (FedA) of Burkholderia sp. strain NF100 and organophosphorus hydrolase (OphB) of Burkholderia sp. strain JBA3. However, the substrate specificities and quaternary structures of the HADs were largely different from those of FedA and OphB. These results show that HADs from sphingomonads are novel members of the bacterial phosphotriesterase family. PMID:25038092

  4. Exopolysaccharide biosynthetic glycoside hydrolases can be utilized to disrupt and prevent Pseudomonas aeruginosa biofilms

    PubMed Central

    Baker, Perrin; Hill, Preston J.; Snarr, Brendan D.; Alnabelseya, Noor; Pestrak, Matthew J.; Lee, Mark J.; Jennings, Laura K.; Tam, John; Melnyk, Roman A.; Parsek, Matthew R.; Sheppard, Donald C.; Wozniak, Daniel J.; Howell, P. Lynne

    2016-01-01

    Bacterial biofilms present a significant medical challenge because they are recalcitrant to current therapeutic regimes. A key component of biofilm formation in the opportunistic human pathogen Pseudomonas aeruginosa is the biosynthesis of the exopolysaccharides Pel and Psl, which are involved in the formation and maintenance of the structural biofilm scaffold and protection against antimicrobials and host defenses. Given that the glycoside hydrolases PelAh and PslGh encoded in the pel and psl biosynthetic operons, respectively, are utilized for in vivo exopolysaccharide processing, we reasoned that these would provide specificity to target P. aeruginosa biofilms. Evaluating these enzymes as potential therapeutics, we demonstrate that these glycoside hydrolases selectively target and degrade the exopolysaccharide component of the biofilm matrix. PelAh and PslGh inhibit biofilm formation over a 24-hour period with a half maximal effective concentration (EC50) of 69.3 ± 1.2 and 4.1 ± 1.1 nM, respectively, and are capable of disrupting preexisting biofilms in 1 hour with EC50 of 35.7 ± 1.1 and 12.9 ± 1.1 nM, respectively. This treatment was effective against clinical and environmental P. aeruginosa isolates and reduced biofilm biomass by 58 to 94%. These noncytotoxic enzymes potentiated antibiotics because the addition of either enzyme to a sublethal concentration of colistin reduced viable bacterial counts by 2.5 orders of magnitude when used either prophylactically or on established 24-hour biofilms. In addition, PelAh was able to increase neutrophil killing by ~50%. This work illustrates the feasibility and benefits of using bacterial exopolysaccharide biosynthetic glycoside hydrolases to develop novel antibiofilm therapeutics. PMID:27386527

  5. New perspective on glycoside hydrolase binding to lignin from pretreated corn stover

    DOE PAGESBeta

    Yarbrough, John M.; Mittal, Ashutosh; Mansfield, Elisabeth; Taylor, II, Larry E.; Hobdey, Sarah E.; Sammond, Deanne W.; Bomble, Yannick J.; Crowley, Michael F.; Decker, Stephen R.; Himmel, Michael E.; et al

    2015-12-18

    In this study, non-specific binding of cellulases to lignin has been implicated as a major factor in the loss of cellulase activity during biomass conversion to sugars. It is believed that this binding may strongly impact process economics through loss of enzyme activities during hydrolysis and enzyme recycling scenarios. The current model suggests glycoside hydrolase activities are lost though non-specific/non-productive binding of carbohydrate-binding domains to lignin, limiting catalytic site access to the carbohydrate components of the cell wall.

  6. Pyrazole phenylcyclohexylcarbamates as inhibitors of human fatty acid amide hydrolases (FAAH).

    PubMed

    Aghazadeh Tabrizi, Mojgan; Baraldi, Pier Giovanni; Ruggiero, Emanuela; Saponaro, Giulia; Baraldi, Stefania; Romagnoli, Romeo; Martinelli, Adriano; Tuccinardi, Tiziano

    2015-06-01

    Fatty acid amide hydrolase (FAAH) inhibitors have gained attention as potential therapeutic targets in the management of neuropathic pain. Here, we report a series of pyrazole phenylcyclohexylcarbamate derivatives standing on the known carbamoyl FAAH inhibitor URB597. Structural modifications led to the recognition of compound 22 that inhibited human recombinant FAAH (hrFAAH) in the low nanomolar range (IC50 = 11 nM). The most active compounds of this series showed significant selectivity toward monoacylglycerol lipase (MAGL) enzyme. In addition, molecular modeling and reversibility behavior of the new class of FAAH inhibitors are presented in this article. PMID:26002335

  7. Novel glycoside hydrolases identified by screening a Chinese Holstein dairy cow rumen-derived metagenome library.

    PubMed

    Zhao, Shengguo; Wang, Jiaqi; Bu, Dengpan; Liu, Kailang; Zhu, Yaxin; Dong, Zhiyang; Yu, Zhongtang

    2010-10-01

    One clone encoding glycoside hydrolases was identified through functional screening of a rumen bacterial artificial chromosome (BAC) library. Of the 68 open reading frames (ORFs) predicted, one ORF encodes a novel endo-β-1,4-xylanase with two catalytic domains of family GH43 and two cellulose-binding modules (CBMs) of family IV. Partial characterization showed that this endo-xylanase has a greater specific activity than a number of other xylanases over a wide temperature range at neutral pH and could be useful in some industrial applications. PMID:20709844

  8. Cloning and analysis of bile salt hydrolase genes from Lactobacillus plantarum CGMCC No. 8198.

    PubMed

    Gu, Xiang-Chao; Luo, Xue-Gang; Wang, Chong-Xi; Ma, De-Yun; Wang, Yan; He, Ying-Ying; Li, Wen; Zhou, Hao; Zhang, Tong-Cun

    2014-05-01

    Genes coding for bile salt hydrolase of Lactobacillus plantarum CGMCC 8198, a novel probiotic strain isolated from silage, were identified, analyzed and cloned. L. plantarum strongly resisted the inhibitory effects of bile salts and also decreased serum cholesterol levels by 20% in mice with hypercholesterolemia. Using RT-PCR analysis, bsh2, bsh3 and bsh4 were upregulated by bile salts in a dose-dependent manner. All three bsh genes had high similarity with those of other Lactobacillus strains. All three recombinant BSHs had high activities for the hydrolysis of glycodeoxycholic acids and taurodeoxycholic acids. PMID:24375235

  9. Detection and determination of lipase (acylglycerol hydrolase) activity from various sources.

    PubMed

    Jensen, R G

    1983-09-01

    Methods for the detection and determination of lipases (acylglycerol hydrolases) and preparation of assays are reviewed including substrates, conditions and screening. Some newer methods for the determination of lipase activity are discussed. Several of these are: (a) titrimetry, (b) colorimetry of Cu soaps of free fatty acids (FFA), (c) colorimetry of chromophores in the acyl chain of FFA or in glycerol, (d) radioassay, (e) gas liquid chromatography, (f) enzymatic treatment of FFA and measurement of the resulting products, and (g) direct immunological determination of the lipase. Examples and sensitivities are given and advantages and disadvantages are described. PMID:6633171

  10. Radiometric assay of ghrelin hydrolase activity and 3H-ghrelin distribution into mouse tissues.

    PubMed

    Chen, Vicky Ping; Gao, Yang; Geng, Liyi; Brimijoin, Stephen

    2015-12-15

    A high-throughput radiometric assay was developed to characterize enzymatic hydrolysis of ghrelin and to track the peptide's fate in vivo. The assay is based on solvent partitioning of [(3)H]-octanoic acid liberated from [(3)H]-octanoyl ghrelin during enzymatic hydrolysis. This simple and cost-effective method facilitates kinetic analysis of ghrelin hydrolase activity of native and mutated butyrylcholinesterases or carboxylesterases from multiple species. In addition, the assay's high sensitivity facilitates ready evaluation of ghrelin's pharmacokinetics and tissue distribution in mice after i.v. bolus administration of radiolabeled peptide. PMID:26514871

  11. Characterization of organophosphorus hydrolases and the genetic manipulation of the phosphotriesterase from pseudomonas diminuta

    SciTech Connect

    Dave, K.I.; Miller, C.E.; Wild, J.R.

    1993-12-31

    There are a variety of enzymes which are specifically capable of hydrolyzing organophosphorus esters with different phosphoryl bonds from the typical phosphotriester bonds of common insecticidal neurotoxins (e.g. paraoxon or coumaphos) to the phosphonate-fluoride bonds of chemical warfare agents (e.g. soman or sarin). These enzymes comprise a diverse set of enzymes whose basic architecture and substrate specificities vary dramatically, yet they appear to be ubiquitous throughout nature. The most thoroughly studied of these enzymes is the organophosphate hydrolase (opd gene product) of Pseudomonas diminuta and Ftavobacterium sp. ATCC 27551, and the heterologous expression, post-translational modification, and genetic engineering studies undertaken with this enzyme are described.

  12. Expression and purification of an engineered, yeast-expressed Leishmania donovani nucleoside hydrolase with immunogenic properties.

    PubMed

    Hudspeth, Elissa M; Wang, Qian; Seid, Christopher A; Hammond, Molly; Wei, Junfei; Liu, Zhuyun; Zhan, Bin; Pollet, Jeroen; Heffernan, Michael J; McAtee, C Patrick; Engler, David A; Matsunami, Risë K; Strych, Ulrich; Asojo, Oluwatoyin A; Hotez, Peter J; Bottazzi, Maria Elena

    2016-07-01

    Leishmania donovani is the major cause of visceral leishmaniasis (kala-azar), now recognized as the parasitic disease with the highest level of mortality second only to malaria. No human vaccine is currently available. A 36 kDa L. donovani nucleoside hydrolase (LdNH36) surface protein has been previously identified as a potential vaccine candidate antigen. Here we present data on the expression of LdNH36 in Pichia pastoris and its purification at the 20 L scale to establish suitability for future pilot scale manufacturing. To improve efficiency of process development and ensure reproducibility, 4 N-linked glycosylation sites shown to contribute to heterogeneous high-mannose glycosylation were mutated to glutamine residues. The mutant LdNH36 (LdNH36-dg2) was expressed and purified to homogeneity. Size exclusion chromatography and light scattering demonstrated that LdNH36-dg2 existed as a tetramer in solution, similar to the wild-type recombinant L. major nucleoside hydrolase. The amino acid mutations do not affect the tetrameric interface as confirmed by theoretical modeling, and the mutated amino acids are located outside the major immunogenic domain. Immunogenic properties of the LdNH36-dg2 recombinant protein were evaluated in BALB/c mice using formulations that included a synthetic CpG oligodeoxynucleotide, together with a microparticle delivery platform (poly(lactic-co-glycolic acid)). Mice exhibited high levels of IgG1, IgG2a, and IgG2b antibodies that were reactive to both LdNH36-dg2 and LdNH36 wild-type. While the point mutations did affect the hydrolase activity of the enzyme, the IgG antibodies elicited by LdNH36-dg2 were shown to inhibit the hydrolase activity of the wild-type LdNH36. The results indicate that LdNH36-dg2 as expressed in and purified from P. pastoris is suitable for further scale-up, manufacturing, and testing in support of future first-in-humans phase 1 clinical trials. PMID:26839079

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

    PubMed

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

    2013-06-13

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

  14. Biosensing Paraoxon in Simulated Environmental Samples by Immobilized Organophosphorus Hydrolase in Functionalized Mesoporous Silica

    SciTech Connect

    Lei, Chenghong; Valenta, Michelle M.; Saripalli, Prasad; Ackerman, Eric J.

    2007-01-01

    There is a critical need for highly sensitive, cost-effective sensors to conduct ecological analyses for environmental and homeland security related applications. We report here on a method which significantly overcomes this difficulty, and demonstrate its application in a biosensor for aquatic environmental applications. A fast-responding and stable biosensor was developed via immobilization of organophosphorus hydrolase (OPH) in functionalized mesoporous silica (FMS) with pore sizes in tens of nanometers. The sensor was tested for detection of paraoxon in simulated environmental samples, under wide ranging physico-chemical conditions.

  15. Detection of aacA-aphD, qacEδ1, marA, floR, and tetA genes from multidrug-resistant bacteria: Comparative analysis of real-time multiplex PCR assays using EvaGreen(®) and SYBR(®) Green I dyes.

    PubMed

    Khan, Saeed A; Sung, Kidon; Nawaz, Mohamed S

    2011-01-01

    We have developed multiplex real-time PCR assays that utilize DNA-intercalating dyes, SYBR Green I (SG) and EvaGreen (EG), with two primer sets (set 1=qacEδ1, tetA and aacA-aphD; set 2=tetA, marA, and floR) to simultaneously amplify the qacEδ1, tetA, aacA-aphD, marA, and floR genes. Validity of the multiplex PCR assays was confirmed by testing 83 bacterial isolates, including Staphylococcus aureus (28 isolates), Enterococcus spp. (17 isolates), Salmonella enterica serovar Typhimurium (8 isolates), Citrobacter spp. (9 isolates), Escherichia coli (14 isolates) and Aeromonas veronii (7 isolates), and performing sequence analysis of representative PCR products. Agarose gel analysis revealed the presence of correct size PCR products, and the differences in their thermal melting (T(m)) curves were used to distinguish various PCR products. Although T(m) peaks of different amplicons after EG-based singleplex and multiplex PCR assays were resolved nicely, only one or two peaks were seen for SG-bound amplicons. EG-based multiplex real-time PCR assays provided better peak resolution. There was a good correlation with a better linear relationship between the C(t) and log input DNA concentration for the set 1 and set 2 genes in EG-based assays (R(EG)(2)=0.9813and0.9803) than in SG-based assays (R(SG)(2)=0.5276and0.6255). The sensitivities of detection were 2.5-25fg and 25-250fg of template DNA in EG and SG-based singleplex and multiplex PCR assays, respectively. The assays, which could be completed in less than 45min, offer sensitive and rapid detection of qacEδ1, aacA-aphD, marA, floR, and tetA genes from a diverse group of multiple antibiotic-resistant bacterial strains. PMID:21256956

  16. Structural genomics analysis of uncharacterized protein families overrepresented in human gut bacteria identifies a novel glycoside hydrolase

    PubMed Central

    2014-01-01

    Background Bacteroides spp. form a significant part of our gut microbiome and are well known for optimized metabolism of diverse polysaccharides. Initial analysis of the archetypal Bacteroides thetaiotaomicron genome identified 172 glycosyl hydrolases and a large number of uncharacterized proteins associated with polysaccharide metabolism. Results BT_1012 from Bacteroides thetaiotaomicron VPI-5482 is a protein of unknown function and a member of a large protein family consisting entirely of uncharacterized proteins. Initial sequence analysis predicted that this protein has two domains, one on the N- and one on the C-terminal. A PSI-BLAST search found over 150 full length and over 90 half size homologs consisting only of the N-terminal domain. The experimentally determined three-dimensional structure of the BT_1012 protein confirms its two-domain architecture and structural analysis of both domains suggests their specific functions. The N-terminal domain is a putative catalytic domain with significant similarity to known glycoside hydrolases, the C-terminal domain has a beta-sandwich fold typically found in C-terminal domains of other glycosyl hydrolases, however these domains are typically involved in substrate binding. We describe the structure of the BT_1012 protein and discuss its sequence-structure relationship and their possible functional implications. Conclusions Structural and sequence analyses of the BT_1012 protein identifies it as a glycosyl hydrolase, expanding an already impressive catalog of enzymes involved in polysaccharide metabolism in Bacteroides spp. Based on this we have renamed the Pfam families representing the two domains found in the BT_1012 protein, PF13204 and PF12904, as putative glycoside hydrolase and glycoside hydrolase-associated C-terminal domain respectively. PMID:24742328

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

    SciTech Connect

    Chou, Jyh-Ching |; Cohen, J.D.; Mulbry, W.W.

    1996-11-01

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

  18. Expression and characterization of an epoxide hydrolase from Anopheles gambiae with high activity on epoxy fatty acids

    PubMed Central

    Xu, Jiawen; Morisseau, Christophe; Hammock, Bruce D.

    2014-01-01

    In insects, epoxide hydrolases (EHs) play critical roles in the metabolism of xenobiotic epoxides from the food resources and in the regulation of endogenous chemical mediators, such as juvenile hormones. Using the baculovirus expression system, we expressed and characterized an epoxide hydrolase from Anopheles gambiae (AgEH) that is distinct in evolutionary history from insect juvenile hormone epoxide hydrolases (JHEHs). We partially purified the enzyme by ion exchange chromatography and isoelectric focusing. The experimentally determined molecular weight and pI were estimated to be 35kD and 6.3 respectively, different than the theoretical ones. The AgEH had the greatest activity on long chain epoxy fatty acids such as 14,15-epoxyeicosatrienoic acids (14,15-EET) and 9,10-epoxy-12Z-octadecenoic acids (9,10-EpOME or leukotoxin) among the substrates evaluated. Juvenile hormone III, a terpenoid insect growth regulator, was the next best substrate tested. The AgEH showed kinetics comparable to the mammalian soluble epoxide hydrolases, and the activity could be inhibited by AUDA [12-(3-adamantan-1-yl-ureido) dodecanoic acid], a urea-based inhibitor designed to inhibit the mammalian soluble epoxide hydrolases. The rabbit serum generated against the soluble epoxide hydrolase of Mus musculus can both cross-react with natural and denatured forms of the AgEH, suggesting immunologically they are similar. The study suggests there are mammalian sEH homologs in insects, and epoxy fatty acids may be important chemical mediators in insects. PMID:25173592

  19. Oxime esters as selective, covalent inhibitors of the serine hydrolase retinoblastoma-binding protein 9 (RBBP9).

    PubMed

    Bachovchin, Daniel A; Wolfe, Monique R; Masuda, Kim; Brown, Steven J; Spicer, Timothy P; Fernandez-Vega, Virneliz; Chase, Peter; Hodder, Peter S; Rosen, Hugh; Cravatt, Benjamin F

    2010-04-01

    We recently described a fluorescence polarization platform for competitive activity-based protein profiling (fluopol-ABPP) that enables high-throughput inhibitor screening for enzymes with poorly characterized biochemical activity. Here, we report the discovery of a class of oxime ester inhibitors for the unannotated serine hydrolase RBBP9 from a full-deck (200,000+ compound) fluopol-ABPP screen conducted in collaboration with the Molecular Libraries Screening Center Network (MLSCN). We show that these compounds covalently inhibit RBBP9 by modifying enzyme's active site serine nucleophile and, based on competitive ABPP in cell and tissue proteomes, are selective for RBBP9 relative to other mammalian serine hydrolases. PMID:20207142

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

    SciTech Connect

    Balotra, Sahil; Newman, Janet; French, Nigel G.; Briggs, Lyndall J.; Peat, Thomas S.; Scott, Colin

    2014-02-19

    The amidase domain of the allophanate hydrolase AtzF from Pseudomonas sp. strain ADP has been crystallized and preliminary X-ray diffraction data have been collected. The allophanate hydrolase from Pseudomonas sp. strain ADP was expressed and purified, and a tryptic digest fragment was subsequently identified, expressed and purified. This 50 kDa construct retained amidase activity and was crystallized. The crystals diffracted to 2.5 Å resolution and adopted space group P2{sub 1}, with unit-cell parameters a = 82.4, b = 179.2, c = 112.6 Å, β = 106.6°.

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

    SciTech Connect

    Pecic, Stevan; Pakhomova, Svetlana; Newcomer, Marcia E.; Morisseau, Christophe; Hammock, Bruce D.; Zhu, Zhengxiang; Rinderspacher, Alison; Deng, Shi-Xian

    2013-09-27

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

  2. Structural insights into the reaction mechanism of S-adenosyl-L-homocysteine hydrolase

    PubMed Central

    Kusakabe, Yoshio; Ishihara, Masaaki; Umeda, Tomonobu; Kuroda, Daisuke; Nakanishi, Masayuki; Kitade, Yukio; Gouda, Hiroaki; Nakamura, Kazuo T.; Tanaka, Nobutada

    2015-01-01

    S-adenosyl-L-homocysteine hydrolase (SAH hydrolase or SAHH) is a highly conserved enzyme that catalyses the reversible hydrolysis of SAH to L-homocysteine (HCY) and adenosine (ADO). High-resolution crystal structures have been reported for bacterial and plant SAHHs, but not mammalian SAHHs. Here, we report the first high-resolution crystal structure of mammalian SAHH (mouse SAHH) in complex with a reaction product (ADO) and with two reaction intermediate analogues—3’-keto-aristeromycin (3KA) and noraristeromycin (NRN)—at resolutions of 1.55, 1.55, and 1.65 Å. Each of the three structures constitutes a structural snapshot of one of the last three steps of the five-step process of SAH hydrolysis by SAHH. In the NRN complex, a water molecule, which is an essential substrate for ADO formation, is structurally identified for the first time as the candidate donor in a Michael addition by SAHH to the 3’-keto-4’,5’-didehydroadenosine reaction intermediate. The presence of the water molecule is consistent with the reaction mechanism proposed by Palmer & Abeles in 1979. These results provide insights into the reaction mechanism of the SAHH enzyme. PMID:26573329

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

    PubMed

    Mellon, Jay E

    2015-08-01

    Several atoxigenic Aspergillus flavus isolates, including some being used as biocontrol agents, and one toxigenic isolate were surveyed for the ability to produce extracellular xylanolytic and pectinolytic hydrolases. All of the tested isolates displayed good production of endoxylanases when grown on a medium utilizing larch xylan as a sole carbon substrate. Four of the tested isolates produced reasonably high levels of esterase activity, while the atoxigenic biocontrol agent NRRL 21882 isolate esterase level was significantly lower than the others. Atoxigenic A. flavus isolates 19, 22, K49, AF36 (the latter two are biocontrol agents) and toxigenic AF13 produced copious levels of pectinolytic activity when grown on a pectin medium. The pectinolytic activity levels of the atoxigenic A. flavus 17 and NRRL 21882 isolates were significantly lower than the other tested isolates. In addition, A. flavus isolates that displayed high levels of pectinolytic activity in the plate assay produced high levels of endopolygalacturonase (pectinase) P2c, as ascertained by isoelectric focusing electrophoresis. Isolate NRRL 21882 displayed low levels of both pectinase P2c and pectin methyl esterase. A. flavus appears capable of producing these hydrolytic enzymes irrespective of aflatoxin production. This ability of atoxigenic isolates to produce xylanolytic and pectinolytic hydrolases mimics that of toxigenic isolates and, therefore, contributes to the ability of atoxigenic isolates to occupy the same niche as A. flavus toxigenic isolates. PMID:26295409

  4. Identification of the Major Prostaglandin Glycerol Ester Hydrolase in Human Cancer Cells*

    PubMed Central

    Manna, Joseph D.; Wepy, James A.; Hsu, Ku-Lung; Chang, Jae Won; Cravatt, Benjamin F.; Marnett, Lawrence J.

    2014-01-01

    Prostaglandin glycerol esters (PG-Gs) are produced as a result of the oxygenation of the endocannabinoid, 2-arachidonoylglycerol, by cyclooxygenase 2. Understanding the role that PG-Gs play in a biological setting has been difficult because of their sensitivity to enzymatic hydrolysis. By comparing PG-G hydrolysis across human cancer cell lines to serine hydrolase activities determined by activity-based protein profiling, we identified lysophospholipase A2 (LYPLA2) as a major enzyme responsible for PG-G hydrolysis. The principal role played by LYPLA2 in PGE2-G hydrolysis was confirmed by siRNA knockdown. Purified recombinant LYPLA2 hydrolyzed PG-Gs in the following order of activity: PGE2-G > PGF2α-G > PGD2-G; LYPLA2 hydrolyzed 1- but not 2-arachidonoylglycerol or arachidonoylethanolamide. Chemical inhibition of LYPLA2 in the mouse macrophage-like cell line, RAW264.7, elicited an increase in PG-G production. Our data indicate that LYPLA2 serves as a major PG-G hydrolase in human cells. Perturbation of this enzyme should enable selective modulation of PG-Gs without alterations in endocannabinoids, thereby providing a means to decipher the unique functions of PG-Gs in biology and disease. PMID:25301951

  5. Crystal structure of the glycosidase family 73 peptidoglycan hydrolase FlgJ

    SciTech Connect

    Hashimoto, Wataru; Ochiai, Akihito; Momma, Keiko; Itoh, Takafumi; Mikami, Bunzo; Maruyama, Yukie; Murata, Kousaku

    2009-03-27

    Glycoside hydrolase (GH) categorized into family 73 plays an important role in degrading bacterial cell wall peptidoglycan. The flagellar protein FlgJ contains N- and C-terminal domains responsible for flagellar rod assembly and peptidoglycan hydrolysis, respectively. A member of family GH-73, the C-terminal domain (SPH1045-C) of FlgJ from Sphingomonas sp. strain A1 was expressed in Escherichia coli, purified, and characterized. SPH1045-C exhibited bacterial cell lytic activity most efficiently at pH 6.0 and 37 deg. C. The X-ray crystallographic structure of SPH1045-C was determined at 1.74 A resolution by single-wavelength anomalous diffraction. The enzyme consists of two lobes, {alpha} and {beta}. A deep cleft located between the two lobes can accommodate polymer molecules, suggesting that the active site is located in the cleft. Although SPH1045-C shows a structural homology with family GH-22 and GH-23 lysozymes, the arrangement of the nucleophile/base residue in the active site is specific to each peptidoglycan hydrolase.

  6. A chemical proteomic atlas of brain serine hydrolases identifies cell type-specific pathways regulating neuroinflammation

    PubMed Central

    Viader, Andreu; Ogasawara, Daisuke; Joslyn, Christopher M; Sanchez-Alavez, Manuel; Mori, Simone; Nguyen, William; Conti, Bruno; Cravatt, Benjamin F

    2016-01-01

    Metabolic specialization among major brain cell types is central to nervous system function and determined in large part by the cellular distribution of enzymes. Serine hydrolases are a diverse enzyme class that plays fundamental roles in CNS metabolism and signaling. Here, we perform an activity-based proteomic analysis of primary mouse neurons, astrocytes, and microglia to furnish a global portrait of the cellular anatomy of serine hydrolases in the brain. We uncover compelling evidence for the cellular compartmentalization of key chemical transmission pathways, including the functional segregation of endocannabinoid (eCB) biosynthetic enzymes diacylglycerol lipase-alpha (DAGLα) and –beta (DAGLβ) to neurons and microglia, respectively. Disruption of DAGLβ perturbed eCB-eicosanoid crosstalk specifically in microglia and suppressed neuroinflammatory events in vivo independently of broader effects on eCB content. Mapping the cellular distribution of metabolic enzymes thus identifies pathways for regulating specialized inflammatory responses in the brain while avoiding global alterations in CNS function. DOI: http://dx.doi.org/10.7554/eLife.12345.001 PMID:26779719

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

    SciTech Connect

    Danielsen, E.M. )

    1990-01-09

    The pig intestinal brush border enzymes aminopeptidase and lactase-phlorizin hydrolase are present in the microvilla membrane as homodimers. Dimethyl adipimidate was used to cross-link the two ({sup 35}S)methionine-labeled brush border enzymes from cultured mucosal explants. For aminopeptidase N, dimerization did not begin until 5-10 min after synthesis, and maximal dimerization by cross-linking of the transient form of the enzyme required 1 h, whereas the mature form of aminopeptidase N cross-linked with unchanged efficiency from 45 min to 3 h of labeling. Formation of dimers of this enzyme therefore occurs prior to the Golgi-associated processing, and the slow rate of dimerization may be the rate-limiting step in the transport from the endoplasmic reticulum to the Golgi complex. For lactase-phlorizin hydrolase, the posttranslational processing includes a proteolytic cleavage of its high molecular weight precursor. Since only the mature form and not the precursor of this enzyme could be cross-linked, formation of tightly associated dimers only takes place after transport out of the endoplasmic reticulum. Dimerization of the two brush border enzymes therefore seems to occur in different organelles of the enterocyte.

  8. Human microsomal epoxide hydrolase: genetic polymorphism and functional expression in vitro of amino acid variants

    PubMed Central

    Hassett, Christopher; Aicher, Lauri; Sidhu, Jaspreet S.

    2016-01-01

    Human microsomal epoxide hydrolase (mEH) is a biotransformation enzyme that metabolizes reactive epoxide intermediates to more water-soluble trans-dihydrodiol derivatives. We compared protein-coding sequences from six full-length human mEH DNA clones and assessed potential amino acid variation at seven positions. The prevalence of these variants was assessed in at least 37 unrelated individuals using polymerase chain reaction experiments. Only Tyr/His 113 (exon 3) and His/Arg 139 (exon 4) variants were observed. The genotype frequencies determined for residue 113 alleles indicate that this locus may not be in Hardy – Weinberg equilibrium, whereas frequencies observed for residue 139 alleles were similar to expected values. Nucleotide sequences coding for the variant amino acids were constructed in an mEH cDNA using site-directed mutagenesis, and each was expressed in vitro by transient transfection of COS-1 cells. Epoxide hydrolase mRNA level, catalytic activity, and immunoreactive protein were evaluated for each construct. The results of these analyses demonstrated relatively uniform levels of mEH RNA expression between the constructs. mEH enzymatic activity and immunoreactive protein were strongly correlated, indicating that mEH specific activity was similar for each variant. However, marked differences were noted in the relative amounts of immunoreactive protein and enzymatic activity resulting from the amino acid substitutions. These data suggest that common human mEH amino acid polymorphisms may alter enzymatic function, possibly by modifying protein stability. PMID:7516776

  9. Heavy chain single-domain antibodies to detect native human soluble epoxide hydrolase.

    PubMed

    Cui, Yongliang; Li, Dongyang; Morisseau, Christophe; Dong, Jie-Xian; Yang, Jun; Wan, Debin; Rossotti, Martín A; Gee, Shirley J; González-Sapienza, Gualberto G; Hammock, Bruce D

    2015-09-01

    The soluble epoxide hydrolase (sEH) is a potential pharmacological target for treating hypertension, vascular inflammation, pain, cancer, and other diseases. However, there is not a simple, inexpensive, and reliable method to estimate levels of active sEH in tissues. Toward developing such an assay, a polyclonal variable domain of heavy chain antibody (VHH) sandwich immunoassay was developed. Ten VHHs, which are highly selective for native human sEH, were isolated from a phage-displayed library. The ten VHHs have no significant cross-reactivity with human microsomal epoxide hydrolase, rat and mouse sEH, and denatured human sEH. There is a high correlation between protein levels of the sEH determined by the enzyme-linked immunosorbent assay (ELISA) and the catalytic activity of the enzyme in S9 fractions of human tissues (liver, kidney, and lung). The VHH-based ELISA appears to be a new reliable method for monitoring the sEH and may be useful as a diagnostic tool for diseases influenced by sEH. This study also demonstrates the broad utility of VHH in biochemical and pharmacological research. PMID:26229025

  10. Soluble epoxide hydrolase as an anti-inflammatory target of the thrombolytic stroke drug SMTP-7.

    PubMed

    Matsumoto, Naoki; Suzuki, Eriko; Ishikawa, Makoto; Shirafuji, Takumi; Hasumi, Keiji

    2014-12-26

    Although ischemic stroke is a major cause of death and disability worldwide, only a small fraction of patients benefit from the current thrombolytic therapy due to a risk of cerebral hemorrhage caused by inflammation. Thus, the development of a new strategy to combat inflammation during thrombolysis is an urgent demand. The small molecule thrombolytic SMTP-7 effectively treats ischemic stroke in several animal models with reducing cerebral hemorrhage. Here we revealed that SMTP-7 targeted soluble epoxide hydrolase (sEH) to suppress inflammation. SMTP-7 inhibited both of the two sEH enzyme activities: epoxide hydrolase (which inactivates anti-inflammatory epoxy-fatty acids) and lipid phosphate phosphatase. SMTP-7 suppressed epoxy-fatty acid hydrolysis in HepG2 cells in culture, implicating the sEH inhibition in the anti-inflammatory mechanism. The sEH inhibition by SMTP-7 was independent of its thrombolytic activity. The simultaneous targeting of thrombolysis and sEH by a single molecule is a promising strategy to revolutionize the current stroke therapy. PMID:25361765

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

    PubMed Central

    Mellon, Jay E.

    2015-01-01

    Several atoxigenic Aspergillus flavus isolates, including some being used as biocontrol agents, and one toxigenic isolate were surveyed for the ability to produce extracellular xylanolytic and pectinolytic hydrolases. All of the tested isolates displayed good production of endoxylanases when grown on a medium utilizing larch xylan as a sole carbon substrate. Four of the tested isolates produced reasonably high levels of esterase activity, while the atoxigenic biocontrol agent NRRL 21882 isolate esterase level was significantly lower than the others. Atoxigenic A. flavus isolates 19, 22, K49, AF36 (the latter two are biocontrol agents) and toxigenic AF13 produced copious levels of pectinolytic activity when grown on a pectin medium. The pectinolytic activity levels of the atoxigenic A. flavus 17 and NRRL 21882 isolates were significantly lower than the other tested isolates. In addition, A. flavus isolates that displayed high levels of pectinolytic activity in the plate assay produced high levels of endopolygalacturonase (pectinase) P2c, as ascertained by isoelectric focusing electrophoresis. Isolate NRRL 21882 displayed low levels of both pectinase P2c and pectin methyl esterase. A. flavus appears capable of producing these hydrolytic enzymes irrespective of aflatoxin production. This ability of atoxigenic isolates to produce xylanolytic and pectinolytic hydrolases mimics that of toxigenic isolates and, therefore, contributes to the ability of atoxigenic isolates to occupy the same niche as A. flavus toxigenic isolates. PMID:26295409

  12. [Inhibition of adherence of Corynebacterium diphtheriae to human buccal epithelium by glycoside hydrolases from marine hydrobiontes].

    PubMed

    Zaporozhets, T S; Makarenkova, I D; Bakunina, I Iu; Burtseva, Iu V; Kusaĭkin, M I; Balabanova, L A; Zviagintseva, T N; Besednova, N N; Rasskazov, V A

    2010-01-01

    A possibility of adhesion inhibition of Corynebacterium diphtheriae to human buccal epithelium by glycoside hydrolases of marine hydrobiontes was investigated using alpha-galactosidase from marine bacterium Pseudoalteromonas sp. KMM 701, total enzyme preparation and beta-1,3-glucanase from marine fungi Chaetomium, total enzyme preparation and beta-1,3-glucanase from marine mollusk Littorina kurila, and total enzyme preparation from crystalline style of marine mollusk Spisula sachalinensis were used. The enzymes were added to test-tubes containing buccal epithelial cells and/or the toxigenic bacterial strain C. diphtheriae No 1129, v. gravis. All the investigated enzymes were able to abort C. diphtheriae adherence, to human buccal epithelocytes. Inhibition of adhesion was more pronounced in the case of treatment of epithelocytes with highly purified enzymes of marine hydrobiontes in comparison with total enzyme preparations. The significant inhibition of C. diphtheriae adhesion was observed when the enzymes were added to the epithelocytes with the attached microorganisms. The results obtained show that glycoside hydrolases of marine hydrobiontes degrade any carbohydrates expressed on cell surface of bacterium or human buccal epithelocytes, impair unique lectin-carbohydrate interaction and prevent the adhesion. PMID:20695214

  13. A multifunctional hybrid glycosyl hydrolase discovered in an uncultured microbial consortium from ruminant gut.

    PubMed

    Palackal, Nisha; Lyon, Christopher S; Zaidi, Seema; Luginbühl, Peter; Dupree, Paul; Goubet, Florence; Macomber, John L; Short, Jay M; Hazlewood, Geoffrey P; Robertson, Dan E; Steer, Brian A

    2007-02-01

    A unique multifunctional glycosyl hydrolase was discovered by screening an environmental DNA library prepared from a microbial consortium collected from cow rumen. The protein consists of two adjacent catalytic domains. Sequence analysis predicted that one domain conforms to glycosyl hydrolase family 5 and the other to family 26. The enzyme is active on several different beta-linked substrates and possesses mannanase, xylanase, and glucanase activities. Site-directed mutagenesis studies on the catalytic residues confirmed the presence of two functionally independent catalytic domains. Using site-specific mutations, it was shown that one catalytic site hydrolyzes beta-1,4-linked mannan substrates, while the second catalytic site hydrolyzes beta-1,4-linked xylan and beta-1,4-linked glucan substrates. Polysaccharide Analysis using Carbohydrate gel Electrophoresis (PACE) also confirmed that the enzyme has discrete domains for binding and hydrolysis of glucan- and mannan-linked polysaccharides. Such multifunctional enzymes have many potential industrial applications in plant processing, including biomass saccharification, animal feed nutritional enhancement, textile, and pulp and paper processing. PMID:17103163

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

    PubMed

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

    2016-06-01

    Triazophos is a broad-spectrum and highly effective insecticide, and the residues of triazophos have been frequently detected in the environment. A triazophos-degrading bacterium, Burkholderia sp. SZL-1, was isolated from a long-term triazophos-polluted soil. Strain SZL-1 could hydrolyze triazophos to 1-phenyl-3-hydroxy-1,2,4-triazole, which was further utilized as the carbon sources for growth. The triazophos hydrolase gene trhA, cloned from strain SZL-1, was expressed and homogenously purified using Ni-nitrilotriacetic acid affinity chromatography. TrhA is 55 kDa and displays maximum activity at 25°C, pH 8.0. This enzyme still has nearly 60% activity at the range of 15°C-50°C for 30 min. TrhA was mutated by sequential error prone PCR and screened for improved activity for triazophos degradation. One purified variant protein (Val89-Gly89) named TrhA-M1 showed up to 3-fold improvement in specific activity against triazophos, and the specificity constants of Kcat and Kcat/Km for TrhA-M1 were improved up to 2.3- and 8.28-fold, respectively, compared to the wild-type enzyme. The results in this paper provided potential material for the contaminated soil remediation and hydrolase genetic structure research. PMID:27190294

  15. Soluble Epoxide Hydrolase as an Anti-inflammatory Target of the Thrombolytic Stroke Drug SMTP-7*

    PubMed Central

    Matsumoto, Naoki; Suzuki, Eriko; Ishikawa, Makoto; Shirafuji, Takumi; Hasumi, Keiji

    2014-01-01

    Although ischemic stroke is a major cause of death and disability worldwide, only a small fraction of patients benefit from the current thrombolytic therapy due to a risk of cerebral hemorrhage caused by inflammation. Thus, the development of a new strategy to combat inflammation during thrombolysis is an urgent demand. The small molecule thrombolytic SMTP-7 effectively treats ischemic stroke in several animal models with reducing cerebral hemorrhage. Here we revealed that SMTP-7 targeted soluble epoxide hydrolase (sEH) to suppress inflammation. SMTP-7 inhibited both of the two sEH enzyme activities: epoxide hydrolase (which inactivates anti-inflammatory epoxy-fatty acids) and lipid phosphate phosphatase. SMTP-7 suppressed epoxy-fatty acid hydrolysis in HepG2 cells in culture, implicating the sEH inhibition in the anti-inflammatory mechanism. The sEH inhibition by SMTP-7 was independent of its thrombolytic activity. The simultaneous targeting of thrombolysis and sEH by a single molecule is a promising strategy to revolutionize the current stroke therapy. PMID:25361765

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

    SciTech Connect

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

    2008-07-08

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

  17. Molecular characterization of human ABHD2 as TAG lipase and ester hydrolase.

    PubMed

    M, Naresh Kumar; V B S C, Thunuguntla; G K, Veeramachaneni; B, Chandra Sekhar; Guntupalli, Swapna; J S, Bondili

    2016-08-01

    Alterations in lipid metabolism have been progressively documented as a characteristic property of cancer cells. Though, human ABHD2 gene was found to be highly expressed in breast and lung cancers, its biochemical functionality is yet uncharacterized. In the present study we report, human ABHD2 as triacylglycerol (TAG) lipase along with ester hydrolysing capacity. Sequence analysis of ABHD2 revealed the presence of conserved motifs G(205)XS(207)XG(209) and H(120)XXXXD(125) Phylogenetic analysis showed homology to known lipases, Drosophila melanogaster CG3488. To evaluate the biochemical role, recombinant ABHD2 was expressed in Saccharomyces cerevisiae using pYES2/CT vector and His-tag purified protein showed TAG lipase activity. Ester hydrolase activity was confirmed with pNP acetate, butyrate and palmitate substrates respectively. Further, the ABHD2 homology model was built and the modelled protein was analysed based on the RMSD and root mean square fluctuation (RMSF) of the 100 ns simulation trajectory. Docking the acetate, butyrate and palmitate ligands with the model confirmed covalent binding of ligands with the Ser(207) of the GXSXG motif. The model was validated with a mutant ABHD2 developed with alanine in place of Ser(207) and the docking studies revealed loss of interaction between selected ligands and the mutant protein active site. Based on the above results, human ABHD2 was identified as a novel TAG lipase and ester hydrolase. PMID:27247428

  18. A remarkable activity of human leukotriene A4 hydrolase (LTA4H) toward unnatural amino acids.

    PubMed

    Byzia, Anna; Haeggström, Jesper Z; Salvesen, Guy S; Drag, Marcin

    2014-05-01

    Leukotriene A4 hydrolase (LTA4H--EC 3.3.2.6) is a bifunctional zinc metalloenzyme, which processes LTA4 through an epoxide hydrolase activity and is also able to trim one amino acid at a time from N-terminal peptidic substrates via its aminopeptidase activity. In this report, we have utilized a library of 130 individual proteinogenic and unnatural amino acid fluorogenic substrates to determine the aminopeptidase specificity of this enzyme. We have found that the best proteinogenic amino acid recognized by LTA4H is arginine. However, we have also observed several unnatural amino acids, which were significantly better in terms of cleavage rate (k cat/K m values). Among them, the benzyl ester of aspartic acid exhibited a k cat/K m value that was more than two orders of magnitude higher (1.75 × 10(5) M(-1) s(-1)) as compared to L-Arg (1.5 × 10(3) M(-1) s(-1)). This information can be used for design of potent inhibitors of this enzyme, but may also suggest yet undiscovered functions or specificities of LTA4H. PMID:24573245

  19. Computational insights into function and inhibition of fatty acid amide hydrolase.

    PubMed

    Palermo, Giulia; Rothlisberger, Ursula; Cavalli, Andrea; De Vivo, Marco

    2015-02-16

    The Fatty Acid Amide Hydrolase (FAAH) enzyme is a membrane-bound serine hydrolase responsible for the deactivating hydrolysis of a family of naturally occurring fatty acid amides. FAAH is a critical enzyme of the endocannabinoid system, being mainly responsible for regulating the level of its main cannabinoid substrate anandamide. For this reason, pharmacological inhibition of FAAH, which increases the level of endogenous anandamide, is a promising strategy to cure a variety of diseases including pain, inflammation, and cancer. Much structural, mutagenesis, and kinetic data on FAAH has been generated over the last couple of decades. This has prompted several informative computational investigations to elucidate, at the atomic-level, mechanistic details on catalysis and inhibition of this pharmaceutically relevant enzyme. Here, we review how these computational studies - based on classical molecular dynamics, full quantum mechanics, and hybrid QM/MM methods - have clarified the binding and reactivity of some relevant substrates and inhibitors of FAAH. We also discuss the experimental implications of these computational insights, which have provided a thoughtful elucidation of the complex physical and chemical steps of the enzymatic mechanism of FAAH. Finally, we discuss how computations have been helpful for building structure-activity relationships of potent FAAH inhibitors. PMID:25240419

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

    SciTech Connect

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

    2009-01-01

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

  1. The Cloning and Characterization of a Soluble Epoxide Hydrolase in Chicken

    PubMed Central

    Harris, T. R.; Morisseau, C.; Walzem, R. L.; Ma, S. J.; Hammock, B. D.

    2006-01-01

    The mammalian soluble epoxide hydrolase (sEH) plays a role in the regulation of blood pressure and vascular homeostasis through its hydrolysis of the endothelial-derived messenger molecules, the epoxyeicosatrienoic acids. This study reports the cloning and expression of a sEH homolog from chicken liver. The resulting 63-kDa protein has an isoelectric point of 6.1. The recombinant enzyme displayed epoxide hydrolase activity when assayed with [3H]-trans-1,3-diphenylpropene oxide (t-DPPO), as well as trans-9,10-epoxystearate and the cis-8,9-, 11,12-, and 14,15- epoxyeicosatrienoic acids. The chicken enzyme displayed a lower kcat:Km for t-DPPO than the mammalian enzymes. The enzyme was sensitive to urea-based inhibitors developed for mammalian sEH. Such compounds could be used to study the role of chicken sEH in conditions in which endothelial-derived vasodilation is believed to be impaired, such as pulmonary hypertension syndrome. PMID:16523628

  2. Substrate recognition and catalysis by LytB, a pneumococcal peptidoglycan hydrolase involved in virulence

    PubMed Central

    Rico-Lastres, Palma; Díez-Martínez, Roberto; Iglesias-Bexiga, Manuel; Bustamante, Noemí; Aldridge, Christine; Hesek, Dusan; Lee, Mijoon; Mobashery, Shahriar; Gray, Joe; Vollmer, Waldemar; García, Pedro; Menéndez, Margarita

    2015-01-01

    Streptococcus pneumoniae is a major cause of life-threatening diseases worldwide. Here we provide an in-depth functional characterization of LytB, the peptidoglycan hydrolase responsible for physical separation of daughter cells. Identified herein as an N-acetylglucosaminidase, LytB is involved also in colonization and invasion of the nasopharynx, biofilm formation and evasion of host immunity as previously demonstrated. We have shown that LytB cleaves the GlcNAc-β-(1,4)-MurNAc glycosidic bond of peptidoglycan building units. The hydrolysis occurs at sites with fully acetylated GlcNAc moieties, with preference for uncross-linked muropeptides. The necessity of GlcN acetylation and the presence of a single acidic moiety (Glu585) essential for catalysis strongly suggest a substrate-assisted mechanism with anchimeric assistance of the acetamido group of GlcNAc moieties. Additionally, modelling of the catalytic region bound to a hexasaccharide tripentapeptide provided insights into substrate-binding subsites and peptidoglycan recognition. Besides, cell-wall digestion products and solubilisation rates might indicate a tight control of LytB activity to prevent unrestrained breakdown of the cell wall. Choline-independent localization at the poles of the cell, mediated by the choline-binding domain, peptidoglycan modification, and choline-mediated (lipo)teichoic-acid attachment contribute to the high selectivity of LytB. Moreover, so far unknown chitin hydrolase and glycosyltransferase activities were detected using GlcNAc oligomers as substrate. PMID:26537571

  3. Engineering of an epoxide hydrolase for efficient bioresolution of bulky pharmaco substrates

    PubMed Central

    Kong, Xu-Dong; Yuan, Shuguang; Li, Lin; Chen, She; Xu, Jian-He; Zhou, Jiahai

    2014-01-01

    Optically pure epoxides are essential chiral precursors for the production of (S)-propranolol, (S)-alprenolol, and other β-adrenergic receptor blocking drugs. Although the enzymatic production of these bulky epoxides has proven difficult, here we report a method to effectively improve the activity of BmEH, an epoxide hydrolase from Bacillus megaterium ECU1001 toward α-naphthyl glycidyl ether, the precursor of (S)-propranolol, by eliminating the steric hindrance near the potential product-release site. Using X-ray crystallography, mass spectrum, and molecular dynamics calculations, we have identified an active tunnel for substrate access and product release of this enzyme. The crystal structures revealed that there is an independent product-release site in BmEH that was not included in other reported epoxide hydrolase structures. By alanine scanning, two mutants, F128A and M145A, targeted to expand the potential product-release site displayed 42 and 25 times higher activities toward α-naphthyl glycidyl ether than the wild-type enzyme, respectively. These results show great promise for structure-based rational design in improving the catalytic efficiency of industrial enzymes for bulky substrates. PMID:25331869

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

    PubMed Central

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

    2008-01-01

    The structure of a structural genomics target protein, Tbru020260AAA from Trypanosoma brucei, has been determined to a resolution of 2.2 Å using multiple-wavelength anomalous diffraction at the Se K edge. This protein belongs to Pfam sequence family PF08538 and is only distantly related to previously studied members of the α/β-hydrolase fold family. Structural superposition onto representative α/β-hydrolase fold proteins of known function indicates that a possible catalytic nucleophile, Ser116 in the T. brucei protein, lies at the expected location. However, the present structure and by extension the other trypanosomatid members of this sequence family have neither sequence nor structural similarity at the location of other active-site residues typical for proteins with this fold. Together with the presence of an additional domain between strands β6 and β7 that is conserved in trypanosomatid genomes, this suggests that the function of these homologs has diverged from other members of the fold family. PMID:18540054

  5. Soluble Epoxide Hydrolase Homologs in Strongylocentrotus purpuratus Suggest a Gene Duplication Event and Subsequent Divergence

    PubMed Central

    Harris, Todd R.; Aronov, Pavel A.

    2008-01-01

    The mammalian soluble epoxide hydrolase (sEH) is a multidomain enzyme composed of C- and N-terminal regions that contain active sites for epoxide hydrolase (EH) and phosphatase activities, respectively. We report the cloning of two 60 kDa multidomain enzymes from the purple sea urchin Strongylocentrotus purpuratus displaying significant sequence similarity to both the N- and C-terminal domains of the mammalian sEH. While one urchin enzyme did not exhibit EH activity, the second enzyme hydrolyzed several lipid messenger molecules metabolized by the mammalian sEH, including the epoxyeicosatrienoic acids. Neither of the urchin enzymes displayed phosphatase activity. The urchin EH was inhibited by small molecule inhibitors of the mammalian sEH and is the likely ancestor of the enzyme. Sequence comparisons suggest that the urchin sEH homologs are the result of a gene fusion event between a gene encoding for an EH and a gene for an enzyme of undetermined function. This fusion event was followed by a duplication event to produce the urchin enzymes. PMID:18554159

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

    PubMed Central

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

    2010-01-01

    Development of cellulosic biofuels from non-food crops is currently an area of intense research interest. Tailoring depolymerizing enzymes to particular feedstocks and pretreatment conditions is one promising avenue of research in this area. Here we added a green-waste compost inoculum to switchgrass (Panicum virgatum) and simulated thermophilic composting in a bioreactor to select for a switchgrass-adapted community and to facilitate targeted discovery of glycoside hydrolases. Small-subunit (SSU) rRNA-based community profiles revealed that the microbial community changed dramatically between the initial and switchgrass-adapted compost (SAC) with some bacterial populations being enriched over 20-fold. We obtained 225 Mbp of 454-titanium pyrosequence data from the SAC community and conservatively identified 800 genes encoding glycoside hydrolase domains that were biased toward depolymerizing grass cell wall components. Of these, ∼10% were putative cellulases mostly belonging to families GH5 and GH9. We synthesized two SAC GH9 genes with codon optimization for heterologous expression in Escherichia coli and observed activity for one on carboxymethyl cellulose. The active GH9 enzyme has a temperature optimum of 50°C and pH range of 5.5 to 8 consistent with the composting conditions applied. We demonstrate that microbial communities adapt to switchgrass decomposition using simulated composting condition and that full-length genes can be identified from complex metagenomic sequence data, synthesized and expressed resulting in active enzyme. PMID:20098679

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

    SciTech Connect

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

    2009-11-15

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

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

    SciTech Connect

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

    2011-05-11

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

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

    PubMed Central

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

    2011-01-01

    Tannin acyl hydrolase also referred as tannase is an enzyme with important applications in several science and technology fields. Due to its hydrolytic and synthetic properties, tannase could be used to reduce the negative effects of tannins in beverages, food, feed, and tannery effluents, for the production of gallic acid from tannin-rich materials, the elucidation of tannin structure, and the synthesis of gallic acid esters in nonaqueous media. However, industrial applications of tannase are still very limited due to its high production cost. Thus, there is a growing interest in the production, recovery, and purification of this enzyme. Recently, there have been published a number of papers on the improvement of upstream and downstream processing of the enzyme. These papers dealt with the search for new tannase producing microorganisms, the application of novel fermentation systems, optimization of culture conditions, the production of the enzyme by recombinant microorganism, and the design of efficient protocols for tannase recovery and purification. The present work reviews the state of the art of basic and biotechnological aspects of tannin acyl hydrolase, focusing on the recent advances in the upstream and downstream processing of the enzyme. PMID:21941633

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

    PubMed

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

    2015-06-01

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

  11. Molecular characterization of human ABHD2 as TAG lipase and ester hydrolase

    PubMed Central

    M., Naresh Kumar; V.B.S.C., Thunuguntla; G.K., Veeramachaneni; B., Chandra Sekhar; Guntupalli, Swapna; J.S., Bondili

    2016-01-01

    Alterations in lipid metabolism have been progressively documented as a characteristic property of cancer cells. Though, human ABHD2 gene was found to be highly expressed in breast and lung cancers, its biochemical functionality is yet uncharacterized. In the present study we report, human ABHD2 as triacylglycerol (TAG) lipase along with ester hydrolysing capacity. Sequence analysis of ABHD2 revealed the presence of conserved motifs G205XS207XG209 and H120XXXXD125. Phylogenetic analysis showed homology to known lipases, Drosophila melanogaster CG3488. To evaluate the biochemical role, recombinant ABHD2 was expressed in Saccharomyces cerevisiae using pYES2/CT vector and His-tag purified protein showed TAG lipase activity. Ester hydrolase activity was confirmed with pNP acetate, butyrate and palmitate substrates respectively. Further, the ABHD2 homology model was built and the modelled protein was analysed based on the RMSD and root mean square fluctuation (RMSF) of the 100 ns simulation trajectory. Docking the acetate, butyrate and palmitate ligands with the model confirmed covalent binding of ligands with the Ser207 of the GXSXG motif. The model was validated with a mutant ABHD2 developed with alanine in place of Ser207 and the docking studies revealed loss of interaction between selected ligands and the mutant protein active site. Based on the above results, human ABHD2 was identified as a novel TAG lipase and ester hydrolase. PMID:27247428

  12. Determinants of Murein Hydrolase Targeting to Cross-wall of Staphylococcus aureus Peptidoglycan*

    PubMed Central

    Frankel, Matthew B.; Schneewind, Olaf

    2012-01-01

    Cells of eukaryotic or prokaryotic origin express proteins with LysM domains that associate with the cell wall envelope of bacteria. The molecular properties that enable LysM domains to interact with microbial cell walls are not yet established. Staphylococcus aureus, a spherical microbe, secretes two murein hydrolases with LysM domains, Sle1 and LytN. We show here that the LysM domains of Sle1 and LytN direct murein hydrolases to the staphylococcal envelope in the vicinity of the cross-wall, the mid-cell compartment for peptidoglycan synthesis. LysM domains associate with the repeating disaccharide β-N-acetylmuramic acid, (1→4)-β-N-acetylglucosamine of staphylococcal peptidoglycan. Modification of N-acetylmuramic acid with wall teichoic acid, a ribitol-phosphate polymer tethered to murein linkage units, prevents the LysM domain from binding to peptidoglycan. The localization of LytN and Sle1 to the cross-wall is abolished in staphylococcal tagO mutants, which are defective for wall teichoic acid synthesis. We propose a model whereby the LysM domain ensures septal localization of LytN and Sle1 followed by processive cleavage of peptidoglycan, thereby exposing new LysM binding sites in the cross-wall and separating bacterial cells. PMID:22303016

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

    SciTech Connect

    French, Jarrod B.; Ealick, Steven E.

    2011-08-01

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

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

    PubMed

    Matsuzawa, Tomohiko; Mitsuishi, Yasushi; Kameyama, Akihiko; Yaoi, Katsuro

    2016-03-01

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

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

    SciTech Connect

    CHRONISTER, G.B.

    2005-06-14

    This paper discusses selecting and Implementing hydrolasing technology to reduce radioactive contamination in preparing to dispose of the K Basins; two highly contaminated concrete basins at the Hanford Site. A large collection of spent nuclear fuel stored for many years underwater at the K Basins has been removed to stable, dry, safe storage. Remediation activities have begun for the remaining highly contaminated water, sludge, and concrete basin structures. Hydrolasing will be used to decontaminate and prepare the basin structures for disposal. The U. S. Department of Energy's (DOE) Hanford Site is considered the world's largest environmental cleanup project. The site covers 1,517 Km{sup 2} (586 square miles) along the Columbia River in an arid region of the northwest United States (U.S.). Hanford is the largest of the US former nuclear defense production sites. From the World War II era of the mid-1940s until the late-1980s when production stopped, Hanford produced 60 percent of the plutonium for nuclear defense and, as a consequence, produced a significant amount of environmental pollution now being addressed. Spent nuclear fuel was among the major challenges for DOE's environmental cleanup mission at Hanford. The end of production left Hanford with about 105,000 irradiated, solid uranium metal fuel assemblies--representing approximately 2,100 metric tons (80 percent of DOE's spent nuclear fuel). The fuel was ultimately stored in the K Basins water-filled, concrete basins attached to Hanford's K East (KE) and K West (KW) reactors. K Basin's fuel accounted for 95 percent of the total radioactivity in Hanford's former reactor production areas. Located about 457 meters (500 yards) from the Columbia River, the K Basins are two indoor, rectangular structures of reinforced concrete; each filled with more than 3.8 million liters (one million gallons) of water that has become highly contaminated with long-lived radionuclides. At the KW Basin, fuel was packaged and

  16. AphID (Lucid key) http://AphID.AphidNet.org

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This peer-reviewed web site concentrates on the 66 adult alate and apterous aphids that are the world's most cosmopolitan and polyphagous species. The site includes fact sheets about the various aphids species, a glossary of terms helpful to the student, hundreds of photographs and illustrations, a...

  17. THE SOLUBLE EPOXIDE HYDROLASE GENE HARBORS SEQUENCE VARIATIONS ASSOCIATED WITH SUSCEPTIBILITY TO AND PROTECTION FROM INCIDENT ISCHEMIC STROKE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Stroke is the leading cause of severe disability and the third leading cause of death, accounting for one of every 15 deaths in the USA. We investigated the association of polymorphisms in the soluble epoxide hydrolase gene (EPHX2) with incident ischemic stroke in African-Americans and Whites. Twelv...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacteriophage lytic enzymes have recently attracted considerable interest as novel antimicrobials against Gram-positive bacteria. In this work, antimicrobial activity in milk of HydH5 [(a virion-associated peptidoglycan hydrolase (VAPGH) encoded by the Staphylococcus aureus bacteriophage vB_SauS-ph...

  19. Hydrolysis of filter-paper cellulose to glucose by two recombinant endogenous glycosyl hydrolases of Coptotermes formosanus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genes encoding for glycosyl hydrolases in multiple families were recovered from an EST library of Coptotermes formosanus, a wood-eating insect. Functional analyses of these genes not only shed light on the mechanisms the insect employs to successfully use cellulosic materials as energy sources, whic...

  20. Cloning, crystallization and preliminary X-ray study of XC1258, a CN-hydrolase superfamily protein from Xanthomonas campestris

    SciTech Connect

    Tsai, Ying-Der; Chin, Ko-Hsin; Shr, Hui-Lin; Gao, Fei Philip; Lyu, Ping-Chiang; Wang, Andrew H.-J.; Chou, Shan-Ho

    2006-10-01

    A CN-hydrolase superfamily protein from the plant pathogen X. campestris has been overexpressed in E. coli, purified and crystallized. CN-hydrolase superfamily proteins are involved in a wide variety of non-peptide carbon–nitrogen hydrolysis reactions, producing some important natural products such as auxin, biotin, precursors of antibiotics etc. These reactions all involve attack on a cyano or carbonyl carbon by a conserved novel catalytic triad Glu-Lys-Cys through a thiol acylenzyme intermediate. However, classification into the CN-hydrolase superfamily based on sequence similarity alone is not straightforward and further structural data are necessary to improve this categorization. Here, the cloning, expression, crystallization and preliminary X-ray analysis of XC1258, a CN-hydrolase superfamily protein from the plant pathogen Xanthomonas campestris (Xcc), are reported. The SeMet-substituted XC1258 crystals diffracted to a resolution of 1.73 Å. They are orthorhombic and belong to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 143.8, b = 154.63, c = 51.3 Å, respectively.

  1. A dual enzyme system composed of a polyester hydrolase and a carboxylesterase enhances the biocatalytic degradation of polyethylene terephthalate films.

    PubMed

    Barth, Markus; Honak, Annett; Oeser, Thorsten; Wei, Ren; Belisário-Ferrari, Matheus R; Then, Johannes; Schmidt, Juliane; Zimmermann, Wolfgang

    2016-08-01

    TfCut2 from Thermobifida fusca KW3 and the metagenome-derived LC-cutinase are bacterial polyester hydrolases capable of efficiently degrading polyethylene terephthalate (PET) films. Since the enzymatic PET hydrolysis is inhibited by the degradation intermediate mono-(2-hydroxyethyl) terephthalate (MHET), a dual enzyme system consisting of a polyester hydrolase and the immobilized carboxylesterase TfCa from Thermobifida fusca KW3 was employed for the hydrolysis of PET films at 60°C. HPLC analysis of the reaction products obtained after 24 h of hydrolysis showed an increased amount of soluble products with a lower proportion of MHET in the presence of the immobilized TfCa. The results indicated a continuous hydrolysis of the inhibitory MHET by the immobilized TfCa and demonstrated its advantage as a second biocatalyst in combination with a polyester hydrolase for an efficient degradation oft PET films. The dual enzyme system with LC-cutinase produced a 2.4-fold higher amount of degradation products compared to TfCut2 after a reaction time of 24 h confirming the superior activity of his polyester hydrolase against PET films. PMID:27214855

  2. Crystal structure analysis of a glycosides hydrolase family 42 cold-adapted ß-galactosidase from Rahnella sp. R3

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ß-galactosidase isolated from a psychrotrophic bacterium, Rahnella sp. R3 (R-ß-Gal), exhibits high activity at low temperature. R-ß-Gal is a member of the glycoside hydrolases family 42 (GH42), and forms a 225 kDa trimeric structure in solution. The X-ray crystal structure of R-ß-Gal was determi...

  3. Purification and characterization of a glycoside hydrolase family 43 Beta-xylosidase from Geobacillus thermoleovorans IT-08

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The gene encoding a glycoside hydrolase family 43 enzyme termed deAX was isolated and subcloned from a culture seeded with a compost starter mixed bacterium population, expressed with a C-terminal His6-tag, and purified to apparent homogeneity. deAX was monomeric in solution, and had a broad pH maxi...

  4. New mode for divalent metal activation of glycoside hydrolases: X-ray structure of ß-xyloisdase-Ca2+

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We report the first X-ray structure of a glycoside hydrolase family 43 ß-xylosidase, RS223BX, which is strongly activated by the addition of divalent metal cations. The 2.69 Å structure reveals that the Ca2+ cation is located at the back of the active site pocket. The Ca2+ coordinates to H274 to sta...

  5. Purification and characterization of a cis-epoxysuccinic acid hydrolase from Nocardia tartaricans CAS-52, and expression in Escherichia coli.

    PubMed

    Wang, Ziqiang; Wang, Yunshan; Su, Zhiguo

    2013-03-01

    A highly enantioselective cis-epoxysuccinic acid hydrolase from Nocardia tartaricans was purified to electrophoretic homogeneity. The enzyme was purified 184-fold with a yield of 18.8 %. The purified cis-epoxysuccinic acid hydrolase had a monomeric molecular weight of 28 kDa, and its optimum conditions were 37 °C and pH 7-9. With sodium cis-epoxysuccinate as the substrate, Michaelis-Menten enzyme kinetics analysis gave a Km value of 35.71 mM and a Vmax of 2.65 mM min(-1). The enzyme was activated by Ni(2+) and Al(3+), while strongly inhibited by Fe(3+), Fe(2+), Cu(2+), and Ag(+). The cis-epoxysuccinic acid hydrolase gene was cloned, and its open reading frame sequence predicted a protein composed of 253 amino acids. A pET11a expression plasmid carrying the gene under the control of the T7 promoter was introduced into Escherichia coli, and the cis-epoxysuccinic acid hydrolase gene was successfully expressed in the recombinant strains. PMID:22552902

  6. 3-D QSAR ANALYSIS OF INHIBITION OF MURINE SOLUBLE EPOXIDE HYDROLASE (MSEH) BY BENZOYLUREAS, ARYLUREAS, AND THEIR ANALOGUES. (R825433)

    EPA Science Inventory

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

  7. Emergent decarboxylase activity and attenuation of α/β-hydrolase activity during the evolution of methylketone biosynthesis in tomato.

    PubMed

    Auldridge, Michele E; Guo, Yongxia; Austin, Michael B; Ramsey, Justin; Fridman, Eyal; Pichersky, Eran; Noel, Joseph P

    2012-04-01

    Specialized methylketone-containing metabolites accumulate in certain plants, in particular wild tomatoes in which they serve as toxic compounds against chewing insects. In Solanum habrochaites f. glabratum, methylketone biosynthesis occurs in the plastids of glandular trichomes and begins with intermediates of de novo fatty acid synthesis. These fatty-acyl intermediates are converted via sequential reactions catalyzed by Methylketone Synthase2 (MKS2) and MKS1 to produce the n-1 methylketone. We report crystal structures of S. habrochaites MKS1, an atypical member of the α/β-hydrolase superfamily. Sequence comparisons revealed the MKS1 catalytic triad, Ala-His-Asn, as divergent to the traditional α/β-hydrolase triad, Ser-His-Asp. Determination of the MKS1 structure points to a novel enzymatic mechanism dependent upon residues Thr-18 and His-243, confirmed by biochemical assays. Structural analysis further reveals a tunnel leading from the active site consisting mostly of hydrophobic residues, an environment well suited for fatty-acyl chain binding. We confirmed the importance of this substrate binding mode by substituting several amino acids leading to an alteration in the acyl-chain length preference of MKS1. Furthermore, we employ structure-guided mutagenesis and functional assays to demonstrate that MKS1, unlike enzymes from this hydrolase superfamily, is not an efficient hydrolase but instead catalyzes the decarboxylation of 3-keto acids. PMID:22523203

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

    ERIC Educational Resources Information Center

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

    2009-01-01

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

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

    EPA Science Inventory

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

  10. [The effect of antioxidants on the activity of acid hydrolases in blood leukocytes from patients with leukoplakia of mouth mucosa].

    PubMed

    Petrovich, Iu A; Mashkilleĭson, A L; Suleĭmanova, G G; Lagunov, A I

    1989-01-01

    Activity of acid hydrolases, alkaline phosphatase and leucine aminopeptidase was studied in leukocytes of patients with leukoplakia of mouth mucosa before and after the treatment involving antioxidant drugs. The enzymatic activity studied was increased in leukoplakia. Cryotherapy combined with antioxidants and the treatment with antioxidants only contributed to a decrease in these enzymes activity. PMID:2617939

  11. Targeted gene inactivation in Clostridium phytofermentans shows that cellulose degradation requires the family 9 hydrolase Cphy3367

    PubMed Central

    Tolonen, Andrew C; Chilaka, Amanda C; Church, George M

    2009-01-01

    Microbial cellulose degradation is a central part of the global carbon cycle and has great potential for the development of inexpensive, carbon-neutral biofuels from non-food crops. Clostridium phytofermentans has a repertoire of 108 putative glycoside hydrolases to break down cellulose and hemicellulose into sugars, which this organism then ferments primarily to ethanol. An understanding of cellulose degradation at the molecular level requires learning the different roles of these hydrolases. In this study, we show that interspecific conjugation with Escherichia coli can be used to transfer a plasmid into C. phytofermentans that has a resistance marker, an origin of replication that can be selectively lost, and a designed group II intron for efficient, targeted chromosomal insertions without selection. We applied these methods to disrupt the cphy3367 gene, which encodes the sole family 9 glycoside hydrolase (GH9) in the C. phytofermentans genome. The GH9-deficient strain grew normally on some carbon sources such as glucose, but had lost the ability to degrade cellulose. Although C. phytofermentans upregulates the expression of numerous enzymes to break down cellulose, this process thus relies upon a single, key hydrolase, Cphy3367. PMID:19775243

  12. Xyloglucan Endotransglucosylase-Hydrolase17 Interacts with Xyloglucan Endotransglucosylase-Hydrolase31 to Confer Xyloglucan Endotransglucosylase Action and Affect Aluminum Sensitivity in Arabidopsis.

    PubMed

    Zhu, Xiao Fang; Wan, Jiang Xue; Sun, Ying; Shi, Yuan Zhi; Braam, Janet; Li, Gui Xin; Zheng, Shao Jian

    2014-06-19

    Previously, we reported that although the Arabidopsis (Arabidopsis thaliana) Xyloglucan Endotransglucosylase-Hydrolase31 (XTH31) has predominately xyloglucan endohydrolase activity in vitro, loss of XTH31 results in remarkably reduced in vivo xyloglucan endotransglucosylase (XET) action and enhanced Al resistance. Here, we report that XTH17, predicted to have XET activity, binds XTH31 in yeast (Saccharomyces cerevisiae) two-hybrid and coimmunoprecipitations assays and that this interaction may be required for XTH17 XET activity in planta. XTH17 and XTH31 may be colocalized in plant cells because tagged XTH17 fusion proteins, like XTH31 fusion proteins, appear to target to the plasma membrane. XTH17 expression, like that of XTH31, was substantially reduced in the presence of aluminum (Al), even at concentrations as low as 10 µm for 24 h or 25 µm for just 30 min. Agrobacterium tumefaciens-mediated transfer DNA insertion mutant of XTH17, xth17, showed low XET action and had moderately shorter roots than the wild type but was more Al resistant than the wild type. Similar to xth31, xth17 had low hemicellulose content and retained less Al in the cell wall. These data suggest a model whereby XTH17 and XTH31 may exist as a dimer at the plasma membrane to confer in vivo XET action, which modulates cell wall Al-binding capacity and thereby affects Al sensitivity in Arabidopsis. PMID:24948835

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

    PubMed

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

    2014-01-01

    We applied a combination of rational design and directed evolution (DE) to Bacillus subtilis p-nitrobenzyl esterase (pNBE) with the goal of enhancing organophosphorus acid anhydride hydrolase (OPAAH) activity. DE started with a designed variant, pNBE A107H, carrying a histidine homologous with human butyrylcholinesterase G117H to find complementary mutations that further enhance its OPAAH activity. Five sites were selected (G105, G106, A107, A190, and A400) within a 6.7 Å radius of the nucleophilic serine Oγ. All 95 variants were screened for esterase activity with a set of five substrates: pNP-acetate, pNP-butyrate, acetylthiocholine, butyrylthiocholine, or benzoylthiocholine. A microscale assay for OPAAH activity was developed for screening DE libraries. Reductions in esterase activity were generally concomitant with enhancements in OPAAH activity. One variant, A107K, showed an unexpected 7-fold increase in its k cat/K m for benzoylthiocholine, demonstrating that it is also possible to enhance the cholinesterase activity of pNBE. Moreover, DE resulted in at least three variants with modestly enhanced OPAAH activity compared to wild type pNBE. A107H/A190C showed a 50-fold increase in paraoxonase activity and underwent a slow time- and temperature-dependent change affecting the hydrolysis of OPAA and ester substrates. Structural analysis suggests that pNBE may represent a precursor leading to human cholinesterase and carboxylesterase 1 through extension of two vestigial specificity loops; a preliminary attempt to transfer the Ω-loop of BChE into pNBE is described. Unlike butyrylcholinesterase and pNBE, introducing a G143H mutation (equivalent to G117H) did not confer detectable OP hydrolase activity on human carboxylesterase 1 (hCE1). We discuss the use of pNBE as a surrogate scaffold for the mammalian esterases, and the importance of the oxyanion-hole residues for enhancing the OPAAH activity of selected serine hydrolases. PMID:25077141

  14. Three-dimensional Structure of Nylon Hydrolase and Mechanism of Nylon-6 Hydrolysis*

    PubMed Central

    Negoro, Seiji; Shibata, Naoki; Tanaka, Yusuke; Yasuhira, Kengo; Shibata, Hiroshi; Hashimoto, Haruka; Lee, Young-Ho; Oshima, Shohei; Santa, Ryuji; Oshima, Shohei; Mochiji, Kozo; Goto, Yuji; Ikegami, Takahisa; Nagai, Keisuke; Kato, Dai-ichiro; Takeo, Masahiro; Higuchi, Yoshiki

    2012-01-01

    We performed x-ray crystallographic analyses of the 6-aminohexanoate oligomer hydrolase (NylC) from Agromyces sp. at 2.0 Å-resolution. This enzyme is a member of the N-terminal nucleophile hydrolase superfamily that is responsible for the degradation of the nylon-6 industry byproduct. We observed four identical heterodimers (27 kDa + 9 kDa), which resulted from the autoprocessing of the precursor protein (36 kDa) and which constitute the doughnut-shaped quaternary structure. The catalytic residue of NylC was identified as the N-terminal Thr-267 of the 9-kDa subunit. Furthermore, each heterodimer is folded into a single domain, generating a stacked αββα core structure. Amino acid mutations at subunit interfaces of the tetramer were observed to drastically alter the thermostability of the protein. In particular, four mutations (D122G/H130Y/D36A/E263Q) of wild-type NylC from Arthrobacter sp. (plasmid pOAD2-encoding enzyme), with a heat denaturation temperature of Tm = 52 °C, enhanced the protein thermostability by 36 °C (Tm = 88 °C), whereas a single mutation (G111S or L137A) decreased the stability by ∼10 °C. We examined the enzymatic hydrolysis of nylon-6 by the thermostable NylC mutant. Argon cluster secondary ion mass spectrometry analyses of the reaction products revealed that the major peak of nylon-6 (m/z 10,000–25,000) shifted to a smaller range, producing a new peak corresponding to m/z 1500–3000 after the enzyme treatment at 60 °C. In addition, smaller fragments in the soluble fraction were successively hydrolyzed to dimers and monomers. Based on these data, we propose that NylC should be designated as nylon hydrolase (or nylonase). Three potential uses of NylC for industrial and environmental applications are also discussed. PMID:22187439

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

    PubMed Central

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

    2014-01-01

    We applied a combination of rational design and directed evolution (DE) to Bacillus subtilis p-nitrobenzyl esterase (pNBE) with the goal of enhancing organophosphorus acid anhydride hydrolase (OPAAH) activity. DE started with a designed variant, pNBE A107H, carrying a histidine homologous with human butyrylcholinesterase G117H to find complementary mutations that further enhance its OPAAH activity. Five sites were selected (G105, G106, A107, A190, and A400) within a 6.7 Å radius of the nucleophilic serine Oγ. All 95 variants were screened for esterase activity with a set of five substrates: pNP-acetate, pNP-butyrate, acetylthiocholine, butyrylthiocholine, or benzoylthiocholine. A microscale assay for OPAAH activity was developed for screening DE libraries. Reductions in esterase activity were generally concomitant with enhancements in OPAAH activity. One variant, A107K, showed an unexpected 7-fold increase in its kcat/Km for benzoylthiocholine, demonstrating that it is also possible to enhance the cholinesterase activity of pNBE. Moreover, DE resulted in at least three variants with modestly enhanced OPAAH activity compared to wild type pNBE. A107H/A190C showed a 50-fold increase in paraoxonase activity and underwent a slow time- and temperature-dependent change affecting the hydrolysis of OPAA and ester substrates. Structural analysis suggests that pNBE may represent a precursor leading to human cholinesterase and carboxylesterase 1 through extension of two vestigial specificity loops; a preliminary attempt to transfer the Ω-loop of BChE into pNBE is described. Unlike butyrylcholinesterase and pNBE, introducing a G143H mutation (equivalent to G117H) did not confer detectable OP hydrolase activity on human carboxylesterase 1 (hCE1). We discuss the use of pNBE as a surrogate scaffold for the mammalian esterases, and the importance of the oxyanion-hole residues for enhancing the OPAAH activity of selected serine hydrolases. PMID:25077141

  16. Isolation and characterization of an extracellular glycosylated protein complex from Clostridium thermosaccharolyticum with pectin methylesterase and polygalacturonate hydrolase activity.

    PubMed

    Van Rijssel, M; Gerwig, G J; Hansen, T A

    1993-03-01

    An extracellular protein complex was isolated from the supernatant of a pectin-limited continuous culture of Clostridium thermosaccharolyticum Haren. The complex possessed both pectin methylesterase (EC 3.1.1.11) and exo-poly-alpha-galacturonate hydrolase (EC 3.2.1.82) activity and produced digalacturonate from the nonreducing end of the pectin chain. The protein consisted of 230- and 25-kDa subunits. The large subunit contained 10% (wt/wt) sugars (N-acetylgalactosamine and galactose). Under physiological conditions both activities acted in a coordinated manner: the ratio between methanol and digalacturonate released during degradation was constant and equal to the degree of esterification of the pectin used. Prolonged incubation of the enzyme with pectin led to a nondialyzable fraction that was enriched in neutral sugars, such as arabinose, rhamnose, and galactose; the high rhamnose/galacturonic acid ratio was indicative of hairy region-like structures. The smallest substrate utilized by the hydrolase was a tetragalacturonate. Vmax with oligogalacturonates increased with increasing chain length. The Km and Vmax for the polygalacturonate hydrolase with citrus pectate as a substrate were 0.8 g liter-1 and 180 mumol min-1 mg of protein-1, respectively. The Km and Vmax for the esterase with citrus pectin as a substrate were 1.2 g liter-1 and 440 mumol min-1 mg of protein-1, respectively. The temperature optima for the hydrolase and esterase were 70 and 60 degrees C, respectively. Both enzyme activities were stable for more than 1 h at 70 degrees C. The exo-polygalacturonate hydrolase of Clostridium thermosulfurogenes was partially purified while the methylesterase was also copurified. PMID:8481009

  17. Efficient Calculation of Enzyme Reaction Free Energy Profiles Using a Hybrid Differential Relaxation Algorithm: Application to Mycobacterial Zinc Hydrolases.

    PubMed

    Romero, Juan Manuel; Martin, Mariano; Ramirez, Claudia Lilián; Dumas, Victoria Gisel; Marti, Marcelo Adrián

    2015-01-01

    Determination of the free energy profile for an enzyme reaction mechanism is of primordial relevance, paving the way for our understanding of the enzyme's catalytic power at the molecular level. Although hybrid, mostly DFT-based, QM/MM methods have been extensively applied to this type of studies, achieving accurate and statistically converged results at a moderate computational cost is still an open challenge. Recently, we have shown that accurate results can be achieved in less computational time, combining Jarzynski's relationship with a hybrid differential relaxation algorithm (HyDRA), which allows partial relaxation of the solvent during the nonequilibrium steering of the reaction. In this work, we have applied this strategy to study two mycobacterial zinc hydrolases. Mycobacterium tuberculosis infections are still a worldwide problem and thus characterization and validation of new drug targets is an intense field of research. Among possible drug targets, recently two essential zinc hydrolases, MshB (Rv1170) and MA-amidase (Rv3717), have been proposed and structurally characterized. Although possible mechanisms have been proposed by analogy to the widely studied human Zn hydrolases, several key issues, particularly those related to Zn coordination sphere and its role in catalysis, remained unanswered. Our results show that mycobacterial Zn hydrolases share a basic two-step mechanism. First, the attacking water becomes deprotonated by the conserved base and establishes the new C-O bond leading to a tetrahedral intermediate. The intermediate requires moderate reorganization to allow for proton transfer to the amide N and C-N bond breaking to occur in the second step. Zn ion plays a key role in stabilizing the tetrahedral intermediate and balancing the negative charge of the substrate during hydroxide ion attack. Finally, comparative analysis of other Zn hydrolases points to a convergent mechanistic evolution. PMID:26415840

  18. Development of a versatile organophosphorous-hydrolase-based assay for organophosphate pesticides

    NASA Astrophysics Data System (ADS)

    Rogers, Kim R.; Wang, Yi; Mulchandani, Ashok; Mulchandani, P.; Chen, Wilfred

    1999-02-01

    We report a rapid and versatile organophosphorus hydrolase (OPH)-based method for measurement of organophosphate pesticides. This assay is based on a substrate-dependant change in pH near the active site of the enzyme. The pH change is monitored using fluorescein isothiocyanate (FITC) which is covalently immobilized to the enzyme. This method employs FITC-labeled enzyme adsorbed to polymethylmethacrylate beads. Analytes were measured using a microbead fluorescence analyzer. The dynamic concentration range for the assay extends from 25 (mu) M to 400 (mu) M for paraoxon with a detection limit of 8 (mu) M. This assay compared favorably to an HPLC method for monitoring the concentration of coumaphos in bioremediation filtrate samples.

  19. Antibiotic growth promoters enhance animal production by targeting intestinal bile salt hydrolase and its producers.

    PubMed

    Lin, Jun

    2014-01-01

    The growth-promoting effect of antibiotic growth promoters (AGPs) was correlated with the decreased activity of bile salt hydrolase (BSH), an intestinal bacteria-produced enzyme that exerts negative impact on host fat digestion and utilization. Consistent with this finding, independent chicken studies have demonstrated that AGP usage significantly reduced population of Lactobacillus species, the major BSH-producers in the intestine. Recent finding also demonstrated that some AGPs, such as tetracycline and roxarsone, display direct inhibitory effect on BSH activity. Therefore, BSH is a promising microbiome target for developing novel alternatives to AGPs. Specifically, dietary supplementation of BSH inhibitor may promote host lipid metabolism and energy harvest, consequently enhancing feed efficiency and body weight gain in food animals. PMID:24575079

  20. Discovery of Leukotriene A4 Hydrolase Inhibitors Using Metabolomics Biased Fragment Crystallography†

    PubMed Central

    2009-01-01

    We describe a novel fragment library termed fragments of life (FOL) for structure-based drug discovery. The FOL library includes natural small molecules of life, derivatives thereof, and biaryl protein architecture mimetics. The choice of fragments facilitates the interrogation of protein active sites, allosteric binding sites, and protein−protein interaction surfaces for fragment binding. We screened the FOL library against leukotriene A4 hydrolase (LTA4H) by X-ray crystallography. A diverse set of fragments including derivatives of resveratrol, nicotinamide, and indole were identified as efficient ligands for LTA4H. These fragments were elaborated in a small number of synthetic cycles into potent inhibitors of LTA4H representing multiple novel chemotypes for modulating leukotriene biosynthesis. Analysis of the fragment-bound structures also showed that the fragments comprehensively recapitulated key chemical features and binding modes of several reported LTA4H inhibitors. PMID:19618939

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

    PubMed Central

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

    1978-01-01

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

  2. Determination of Organophosphate Pesticides at a Carbon Nanotube/Organophosphorus Hydrolase Electrochemical Biosensor

    SciTech Connect

    Deo, R P.; Wang, Joseph; Block, I; Mulchandani, Ashok; Joshi, K; Trojanowicz, M; Scholz, F; Chen, Wilfred; Lin, Yuehe

    2005-02-08

    An amperometric biosensor for organophosphorus (OP) pesticides based on a carbon-nanotube (CNT) modified transducer and an organophosphorus hydrolase (OPH) biocatalyst is described. A bilayer approach with the OPH layer atop of the CNT film was used for preparing the CNT/OPH biosensor. The CNT layer leads to a greatly improved anodic detection of the enzymatically-generated p-nitrophenol product, including higher sensitivity and stability. The sensor performance was optimized with respect to the surface modification and operating conditions. Under the optimal conditions the biosensor was used to measure as low as 0.15 {micro}M paraoxon and 0.8 {micro}M methyl parathion with sensitivities of 25 and 6 nA/{micro}M, respectively.

  3. Use of Nanostructure-Initiator Mass Spectrometry to Deduce Selectivity of Reaction in Glycoside Hydrolases

    PubMed Central

    Deng, Kai; Takasuka, Taichi E.; Bianchetti, Christopher M.; Bergeman, Lai F.; Adams, Paul D.; Northen, Trent R.; Fox, Brian G.

    2015-01-01

    Chemically synthesized nanostructure-initiator mass spectrometry (NIMS) probes derivatized with tetrasaccharides were used to study the reactivity of representative Clostridium thermocellum β-glucosidase, endoglucanases, and cellobiohydrolase. Diagnostic patterns for reactions of these different classes of enzymes were observed. Results show sequential removal of glucose by the β-glucosidase and a progressive increase in specificity of reaction from endoglucanases to cellobiohydrolase. Time-dependent reactions of these polysaccharide-selective enzymes were modeled by numerical integration, which provides a quantitative basis to make functional distinctions among a continuum of naturally evolved catalytic properties. Consequently, our method, which combines automated protein translation with high-sensitivity and time-dependent detection of multiple products, provides a new approach to annotate glycoside hydrolase phylogenetic trees with functional measurements. PMID:26579511

  4. Dual inhibition of cyclooxygenase-2 and soluble epoxide hydrolase synergistically suppresses primary tumor growth and metastasis

    PubMed Central

    Zhang, Guodong; Panigrahy, Dipak; Hwang, Sung Hee; Yang, Jun; Mahakian, Lisa M.; Wettersten, Hiromi I.; Liu, Jun-Yan; Wang, Yanru; Ingham, Elizabeth S.; Tam, Sarah; Kieran, Mark W.; Weiss, Robert H.; Ferrara, Katherine W.; Hammock, Bruce D.

    2014-01-01

    Prostaglandins derived from the cyclooxygenase (COX) pathway and epoxyeicosatrienoic acids (EETs) from the cytochrome P450/soluble epoxide hydrolase (sEH) pathway are important eicosanoids that regulate angiogenesis and tumorigenesis. COX-2 inhibitors, which block the formation of prostaglandins, suppress tumor growth, whereas sEH inhibitors, which increase endogenous EETs, stimulate primary tumor growth and metastasis. However, the functional interactions of these two pathways in cancer are unknown. Using pharmacological inhibitors as probes, we show here that dual inhibition of COX-2 and sEH synergistically inhibits primary tumor growth and metastasis by suppressing tumor angiogenesis. COX-2/sEH dual pharmacological inhibitors also potently suppress primary tumor growth and metastasis by inhibiting tumor angiogenesis via selective inhibition of endothelial cell proliferation. These results demonstrate a critical interaction of these two lipid metabolism pathways on tumorigenesis and suggest dual inhibition of COX-2 and sEH as a potential therapeutic strategy for cancer therapy. PMID:25024195

  5. A glycoside hydrolase family 31 dextranase with high transglucosylation activity from Flavobacterium johnsoniae.

    PubMed

    Gozu, Yoshifumi; Ishizaki, Yuichi; Hosoyama, Yuhei; Miyazaki, Takatsugu; Nishikawa, Atsushi; Tonozuka, Takashi

    2016-08-01

    Glycoside hydrolase family (GH) 31 enzymes exhibit various substrate specificities, although the majority of members are α-glucosidases. Here, we constructed a heterologous expression system of a GH31 enzyme, Fjoh_4430, from Flavobacterium johnsoniae NBRC 14942, using Escherichia coli, and characterized its enzymatic properties. The enzyme hydrolyzed dextran and pullulan to produce isomaltooligosaccharides and isopanose, respectively. When isomaltose was used as a substrate, the enzyme catalyzed disproportionation to form isomaltooligosaccharides. The enzyme also acted, albeit inefficiently, on p-nitrophenyl α-D-glucopyranoside, and p-nitrophenyl α-isomaltoside was the main product of the reaction. In contrast, Fjoh_4430 did not act on trehalose, kojibiose, nigerose, maltose, maltotriose, or soluble starch. The optimal pH and temperature were pH 6.0 and 60 °C, respectively. Our results indicate that Fjoh_4430 is a novel GH31 dextranase with high transglucosylation activity. PMID:27170214

  6. Cytochemical localization of some hydrolases in the pollen and pollen tubes of Amaryllis vittata Ait.

    PubMed

    Sharma, D

    1982-01-01

    Some hydrolases are localized cytochemically in the pollen and pollen tubes of Amaryllis vittata Ait. The function of different enzymes is discussed in relation to pollen tubes morphogenesis. Activity of most of the enzymes was confined to colpus region, pollen wall and general cytoplasm of pollen and pollen tube. The activity of hydrolytic enzymes like acid monophosphoesterase and lipase and was nil in the exine of both germinated and ungerminated pollen, whereas intense reaction for esterase was observed in exine. Enzyme activity increased after germination which suggest the hydrolysis of stored metabolites and synthesis of proteins and other metabolites for the active growth of pollen tube. Intense reaction for enzymes like alkaline phosphomonoesterase, ATP-ase, 5-nucleotidase etc. at the tip region of pollen tube suggest their role in physiological processes associated with exchange of materials through intercellular transport during tube wall polysaccharide biogenesis. PMID:6298081

  7. Structural and Mechanistic Insights into C-P Bond Hydrolysis by Phosphonoacetate Hydrolase

    SciTech Connect

    Agarwal, Vinayak; Borisova, Svetlana A.; Metcalf, William W.; van der Donk, Wilfred A.; Nair, Satish K.

    2011-12-22

    Bacteria have evolved pathways to metabolize phosphonates as a nutrient source for phosphorus. In Sinorhizobium meliloti 1021, 2-aminoethylphosphonate is catabolized to phosphonoacetate, which is converted to acetate and inorganic phosphate by phosphonoacetate hydrolase (PhnA). Here we present detailed biochemical and structural characterization of PhnA that provides insights into the mechanism of C-P bond cleavage. The 1.35 {angstrom} resolution crystal structure reveals a catalytic core similar to those of alkaline phosphatases and nucleotide pyrophosphatases but with notable differences, such as a longer metal-metal distance. Detailed structure-guided analysis of active site residues and four additional cocrystal structures with phosphonoacetate substrate, acetate, phosphonoformate inhibitor, and a covalently bound transition state mimic provide insight into active site features that may facilitate cleavage of the C-P bond. These studies expand upon the array of reactions that can be catalyzed by enzymes of the alkaline phosphatase superfamily.

  8. Searching for monooxygenases and hydrolases in bacteria from an extreme environment.

    PubMed

    da Cruz, Georgiana F; Angolini, Célio F F; de Oliveira, Luciana G; Lopes, Patrícia F; de Vasconcellos, Suzan P; Crespim, Elaine; de Oliveira, Valéria M; dos Santos Neto, Eugênio V; Marsaioli, Anita J

    2010-06-01

    Microbial oxidation potentials of extremophiles recovered from Pampo Sul oil field, Campos Basin, Brazil, in pure culture or in consortia, were investigated using high-throughput screening (HTS) and multibioreactions. Camphor (1), cis-jasmone (2), 2-methyl-cyclohexanone (3), 1,2-epoxyoctane (4), phenylethyl acetate (5), phenylethyl propionate (6), and phenylethyl octanoate (7) were used to perform multibioreaction assays. Eighty-two bacterial isolates were recovered from oil and formation water samples and those presenting outstanding activities in HTS assays were identified by sequencing their 16S rRNA genes. These results revealed that most microorganisms belonged to the genus Bacillus and presented alcohol dehydrogenase, monooxygenase, epoxide hydrolase, esterase, and lipase activities. PMID:20204614

  9. Effect of Bleomycin Hydrolase Gene Polymorphism on Late Pulmonary Complications of Treatment for Hodgkin Lymphoma

    PubMed Central

    Miltényi, Zsófia; Póliska, Szilárd; Bálint, Bálint László; Illés, Árpád

    2016-01-01

    Background Bleomycin hydrolase (BLMH), an enzyme that inactivates bleomycin, may be a potential candidate that could influence pulmonary function in ABVD (doxorubicin, bleomycin, vinblastin, dacarbasine)–treated Hodgkin lymphoma (HL) patients. Patients and Methods We hypothesized that the BLMH gene SNP A1450G (rs1050565) influences BLMH activity and late pulmonary toxicity. St. George Respiratory Questionnaire, lung scintigraphy and spirometry were used to determine lung function. TaqMan genotyping assay was used to determine genotype distribution of 131 previously treated HL patients. Results Significantly more favorable results were seen in the wild-type A/A genotype group than those in the group containing the mutated allele: A/G+G/G in retrospective pulmonary tests of ABVD treated patients. Conclusion Besides limitations of the current study, bleomycin pharmacokinetics should be further evaluated in patients with BLMH variations, hence identify those cases even in the frontline setting, where bleomycin should be omitted and replaced with targeted therapy. PMID:27327270

  10. A formin-nucleated actin aster concentrates cell wall hydrolases for cell fusion in fission yeast

    PubMed Central

    Dudin, Omaya; Bendezú, Felipe O.; Groux, Raphael; Laroche, Thierry; Seitz, Arne

    2015-01-01

    Cell–cell fusion is essential for fertilization. For fusion of walled cells, the cell wall must be degraded at a precise location but maintained in surrounding regions to protect against lysis. In fission yeast cells, the formin Fus1, which nucleates linear actin filaments, is essential for this process. In this paper, we show that this formin organizes a specific actin structure—the actin fusion focus. Structured illumination microscopy and live-cell imaging of Fus1, actin, and type V myosins revealed an aster of actin filaments whose barbed ends are focalized near the plasma membrane. Focalization requires Fus1 and type V myosins and happens asynchronously always in the M cell first. Type V myosins are essential for fusion and concentrate cell wall hydrolases, but not cell wall synthases, at the fusion focus. Thus, the fusion focus focalizes cell wall dissolution within a broader cell wall synthesis zone to shift from cell growth to cell fusion. PMID:25825517

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

    PubMed

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

    2016-05-01

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

  12. Peroxisomal translocation of soluble epoxide hydrolase protects against ischemic stroke injury

    PubMed Central

    Nelson, Jonathan W; Zhang, Wenri; Alkayed, Nabil J; Koerner, Ines P

    2015-01-01

    Soluble epoxide hydrolase (sEH) contributes to cardiovascular disease, including stroke, although the exact mechanism remains unclear. While primarily a cytosolic enzyme, sEH can translocate into peroxisomes. The relevance of this for stroke injury is not understood. We tested the hypothesis that sEH-mediated injury is tied to the cytoplasmic localization. We found that a human sEH variant possessing increased affinity to peroxisomes reduced stroke injury in sEH-null mice, whereas infarcts were significantly larger when peroxisomal translocation of sEH was disrupted. We conclude that sEH contributes to stroke injury only when localized in the cytoplasm, while peroxisomal sEH may be protective. PMID:26126869

  13. [Molecular engineering of cellulase catalytic domain based on glycoside hydrolase family].

    PubMed

    Zhang, Xiaomei; Li, Dandan; Wang, Lushan; Zhao, Yue; Chen, Guanjun

    2013-04-01

    Molecular engineering of cellulases can improve enzymatic activity and efficiency. Recently, the Carbohydrate-Active enZYmes Database (CAZy), including glycoside hydrolase (GH) families, has been established with the development of Omics and structural measurement technologies. Molecular engineering based on GH families can obviously decrease the probing space of target sequences and structures, and increase the odds of experimental success. Besides, the study of cellulase active-site architecture paves the way toward the explanation of catalytic mechanism. This review focuses on the main GH families and the latest progresses in molecular engineering of catalytic domain. Based on the combination of analysis of a large amount of data in the same GH family and their conservative active-site architecture information, rational design will be an important direction for molecular engineering and promote the rapid development of the conversion of biomass. PMID:23894816

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

    PubMed Central

    Viens, Pascal; Lacombe-Harvey, Marie-Ève; Brzezinski, Ryszard

    2015-01-01

    Chitosanases, enzymes that catalyze the endo-hydrolysis of glycolytic links in chitosan, are the subject of numerous studies as biotechnological tools to generate low molecular weight chitosan (LMWC) or chitosan oligosaccharides (CHOS) from native, high molecular weight chitosan. Glycoside hydrolases belonging to family GH46 are among the best-studied chitosanases, with four crystallography-derived structures available and more than forty enzymes studied at the biochemical level. They were also subjected to numerous site-directed mutagenesis studies, unraveling the molecular mechanisms of hydrolysis. This review is focused on the taxonomic distribution of GH46 proteins, their multi-modular character, the structure-function relationships and their biological functions in the host organisms. PMID:26516868

  15. Design and Synthesis of Activity-Based Probes and Inhibitors for Bleomycin Hydrolase.

    PubMed

    van der Linden, Wouter A; Segal, Ehud; Child, Matthew A; Byzia, Anna; Drąg, Marcin; Bogyo, Matthew

    2015-08-20

    Bleomycin hydrolase (BLMH) is a neutral cysteine aminopeptidase that has been ascribed roles in many physiological and pathological processes, yet its primary biological function remains enigmatic. In this work, we describe the results of screening of a library of fluorogenic substrates to identify non-natural amino acids that are optimally recognized by BLMH. This screen identified several substrates with kcat/KM values that are substantially improved over the previously reported fluorogenic substrates for this enzyme. The substrate sequences were used to design activity-based probes that showed potent labeling of recombinant BLMH as well as endogenously expressed BLMH in cell extracts, and in intact cells. Importantly, we identify potent BLMH inhibitors that are able to fully inhibit endogenous BLMH activity in intact cells. These probes and inhibitors will be valuable new reagents to study BLMH function in cellular and animal models of human diseases where BLMH is likely to be involved. PMID:26256478

  16. Chemical constituents from the root of Polygonum multiflorum and their soluble epoxide hydrolase inhibitory activity.

    PubMed

    Sun, Ya Nan; Li, Wei; Kim, Jang Hoon; Yan, Xi Tao; Kim, Ji Eun; Yang, Seo Young; Kim, Young Ho

    2015-06-01

    Fourteen compounds were isolated from a methanol extract of Polygonum multiflorum roots, and their structures were elucidated by comparing spectroscopic data to published spectra. The inhibitory effects of the isolated compounds on soluble epoxide hydrolase (sEH) were then evaluated. Compounds 1-7 inhibited sEH activity potently, with IC50 values ranging from 6.2 ± 0.5 to 48.6 ± 3.1 μM. Moreover, a kinetic analysis of compounds 1-7 revealed that the inhibitory actions of compounds 1, 3 and 4 were non-competitive, whereas those of compounds 2 and 5-7 were mixed-type. PMID:25413971

  17. Inhibiting an Epoxide Hydrolase Virulence Factor from Pseudomonas aeruginosa Protects CFTR.

    PubMed

    Bahl, Christopher D; Hvorecny, Kelli L; Bomberger, Jennifer M; Stanton, Bruce A; Hammock, Bruce D; Morisseau, Christophe; Madden, Dean R

    2015-08-17

    Opportunistic pathogens exploit diverse strategies to sabotage host defenses. Pseudomonas aeruginosa secretes the CFTR inhibitory factor Cif and thus triggers loss of CFTR, an ion channel required for airway mucociliary defense. However, the mechanism of action of Cif has remained unclear. It catalyzes epoxide hydrolysis, but there is no known role for natural epoxides in CFTR regulation. It was demonstrated that the hydrolase activity of Cif is strictly required for its effects on CFTR. A small-molecule inhibitor that protects this key component of the mucociliary defense system was also uncovered. These results provide a basis for targeting the distinctive virulence chemistry of Cif and suggest an unanticipated role of physiological epoxides in intracellular protein trafficking. PMID:26136396

  18. Use of Nanostructure-Initiator Mass Spectrometry to Deduce Selectivity of Reaction in Glycoside Hydrolases.

    PubMed

    Deng, Kai; Takasuka, Taichi E; Bianchetti, Christopher M; Bergeman, Lai F; Adams, Paul D; Northen, Trent R; Fox, Brian G

    2015-01-01

    Chemically synthesized nanostructure-initiator mass spectrometry (NIMS) probes derivatized with tetrasaccharides were used to study the reactivity of representative Clostridium thermocellum β-glucosidase, endoglucanases, and cellobiohydrolase. Diagnostic patterns for reactions of these different classes of enzymes were observed. Results show sequential removal of glucose by the β-glucosidase and a progressive increase in specificity of reaction from endoglucanases to cellobiohydrolase. Time-dependent reactions of these polysaccharide-selective enzymes were modeled by numerical integration, which provides a quantitative basis to make functional distinctions among a continuum of naturally evolved catalytic properties. Consequently, our method, which combines automated protein translation with high-sensitivity and time-dependent detection of multiple products, provides a new approach to annotate glycoside hydrolase phylogenetic trees with functional measurements. PMID:26579511

  19. A classification of glycosyl hydrolases based on amino acid sequence similarities.

    PubMed Central

    Henrissat, B

    1991-01-01

    The amino acid sequences of 301 glycosyl hydrolases and related enzymes have been compared. A total of 291 sequences corresponding to 39 EC entries could be classified into 35 families. Only ten sequences (less than 5% of the sample) could not be assigned to any family. With the sequences available for this analysis, 18 families were found to be monospecific (containing only one EC number) and 17 were found to be polyspecific (containing at least two EC numbers). Implications on the folding characteristics and mechanism of action of these enzymes and on the evolution of carbohydrate metabolism are discussed. With the steady increase in sequence and structural data, it is suggested that the enzyme classification system should perhaps be revised. PMID:1747104

  20. Towards a molecular-level theory of carbohydrate processivity in glycoside hydrolases.

    PubMed

    Beckham, Gregg T; Ståhlberg, Jerry; Knott, Brandon C; Himmel, Michael E; Crowley, Michael F; Sandgren, Mats; Sørlie, Morten; Payne, Christina M

    2014-06-01

    Polysaccharide depolymerization in nature is primarily accomplished by processive glycoside hydrolases (GHs), which abstract single carbohydrate chains from polymer crystals and cleave glycosidic linkages without dissociating after each catalytic event. Understanding the molecular-level features and structural aspects of processivity is of importance due to the prevalence of processive GHs in biomass-degrading enzyme cocktails. Here, we describe recent advances towards the development of a molecular-level theory of processivity for cellulolytic and chitinolytic enzymes, including the development of novel methods for measuring rates of key steps in processive action and insights gained from structural and computational studies. Overall, we present a framework for developing structure-function relationships in processive GHs and outline additional progress towards developing a fundamental understanding of these industrially important enzymes. PMID:24863902

  1. Antibiotic growth promoters enhance animal production by targeting intestinal bile salt hydrolase and its producers

    PubMed Central

    Lin, Jun

    2014-01-01

    The growth-promoting effect of antibiotic growth promoters (AGPs) was correlated with the decreased activity of bile salt hydrolase (BSH), an intestinal bacteria-produced enzyme that exerts negative impact on host fat digestion and utilization. Consistent with this finding, independent chicken studies have demonstrated that AGP usage significantly reduced population of Lactobacillus species, the major BSH-producers in the intestine. Recent finding also demonstrated that some AGPs, such as tetracycline and roxarsone, display direct inhibitory effect on BSH activity. Therefore, BSH is a promising microbiome target for developing novel alternatives to AGPs. Specifically, dietary supplementation of BSH inhibitor may promote host lipid metabolism and energy harvest, consequently enhancing feed efficiency and body weight gain in food animals. PMID:24575079

  2. Discovery of Leukotriene A4 Hydrolase Inhibitors Using Metabolomics Biased Fragment Crystallography

    SciTech Connect

    Davies, D.; Mamat, B; Magnusson, O; Christensen, J; Haraldsson, M; Mishra, R; Pease, B; Hansen, E; Singh, J; et. al.

    2009-01-01

    We describe a novel fragment library termed fragments of life (FOL) for structure-based drug discovery. The FOL library includes natural small molecules of life, derivatives thereof, and biaryl protein architecture mimetics. The choice of fragments facilitates the interrogation of protein active sites, allosteric binding sites, and protein-protein interaction surfaces for fragment binding. We screened the FOL library against leukotriene A4 hydrolase (LTA4H) by X-ray crystallography. A diverse set of fragments including derivatives of resveratrol, nicotinamide, and indole were identified as efficient ligands for LTA4H. These fragments were elaborated in a small number of synthetic cycles into potent inhibitors of LTA4H representing multiple novel chemotypes for modulating leukotriene biosynthesis. Analysis of the fragment-bound structures also showed that the fragments comprehensively recapitulated key chemical features and binding modes of several reported LTA4H inhibitors.

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

    PubMed

    Nekiunaite, Laura; Isaksen, Trine; Vaaje-Kolstad, Gustav; Abou Hachem, Maher

    2016-08-01

    Starch-binding modules of family 20 (CBM20) are present in 60% of lytic polysaccharide monooxygenases (LPMOs) catalyzing the oxidative breakdown of starch, which highlights functional importance in LPMO activity. The substrate-binding properties of starch-active LMPOs, however, are currently unexplored. Affinities and binding-thermodynamics of two recombinant fungal LPMOs toward starch and β-cyclodextrin were shown to be similar to fungal CBM20s. Amplex Red assays showed ascorbate and Cu-dependent activity, which was inhibited in the presence of β-cylodextrin and amylose. Phylogenetically, the clustering of CBM20s from starch-targeting LPMOs and hydrolases was in accord with taxonomy and did not correlate to appended catalytic activity. Altogether, these results demonstrate that the CBM20-binding scaffold is retained in the evolution of hydrolytic and oxidative starch-degrading activities. PMID:27397613

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

    2011-01-01

    The endocannabinoid anandamide (ANA) participates in the control of cell death inducing the formation of apoptotic bodies and DNA fragmentation. The aim of this study was to evaluate whether the ANA degrading enzyme, the fatty acid amide hydrolase (FAAH), would induce cellular death. Experiments were performed in cerebellar granule neurons cultured with the FAAH inhibitor, URB597 (25, 50 or 100 nM) as well as endogenous lipids such as oleoylethanolamide (OEA) or palmitoylethanolamide (PEA) and cellular viability was determined by MTT test. Neurons cultured with URB597 (25, 50 or 100 nM) displayed a decrease in cellular viability. In addition, if cultured with OEA (25 nM) or PEA (100 nM), cellular death was found. These results further suggest that URB597, OEA or PEA promote cellular death. PMID:21854612

  6. Extensive hydrolysis of phosphonates as unexpected behaviour of the known His6-organophosphorus hydrolase.

    PubMed

    Lyagin, Ilya V; Andrianova, Mariia S; Efremenko, Elena N

    2016-07-01

    The catalytic activity of hexahistidine-tagged organophosphorus hydrolase (His6-OPH) in hydrolytic reactions of methylphosphonic acid (MPA) and its monoesters and diesters being decomposition products of R-VX was demonstrated for the first time. The catalytic constants of enzyme in such reactions were determined. The mechanism of C-P bond cleavage in the MPA by His6-OPH was proposed. Such reaction was estimated to be carried out with the soluble and nanocapsulated forms of His6-OPH. His6-OPH was demonstrated to be capable of degrading the key organophosphorus components of reaction masses (RMs) that are produced by the chemical detoxification of R-VX and RMs are multi-substrate mixtures for this enzyme. The kinetic model describing the behaviour of His6-OPH in RMs was proposed and was shown to adequately fit experimental points during degradation of the real samples of RMs. PMID:26932546

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

    PubMed

    Viens, Pascal; Lacombe-Harvey, Marie-Ève; Brzezinski, Ryszard

    2015-11-01

    Chitosanases, enzymes that catalyze the endo-hydrolysis of glycolytic links in chitosan, are the subject of numerous studies as biotechnological tools to generate low molecular weight chitosan (LMWC) or chitosan oligosaccharides (CHOS) from native, high molecular weight chitosan. Glycoside hydrolases belonging to family GH46 are among the best-studied chitosanases, with four crystallography-derived structures available and more than forty enzymes studied at the biochemical level. They were also subjected to numerous site-directed mutagenesis studies, unraveling the molecular mechanisms of hydrolysis. This review is focused on the taxonomic distribution of GH46 proteins, their multi-modular character, the structure-function relationships and their biological functions in the host organisms. PMID:26516868

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

    SciTech Connect

    French, Jarrod B.; Ealick, Steven E.

    2011-07-19

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

  9. Structure of Acidothermus cellulolyticus family 74 glycoside hydrolase at 1.82 Å resolution

    PubMed Central

    Alahuhta, Markus; Adney, William S.; Himmel, Michael E.; Lunin, Vladimir V.

    2013-01-01

    Here, a 1.82 Å resolution X-ray structure of a glycoside hydrolase family 74 (GH74) enzyme from Acidothermus cellulolyticus is reported. The resulting structure was refined to an R factor of 0.150 and an R free of 0.196. Structural analysis shows that five related structures have been reported with a secondary-structure similarity of between 75 and 89%. The five similar structures were all either Clostridium thermocellum or Geotrichum sp. M128 GH74 xyloglucanases. Structural analysis indicates that the A. cellulolyticus GH74 enzyme is an endoxyloglucanase, as it lacks a characteristic loop that blocks one end of the active site in exoxyloglucanases. Superimposition with the C. thermocellum GH74 shows that Asp451 and Asp38 are the catalytic residues. PMID:24316824

  10. First Structural Insights into α-l-Arabinofuranosidases from the Two GH62 Glycoside Hydrolase Subfamilies*

    PubMed Central

    Siguier, Béatrice; Haon, Mireille; Nahoum, Virginie; Marcellin, Marlène; Burlet-Schiltz, Odile; Coutinho, Pedro M.; Henrissat, Bernard; Mourey, Lionel; O'Donohue, Michael J.; Berrin, Jean-Guy; Tranier, Samuel; Dumon, Claire

    2014-01-01

    α-l-Arabinofuranosidases are glycoside hydrolases that specifically hydrolyze non-reducing residues from arabinose-containing polysaccharides. In the case of arabinoxylans, which are the main components of hemicellulose, they are part of microbial xylanolytic systems and are necessary for complete breakdown of arabinoxylans. Glycoside hydrolase family 62 (GH62) is currently a small family of α-l-arabinofuranosidases that contains only bacterial and fungal members. Little is known about the GH62 mechanism of action, because only a few members have been biochemically characterized and no three-dimensional structure is available. Here, we present the first crystal structures of two fungal GH62 α-l-arabinofuranosidases from the basidiomycete Ustilago maydis (UmAbf62A) and ascomycete Podospora anserina (PaAbf62A). Both enzymes are able to efficiently remove the α-l-arabinosyl substituents from arabinoxylan. The overall three-dimensional structure of UmAbf62A and PaAbf62A reveals a five-bladed β-propeller fold that confirms their predicted classification into clan GH-F together with GH43 α-l-arabinofuranosidases. Crystallographic structures of the complexes with arabinose and cellotriose reveal the important role of subsites +1 and +2 for sugar binding. Intriguingly, we observed that PaAbf62A was inhibited by cello-oligosaccharides and displayed binding affinity to cellulose although no activity was observed on a range of cellulosic substrates. Bioinformatic analyses showed that UmAbf62A and PaAbf62A belong to two distinct subfamilies within the GH62 family. The results presented here provide a framework to better investigate the structure-function relationships within the GH62 family. PMID:24394409

  11. Cloning and characterization of an epoxide hydrolase from Cupriavidus metallidurans-CH34.

    PubMed

    Kumar, Ranjai; Wani, Shadil Ibrahim; Chauhan, Nar Singh; Sharma, Rakesh; Sareen, Dipti

    2011-09-01

    A putative epoxide hydrolase-encoding gene was identified from the genome sequence of Cupriavidus metallidurans CH34. The gene was cloned and overexpressed in Escherichia coli with His(6)-tag at its N-terminus. The epoxide hydrolase (CMEH) was purified to near homogeneity and was found to be a homodimer, with subunit molecular weight of 36 kDa. The CMEH had broad substrate specificity as it could hydrolyze 13 epoxides, out of 15 substrates tested. CMEH had high specific activity with 1,2-epoxyoctane, 1,2-epoxyhexane, styrene oxide (SO) and was also found to be active with meso-epoxides. The enzyme had optimum pH and temperature of 7.5 and 37°C respectively, with racemic SO. Biotransformation of 80 mM SO with recombinant whole E. coli cells expressing CMEH led to 56% ee(P) of (R)-diol with 77.23% conversion in 30 min. The enzyme could hydrolyze (R)-SO, ∼2-fold faster than (S)-SO, though it accepted both (R)- and (S)-SO with similar affinity as K(m)(R) and K(m)(S) of CMEH were 2.05±0.42 and 2.11±0.16 mM, respectively. However, the k(cat)(R) and k(cat)(S) for the two enantiomers of SO were 4.80 and 3.34 s(-1), respectively. The wide substrate spectrum exhibited by CMEH combined with the fast conversion rate makes it a robust biocatalyst for industrial use. Regioselectivity studies with enantiopure (R)- and (S)-SO revealed that with slightly altered regioselectivity, CMEH has a high potential to synthesize an enantiopure (R)-PED, through an enantioconvergent hydrolytic process. PMID:21515382

  12. The Molecular Structure of Epoxide Hydrolase B From And Its Complex With Urea-Based Inhibitor

    SciTech Connect

    Biswal, B.K.; Morisseau, C.; Garen, G.; Cherney, M.M.; Garen, C.; Niu, C.; Hammock, B.D.; James, M.N.G.

    2009-05-11

    Mycobacterium tuberculosis (Mtb), the intracellular pathogen that infects macrophages primarily, is the causative agent of the infectious disease tuberculosis in humans. The Mtb genome encodes at least six epoxide hydrolases (EHs A to F). EHs convert epoxides to trans-dihydrodiols and have roles in drug metabolism as well as in the processing of signaling molecules. Herein, we report the crystal structures of unbound Mtb EHB and Mtb EHB bound to a potent, low-nanomolar (IC(50) approximately 19 nM) urea-based inhibitor at 2.1 and 2.4 A resolution, respectively. The enzyme is a homodimer; each monomer adopts the classical alpha/beta hydrolase fold that composes the catalytic domain; there is a cap domain that regulates access to the active site. The catalytic triad, comprising Asp104, His333 and Asp302, protrudes from the catalytic domain into the substrate binding cavity between the two domains. The urea portion of the inhibitor is bound in the catalytic cavity, mimicking, in part, the substrate binding; the two urea nitrogen atoms donate hydrogen bonds to the nucleophilic carboxylate of Asp104, and the carbonyl oxygen of the urea moiety receives hydrogen bonds from the phenolic oxygen atoms of Tyr164 and Tyr272. The phenolic oxygen groups of these two residues provide electrophilic assistance during the epoxide hydrolytic cleavage. Upon inhibitor binding, the binding-site residues undergo subtle structural rearrangement. In particular, the side chain of Ile137 exhibits a rotation of around 120 degrees about its C(alpha)-C(beta) bond in order to accommodate the inhibitor. These findings have not only shed light on the enzyme mechanism but also have opened a path for the development of potent inhibitors with good pharmacokinetic profiles against all Mtb EHs of the alpha/beta type.

  13. Novel β-1,4-Mannanase Belonging to a New Glycoside Hydrolase Family in Aspergillus nidulans*

    PubMed Central

    Shimizu, Motoyuki; Kaneko, Yuhei; Ishihara, Saaya; Mochizuki, Mai; Sakai, Kiyota; Yamada, Miyuki; Murata, Shunsuke; Itoh, Eriko; Yamamoto, Tatsuya; Sugimura, Yu; Hirano, Tatsuya; Takaya, Naoki; Kobayashi, Tetsuo; Kato, Masashi

    2015-01-01

    Many filamentous fungi produce β-mannan-degrading β-1,4-mannanases that belong to the glycoside hydrolase 5 (GH5) and GH26 families. Here we identified a novel β-1,4-mannanase (Man134A) that belongs to a new glycoside hydrolase (GH) family (GH134) in Aspergillus nidulans. Blast analysis of the amino acid sequence using the NCBI protein database revealed that this enzyme had no similarity to any sequences and no putative conserved domains. Protein homologs of the enzyme were distributed to limited fungal and bacterial species. Man134A released mannobiose (M2), mannotriose (M3), and mannotetraose (M4) but not mannopentaose (M5) or higher manno-oligosaccharides when galactose-free β-mannan was the substrate from the initial stage of the reaction, suggesting that Man134A preferentially reacts with β-mannan via a unique catalytic mode. Man134A had high catalytic efficiency (kcat/Km) toward mannohexaose (M6) compared with the endo-β-1,4-mannanase Man5C and notably converted M6 to M2, M3, and M4, with M3 being the predominant reaction product. The action of Man5C toward β-mannans was synergistic. The growth phenotype of a Man134A disruptant was poor when β-mannans were the sole carbon source, indicating that Man134A is involved in β-mannan degradation in vivo. These findings indicate a hitherto undiscovered mechanism of β-mannan degradation that is enhanced by the novel β-1,4-mannanase, Man134A, when combined with other mannanolytic enzymes including various endo-β-1,4-mannanases. PMID:26385921

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

    PubMed

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

    2016-08-01

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

  15. Dysregulation of Soluble Epoxide Hydrolase and Lipidomic Profiles in Anorexia Nervosa

    PubMed Central

    Shih, Pei-an Betty; Yang, Jun; Morisseau, Christophe; German, J. Bruce; Van Zeeland, Ashley; Armando, Aaron M.; Quehenberger, Oswald; Bergen, Andrew W.; Magistretti, Pierre; Berrettini, Wade; Halmi, Katherine Ann; Schork, Nicholas; Hammock, Bruce D.; Kaye, Walter

    2015-01-01

    Individuals with anorexia nervosa (AN) restrict eating and become emaciated. AN tend to have an aversion to foods rich in fat. Because Epoxide Hydrolase 2 (EPHX2) was identified as a novel AN susceptibility gene, and because its protein product, soluble epoxide hydrolase (sEH), converts bioactive epoxides of polyunsaturated fatty acid (PUFA) to the corresponding diols, lipidomic and metabolomic targets of EPHX2 were assessed to evaluate the biological functions of EPHX2 and their role in AN. Epoxide substrates of sEH and associated oxylipins were measured in ill AN, recovered AN, and gender- and race-matched controls. PUFA and oxylipin markers were tested as potential biomarkers for AN. Oxylipin ratios were calculated as proxy markers of in vivo sEH activity. Several free- and total PUFAs were associated with AN diagnosis and with AN recovery. AN displayed elevated n-3 PUFAs and may differ from controls in PUFA elongation and desaturation processes. Cytochrome P450 pathway oxylipins from arachidonic acid, linoleic acid, alpha-linolenic acid, and docosahexaenoic acid PUFAs are associated with AN diagnosis. The diol:epoxide ratios suggest the sEH activity is higher in AN compared to controls. Multivariate analysis illustrates normalization of lipidomic profiles in recovered ANs. EPHX2 influences AN risk through in vivo interaction with dietary PUFAs. PUFA composition and concentrations as well as sEH activity may contribute to the pathogenesis and prognosis of AN. Our data support the involvement of EPHX2-associated lipidomic and oxylipin dysregulations in AN, and reveal their potential as biomarkers to assess responsiveness to future intervention or treatment. PMID:25824304

  16. Determinants of Reactivity and Selectivity in Soluble Epoxide Hydrolase from Quantum Mechanics/Molecular Mechanics Modeling

    PubMed Central

    2012-01-01

    Soluble epoxide hydrolase (sEH) is an enzyme involved in drug metabolism that catalyzes the hydrolysis of epoxides to form their corresponding diols. sEH has a broad substrate range and shows high regio- and enantioselectivity for nucleophilic ring opening by Asp333. Epoxide hydrolases therefore have potential synthetic applications. We have used combined quantum mechanics/molecular mechanics (QM/MM) umbrella sampling molecular dynamics (MD) simulations (at the AM1/CHARMM22 level) and high-level ab initio (SCS-MP2) QM/MM calculations to analyze the reactions, and determinants of selectivity, for two substrates: trans-stilbene oxide (t-SO) and trans-diphenylpropene oxide (t-DPPO). The calculated free energy barriers from the QM/MM (AM1/CHARMM22) umbrella sampling MD simulations show a lower barrier for phenyl attack in t-DPPO, compared with that for benzylic attack, in agreement with experiment. Activation barriers in agreement with experimental rate constants are obtained only with the highest level of QM theory (SCS-MP2) used. Our results show that the selectivity of the ring-opening reaction is influenced by several factors, including proximity to the nucleophile, electronic stabilization of the transition state, and hydrogen bonding to two active site tyrosine residues. The protonation state of His523 during nucleophilic attack has also been investigated, and our results show that the protonated form is most consistent with experimental findings. The work presented here illustrates how determinants of selectivity can be identified from QM/MM simulations. These insights may also provide useful information for the design of novel catalysts for use in the synthesis of enantiopure compounds. PMID:22280021

  17. Role of leukotriene A4 hydrolase aminopeptidase in the pathogenesis of emphysema1

    PubMed Central

    Paige, Mikell; Wang, Kan; Burdick, Marie; Park, Sunhye; Cha, Josiah; Jeffrey, Erin; Sherman, Nicholas; Shim, Y. Michael

    2014-01-01

    The leukotriene A4 hydrolase (LTA4H) is a bi-functional enzyme with an epoxy hydrolase and aminopeptidase activities. We hypothesize that the LTA4H aminopeptidase activity alleviates neutrophilic inflammation, which contributes to cigarette smoke (CS)-induced emphysema by clearing Proline-Glycine-Proline (PGP), a tri-amino acid chemokine known to induce chemotaxis of neutrophils. To investigate the biological contributions made by the LTA4H aminopeptidase activity in CS-induced emphysema, we exposed wild type mice to CS over five months while treating them with a vehicle or a pharmaceutical agent (4MDM) that selectively augments the LTA4H aminopeptidase without affecting the bio-production of leukotriene B4 (LTB4). Emphysematous phenotypes were assessed by pre mortem lung physiology with a small animal ventilator and by postmortem histologic morphometry. CS exposure acidified the airspaces and induced localization of the LTA4H protein into the nuclei of the epithelial cells. This resulted in accumulation of PGP in the airspaces by suppressing the LTA4H aminopeptidase activity. When the LTA4H aminopeptidase activity was selectively augmented by 4MDM, the levels of PGP in the BALF and infiltration of neutrophils into the lungs were significant reduced without affecting the levels of LTB4. This protected murine lungs from CS-induced emphysematous alveolar remodeling. In conclusion, CS exposure promotes the development of CS-induced emphysema by suppressing the enzymatic activities of the LTA4H aminopeptidase in lung tissues and accumulating PGP and neutrophils in the airspaces. However, restoring the LTA4 aminopeptidase activity with a pharmaceutical agent protected murine lungs from developing CS-induced emphysema. PMID:24771855

  18. Inhibition of endocannabinoid-degrading enzyme fatty acid amide hydrolase increases atherosclerotic plaque vulnerability in mice.

    PubMed

    Hoyer, Friedrich Felix; Khoury, Mona; Slomka, Heike; Kebschull, Moritz; Lerner, Raissa; Lutz, Beat; Schott, Hans; Lütjohann, Dieter; Wojtalla, Alexandra; Becker, Astrid; Zimmer, Andreas; Nickenig, Georg

    2014-01-01

    The role of endocannabinoids such as anandamide during atherogenesis remains largely unknown. Fatty acid amide hydrolase (FAAH) represents the key enzyme in anandamide degradation, and its inhibition is associated with subsequent higher levels of anandamide. Here, we tested whether selective inhibition of FAAH influences the progression of atherosclerosis in mice. Selective inhibition of FAAH using URB597 resulted in significantly increased plasma levels of anandamide compared to control, as assessed by mass spectrometry experiments in mice. Apolipoprotein E-deficient (ApoE(-/-)) mice were fed a high-fat, cholesterol-rich diet to induce atherosclerotic conditions. Simultaneously, mice received either the pharmacological FAAH inhibitor URB597 1mg/kg body weight (n=28) or vehicle (n=25) via intraperitoneal injection three times a week. After eight weeks, mice were sacrificed, and experiments were performed. Vascular superoxide generation did not differ between both groups, as measured by L012 assay. To determine whether selective inhibition of FAAH affects atherosclerotic plaque inflammation, immunohistochemical staining of the aortic root was performed. Atherosclerotic plaque formation, vascular macrophage accumulation, as well as vascular T cell infiltration did not differ between both groups. Interestingly, neutrophil cell accumulation was significantly increased in mice receiving URB597 compared to control. Vascular collagen structures in atherosclerotic plaques were significantly diminished in mice treated with URB597 compared to control, as assessed by picro-sirius-red staining. This was accompanied by an increased aortic expression of matrix metalloproteinase-9, as determined by quantitative RT-PCR and western blot analysis. Inhibition of fatty acid amide hydrolase does not influence plaque size but increases plaque vulnerability in mice. PMID:24286707

  19. Characterization of a Serine Hydrolase Targeted by Acyl-protein Thioesterase Inhibitors in Toxoplasma gondii

    PubMed Central

    Kemp, Louise E.; Rusch, Marion; Adibekian, Alexander; Bullen, Hayley E.; Graindorge, Arnault; Freymond, Céline; Rottmann, Matthias; Braun-Breton, Catherine; Baumeister, Stefan; Porfetye, Arthur T.; Vetter, Ingrid R.; Hedberg, Christian; Soldati-Favre, Dominique

    2013-01-01

    In eukaryotic organisms, cysteine palmitoylation is an important reversible modification that impacts protein targeting, folding, stability, and interactions with partners. Evidence suggests that protein palmitoylation contributes to key biological processes in Apicomplexa with the recent palmitome of the malaria parasite Plasmodium falciparum reporting over 400 substrates that are modified with palmitate by a broad range of protein S-acyl transferases. Dynamic palmitoylation cycles require the action of an acyl-protein thioesterase (APT) that cleaves palmitate from substrates and conveys reversibility to this posttranslational modification. In this work, we identified candidates for APT activity in Toxoplasma gondii. Treatment of parasites with low micromolar concentrations of β-lactone- or triazole urea-based inhibitors that target human APT1 showed varied detrimental effects at multiple steps of the parasite lytic cycle. The use of an activity-based probe in combination with these inhibitors revealed the existence of several serine hydrolases that are targeted by APT1 inhibitors. The active serine hydrolase, TgASH1, identified as the homologue closest to human APT1 and APT2, was characterized further. Biochemical analysis of TgASH1 indicated that this enzyme cleaves substrates with a specificity similar to APTs, and homology modeling points toward an APT-like enzyme. TgASH1 is dispensable for parasite survival, which indicates that the severe effects observed with the β-lactone inhibitors are caused by the inhibition of non-TgASH1 targets. Other ASH candidates for APT activity were functionally characterized, and one of them was found to be resistant to gene disruption due to the potential essential nature of the protein. PMID:23913689

  20. Phylogenetic analyses suggest multiple changes of substrate specificity within the Glycosyl hydrolase 20 family

    PubMed Central

    2008-01-01

    Background Beta-N-acetylhexosaminidases belonging to the glycosyl hydrolase 20 (GH20) family are involved in the removal of terminal β-glycosidacally linked N-acetylhexosamine residues. These enzymes, widely distributed in microorganisms, animals and plants, are involved in many important physiological and pathological processes, such as cell structural integrity, energy storage, pathogen defence, viral penetration, cellular signalling, fertilization, development of carcinomas, inflammatory events and lysosomal storage diseases. Nevertheless, only limited analyses of phylogenetic relationships between GH20 genes have been performed until now. Results Careful phylogenetic analyses of 233 inferred protein sequences from eukaryotes and prokaryotes reveal a complex history for the GH20 family. In bacteria, multiple gene duplications and lineage specific gene loss (and/or horizontal gene transfer) are required to explain the observed taxonomic distribution. The last common ancestor of extant eukaryotes is likely to have possessed at least one GH20 family member. At least one gene duplication before the divergence of animals, plants and fungi as well as other lineage specific duplication events have given rise to multiple paralogous subfamilies in eukaryotes. Phylogenetic analyses also suggest that a second, divergent subfamily of GH20 family genes present in animals derive from an independent prokaryotic source. Our data suggest multiple convergent changes of functional roles of GH20 family members in eukaryotes. Conclusion This study represents the first detailed evolutionary analysis of the glycosyl hydrolase GH20 family. Mapping of data concerning physiological function of GH20 family members onto the phylogenetic tree reveals that apparently convergent and highly lineage specific changes in substrate specificity have occurred in multiple GH20 subfamilies. PMID:18647384

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

    PubMed

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

    2016-04-01

    Individuals with anorexia nervosa (AN) restrict eating and become emaciated. They tend to have an aversion to foods rich in fat. Because epoxide hydrolase 2 (EPHX2) was identified as a novel AN susceptibility gene, and because its protein product, soluble epoxide hydrolase (sEH), converts bioactive epoxides of polyunsaturated fatty acid (PUFA) to the corresponding diols, lipidomic and metabolomic targets of EPHX2 were assessed to evaluate the biological functions of EPHX2 and their role in AN. Epoxide substrates of sEH and associated oxylipins were measured in ill AN, recovered AN and gender- and race-matched controls. PUFA and oxylipin markers were tested as potential biomarkers for AN. Oxylipin ratios were calculated as proxy markers of in vivo sEH activity. Several free- and total PUFAs were associated with AN diagnosis and with AN recovery. AN displayed elevated n-3 PUFAs and may differ from controls in PUFA elongation and desaturation processes. Cytochrome P450 pathway oxylipins from arachidonic acid, linoleic acid, alpha-linolenic acid and docosahexaenoic acid PUFAs are associated with AN diagnosis. The diol:epoxide ratios suggest the sEH activity is higher in AN compared with controls. Multivariate analysis illustrates normalization of lipidomic profiles in recovered ANs. EPHX2 influences AN risk through in vivo interaction with dietary PUFAs. PUFA composition and concentrations as well as sEH activity may contribute to the pathogenesis and prognosis of AN. Our data support the involvement of EPHX2-associated lipidomic and oxylipin dysregulations in AN, and reveal their potential as biomarkers to assess responsiveness to future intervention or treatment. PMID:25824304

  2. Molecular Dynamics of Organophosphorous Hydrolases Bound to the Nerve Agent Soman

    SciTech Connect

    Soares, Thereza A.; Osman, Mohamed A.; Straatsma, TP

    2007-07-01

    The organophosphorous hydrolase (OPH) from Pseudomonas diminuta is capable of degrading extremely toxic organophosphorous compounds with a high catalytic turnover and broad substrate specificity. The potential use of this enzyme for the detection and detoxification of warfare nerve agents has spurred efforts to engineer mutants of enhanced catalytic activity and modified stereospecificity towards the most toxic forms of organophosphate nerve agents. Molecular dynamics simulations of the wild-type OPH and the complexes between the wild-type and the triple-mutant H254G/H257W/L303R forms and the substrate SpSc-soman have been carried out to enhance our molecular level understanding of its reaction mechanism. Comparison of the three simulations indicate that substrate binding induces conformational changes of the loops near the active site, suggesting an induced-fit mechanism. Likewise, the coordination of the zinc cations in the active site of the enzyme differs between the free enzyme and the complexes. In the absence of the substrate, the more exposed b-zinc is hexa-coordinated and the less exposed a-zinc is penta-coordinated. In the presence of the substrate, the b- zinc atom can be both penta- or hexa-coordinated while the a-zinc atom is tetra-coordinated. In addition, binding energies were calculated from electrostatic properties obtained by solution of the Poisson-Boltzmann equation combined with a surface area-dependent apolar contribution. The calculations indicate that the binding of SpSc-soman to OPH is driven by nonpolar interactions while electrostatic interactions determine binding specificity. These results provide a qualitative, molecular-level explanation for 2 the three-fold increase in catalytic efficiency of the triple-mutant towards SpSc-soman. Keywords: organophosphorous hydrolase, phosphotriesterase, nerve agents, soman, molecular dynamics, Poisson-Boltzmann equation, continuum electrostatics, metalloprotein.

  3. Product formation controlled by substrate dynamics in leukotriene A4 hydrolase.

    PubMed

    Stsiapanava, Alena; Tholander, Fredrik; Kumar, Ramakrishnan B; Qureshi, Abdul Aziz; Niegowski, Damian; Hasan, Mahmudul; Thunnissen, Marjolein; Haeggström, Jesper Z; Rinaldo-Matthis, Agnes

    2014-02-01

    Leukotriene A4 hydrolase/aminopeptidase (LTA4H) (EC 3.3.2.6) is a bifunctional zinc metalloenzyme with both an epoxide hydrolase and an aminopeptidase activity. LTA4H from the African claw toad, Xenopus laevis (xlLTA4H) has been shown to, unlike the human enzyme, convert LTA4 to two enzymatic metabolites, LTB4 and another biologically active product Δ(6)-trans-Δ(8)-cis-LTB4 (5(S),12R-dihydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid). In order to study the molecular aspect of the formation of this product we have characterized the structure and function of xlLTA4H. We solved the structure of xlLTA4H to a resolution of 2.3Å. It is a dimeric structure where each monomer has three domains with the active site in between the domains, similar as to the human structure. An important difference between the human and amphibian enzyme is the phenylalanine to tyrosine exchange at position 375. Our studies show that mutating F375 in xlLTA4H to tyrosine abolishes the formation of the LTB4 isomeric product Δ(6)-trans-Δ(8)-cis-LTB4. In an attempt to understand how one amino acid exchange leads to a new product profile as seen in the xlLTA4H, we performed a conformer analysis of the triene part of the substrate LTA4. Our results show that the Boltzmann distribution of substrate conformers correlates with the observed distribution of products. We suggest that the observed difference in product profile between the human and the xlLTA4H arises from different level of discrimination between substrate LTA4 conformers. PMID:24333438

  4. Long-term consequences of perinatal fatty acid amino hydrolase inhibition

    PubMed Central

    Wu, Chia-Shan; Morgan, Daniel; Jew, Chris P; Haskins, Chris; Andrews, Mary-Jeanette; Leishman, Emma; Spencer, Corinne M; Czyzyk, Traci; Bradshaw, Heather; Mackie, Ken; Lu, Hui-Chen

    2014-01-01

    Background and PurposeFatty acid amide hydrolase inhibitors show promise as a treatment for anxiety, depression and pain. Here we investigated whether perinatal exposure to URB597, a fatty acid amide hydrolase inhibitor, alters brain development and affects behaviour in adult mice. Experimental ApproachMouse dams were treated daily from gestational day 10.5 to 16.5 with 1, 3 or 10 mg kg−1 URB597. MS was used to measure a panel of endocannabinoids and related lipid compounds and brain development was assessed at embryonic day 16.5. Separate cohorts of mouse dams were treated with 10 mg kg−1 URB597, from gestational day 10.5 to postnatal day 7, and the adult offspring were assessed with a battery of behavioural tests. Key ResultsPerinatal URB597 exposure elevated anandamide and related N-acyl amides. URB597 did not induce signs of toxicity or affect dam weight gain, neurogenesis or axonal development at embryonic day 16.5. It did lead to subtle behavioural deficits in adult offspring, manifested by reduced cocaine-conditioned preference, increased depressive behaviours and impaired working memory. Anxiety levels, motor function and sensory-motor gating were not significantly altered. Conclusions and ImplicationsTaken together, the present results highlight how exposure to elevated levels of anandamide and related N-acyl amides during brain development can lead to subtle alterations in behaviour in adulthood. Linked ArticlesThis article is part of a themed section on Cannabinoids 2013. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-6 PMID:24730060

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

    SciTech Connect

    Ito, Tasuku; Saikawa, Kyo; Kim, Seonah; Fujita, Kiyotaka; Ishiwata, Akihiro; Kaeothip, Sophon; Arakawa, Takatoshi; Wakagi, Takayoshi; Beckham, Gregg T.; Ito, Yukishige; Fushinobu, Shinya

    2014-04-25

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

  6. Soluble epoxide hydrolase expression in a porcine model of arteriovenous graft stenosis and anti-inflammatory effects of a soluble epoxide hydrolase inhibitor

    PubMed Central

    Sanders, William G.; Morisseau, Christophe; Hammock, Bruce D.; Cheung, Alfred K.

    2012-01-01

    Synthetic arteriovenous (AV) grafts, placed between an artery and vein, are used for hemodialysis but often fail due to stenosis, typically at the vein-graft anastomosis. This study recorded T lymphocyte and macrophage accumulation at the vein-graft anastomosis, suggesting a role for inflammation in stenosis development. Epoxyeicosatrienoic acids (EETs), products of cytochrome P-450 epoxidation of arachidonic acid, have vasculoprotective and anti-inflammatory effects including inhibition of platelet activation, cell migration, and adhesion. EETs are hydrolyzed by soluble epoxide hydrolase (sEH) to less active diols. The effects of a specific inhibitor of sEH (sEHI) on cytokine release from human monocytes and mouse bone marrow–derived macrophages (BMMΦ) from wild-type (WT) and sEH knockout (KO) animals were investigated. Expression of sEH protein increased over time at the anastomosis as evaluated by immunohistochemistry. Pre-exposure of adherent human monocytes to sEHI (5 μM) significantly inhibited lipopolysaccharide-induced release of monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor-α and enhanced the EET-to-diol ratio. Release of MCP-1 from WT BMMΦ was significantly inhibited but release from sEH KO BMMΦ was not attenuated indicating the specificity of the sEHI. In contrast, sEHI did not inhibit the release of macrophage inflammatory protein-1 or interleukin-6. Nuclear translocation of NF-κB, as assessed by immunocytochemical staining, was not decreased with sEHI in monocytes, but the phosphorylation of JNK was completely abrogated, suggesting this pathway is the target of sEHI effects in monocytes. These results suggest that sEHI may be useful for inhibition of inflammation and subsequently stenosis in AV grafts. PMID:22621785

  7. Oxime esters as selective, covalent inhibitors of the serine hydrolase retinoblastoma-binding protein 9 (RBBP9)

    PubMed Central

    Bachovchin, Daniel A.; Wolfe, Monique R.; Masuda, Kim; Brown, Steven J.; Spicer, Timothy P.; Fernandez-Vega, Virneliz; Chase, Peter; Hodder, Peter S.; Rosen, Hugh

    2010-01-01

    We recently described a fluorescence polarization platform for competitive activity-based protein profiling (fluopol-ABPP) that enables high-throughput inhibitor screening for enzymes with poorly characterized biochemical activity. Here, we report the discovery of a class of oxime ester inhibitors for the unannotated serine hydrolase RBBP9 from a full-deck (200,000+ compound) fluopol-ABPP screen conducted in collaboration with the Molecular Libraries Screening Center Network (MLSCN). We show that these compounds covalently inhibit RBBP9 by modifying the enzyme’s active site serine nucleophile and, based on competitive ABPP in cell and tissue proteomes, are selective for RBBP9 relative to other mammalian serine hydrolases. PMID:20207142

  8. Activity-Based Protein Profiling of Organophosphorus and Thiocarbamate Pesticides Reveals Multiple Serine Hydrolase Targets in Mouse Brain

    PubMed Central

    NOMURA, DANIEL K.; CASIDA, JOHN E.

    2010-01-01

    Organophosphorus (OP) and thiocarbamate (TC) agrochemicals are used worldwide as insecticides, herbicides, and fungicides, but their safety assessment in terms of potential off-targets remains incomplete. In this study, we used a chemoproteomic platform, termed activity-based protein profiling, to broadly define serine hydrolase targets in mouse brain of a panel of 29 OP and TC pesticides. Among the secondary targets identified, enzymes involved in degradation of endocannabinoid signaling lipids, monoacylglycerol lipase and fatty acid amide hydrolase, were inhibited by several OP and TC pesticides. Blockade of these two enzymes led to elevations in brain endocannabinoid levels and dysregulated brain arachidonate metabolism. Other secondary targets include enzymes thought to also play important roles in the nervous system and unannotated proteins. This study reveals a multitude of secondary targets for OP and TC pesticides and underscores the utility of chemoproteomic platforms in gaining insights into biochemical pathways that are perturbed by these toxicants. PMID:21341672

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

    SciTech Connect

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

    2011-05-01

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

  10. Expression, purification, and buffer solubility optimization of the putative human peptidyl-tRNA hydrolase PTRHD1.

    PubMed

    Burks, Geordan L; McFeeters, Hana; McFeeters, Robert L

    2016-10-01

    Performing the essential function of recycling peptidyl-tRNAs, peptidyl-tRNA hydrolases are ubiquitous in all domains of life. The multicomponent eukaryotic Pth system differs greatly from the bacterial system composed predominantly of a single Pth1 enzyme. While bacterial Pth1s are structurally well characterized and promising new targets for antibiotic development, eukaryotic Pths are largely understudied. From amino acid sequence alignment and secondary structure predictions, the human gene product PTRHD1 was classified as a eukaryotic Pth. Herein, we report cloning, recombinant bacterial expression, and weak binding to peptidyl-tRNA for PTRHD1. Additionally, we report binding to tRNA but absence of peptidyl-tRNA hydrolase activity. Thus, PTRHD1 is not a Pth and the functional consequence of nucleotide binding remains undefined. PMID:27235175

  11. The Peptidoglycan Hydrolase of Staphylococcus aureus Bacteriophage ϕ11 Plays a Structural Role in the Viral Particle

    PubMed Central

    Rodríguez-Rubio, Lorena; Quiles-Puchalt, Nuria; Martínez, Beatriz; Rodríguez, Ana; Penadés, José R.

    2013-01-01

    The role of virion-associated peptidoglycan hydrolases (VAPGHs) in the phage infection cycle is not clear. gp49, the VAPGH from Staphylococcus aureus phage ϕ11, is not essential for phage growth but stabilizes the viral particles. ϕ11Δ49 phages showed a reduced burst size and delayed host lysis. Complementation of gp49 with HydH5 from bacteriophage vB_SauS-phiIPLA88 restored the wild-type phenotype. PMID:23892745

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

    PubMed Central

    Balotra, Sahil; Newman, Janet; French, Nigel G.; Briggs, Lyndall J.; Peat, Thomas S.; Scott, Colin

    2014-01-01

    The allophanate hydrolase from Pseudomonas sp. strain ADP was expressed and purified, and a tryptic digest fragment was subsequently identified, expressed and purified. This 50 kDa construct retained amidase activity and was crystallized. The crystals diffracted to 2.5 Å resolution and adopted space group P21, with unit-cell parameters a = 82.4, b = 179.2, c = 112.6 Å, β = 106.6°. PMID:24598916

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

    SciTech Connect

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

    2007-10-01

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

  14. The lid domain of the MCP hydrolase DxnB2 contributes to the reactivity towards recalcitrant PCB metabolites

    PubMed Central

    Yam, Katherine C.; Ghosh, Subhangi; Bolin, Jeffrey T.; Eltis, Lindsay D.

    2013-01-01

    DxnB2 and BphD are meta-cleavage product (MCP) hydrolases that catalyze C-C bond hydrolysis of the biphenyl metabolite 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA). BphD is a bottleneck in the bacterial degradation of polychlorinated biphenyls (PCBs) by the Bph catabolic pathway due in part to inhibition by 3-Cl HOPDAs. By contrast, DxnB2 from Sphingomonas wittichii RW1 catalyzes the hydrolysis of 3-Cl HOPDAs more efficiently. X-ray crystallographic studies of the catalytically inactive S105A variant of DxnB2 complexed with 3-Cl HOPDA revealed a binding mode in which C1 through C6 of the dienoate are coplanar. The chlorine substituent is accommodated by a hydrophobic pocket that is larger than the homologous site in BphDLB400 from Burkholderia xenovorans LB400. The planar binding mode observed in the crystalline complex was consistent with the hyper- and hypsochromically-shifted absorption spectra of 3-Cl and 3,9,11-triCl HOPDA, respectively, bound to S105A in solution. Moreover, ESred, an intermediate possessing a bathochromically-shifted spectrum observed in the turnover of HOPDA, was not detected, suggesting that substrate destabilization was rate-limiting in the turnover of these PCB metabolites. Interestingly, electron density for the first α-helix of the lid domain was poorly defined in the dimeric DxnB2 structures, unlike in the tetrameric BphDLB400. Structural comparison of MCP hydrolases identified the NC-loop, connecting the lid to the α/β-hydrolase core domain, as a determinant in oligomeric state and suggests its involvement in catalysis. Finally, an increased mobility of the DxnB2 lid may contribute to the enzyme’s ability to hydrolyze PCB metabolites, highlighting how lid architecture contributes to substrate specificity in α/β-hydrolases. PMID:23879719

  15. Structural Insights into an Oxalate-producing Serine Hydrolase with an Unusual Oxyanion Hole and Additional Lyase Activity.

    PubMed

    Oh, Juntaek; Hwang, Ingyu; Rhee, Sangkee

    2016-07-15

    In Burkholderia species, the production of oxalate, an acidic molecule, is a key event for bacterial growth in the stationary phase. Oxalate plays a central role in maintaining environmental pH, which counteracts inevitable population-collapsing alkaline toxicity in amino acid-based culture medium. In the phytopathogen Burkholderia glumae, two enzymes are responsible for oxalate production. First, the enzyme oxalate biosynthetic component A (ObcA) catalyzes the formation of a tetrahedral C6-CoA adduct from the substrates acetyl-CoA and oxaloacetate. Then the ObcB enzyme liberates three products from the C6-CoA adduct: oxalate, acetoacetate, and CoA. Interestingly, these two stepwise reactions are catalyzed by a single bifunctional enzyme, Obc1, from Burkholderia thailandensis and Burkholderia pseudomallei Obc1 has an ObcA-like N-terminal domain and shows ObcB activity in its C-terminal domain despite no sequence homology with ObcB. We report the crystal structure of Obc1 in its apo and glycerol-bound form at 2.5 Å and 2.8 Å resolution, respectively. The Obc1 N-terminal domain is essentially identical both in structure and function to that of ObcA. Its C-terminal domain has an α/β hydrolase fold that has a catalytic triad for oxalate production and a novel oxyanion hole distinct from the canonical HGGG motif in other α/β hydrolases. Functional analyses through mutagenesis studies suggested that His-934 is an additional catalytic acid/base for its lyase activity and liberates two additional products, acetoacetate and CoA. These results provide structural and functional insights into bacterial oxalogenesis and an example of divergent evolution of the α/β hydrolase fold, which has both hydrolase and lyase activity. PMID:27226606

  16. A Substrate-Assisted Mechanism of Nucleophile Activation in a Ser-His-Asp Containing C-C Bond Hydrolase

    SciTech Connect

    Ruzzini, Antonio C.; Bhowmik, Shiva; Ghosh, Subhangi; Yam, Katherine C.; Bolin, Jeffrey T.; Eltis, Lindsay D.

    2013-11-12

    The meta-cleavage product (MCP) hydrolases utilize a Ser–His–Asp triad to hydrolyze a carbon–carbon bond. Hydrolysis of the MCP substrate has been proposed to proceed via an enol-to-keto tautomerization followed by a nucleophilic mechanism of catalysis. Ketonization involves an intermediate, ESred, which possesses a remarkable bathochromically shifted absorption spectrum. We investigated the catalytic mechanism of the MCP hydrolases using DxnB2 from Sphingomonas wittichii RW1. Pre-steady-state kinetic and LC ESI/MS evaluation of the DxnB2-mediated hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid to 2-hydroxy-2,4-pentadienoic acid and benzoate support a nucleophilic mechanism catalysis. In DxnB2, the rate of ESred decay and product formation showed a solvent kinetic isotope effect of 2.5, indicating that a proton transfer reaction, assigned here to substrate ketonization, limits the rate of acylation. For a series of substituted MCPs, this rate was linearly dependent on MCP pKa2nuc ~ 1). Structural characterization of DxnB2 S105A:MCP complexes revealed that the catalytic histidine is displaced upon substrate-binding. The results provide evidence for enzyme-catalyzed ketonization in which the catalytic His–Asp pair does not play an essential role. The data further suggest that ESred represents a dianionic intermediate that acts as a general base to activate the serine nucleophile. This substrate-assisted mechanism of nucleophilic catalysis distinguishes MCP hydrolases from other serine hydrolases.

  17. Local structure based method for prediction of the biochemical function of proteins: Applications to glycoside hydrolases.

    PubMed

    Parasuram, Ramya; Mills, Caitlyn L; Wang, Zhouxi; Somasundaram, Saroja; Beuning, Penny J; Ondrechen, Mary Jo

    2016-01-15

    Thousands of protein structures of unknown or uncertain function have been reported as a result of high-throughput structure determination techniques developed by Structural Genomics (SG) projects. However, many of the putative functional assignments of these SG proteins in the Protein Data Bank (PDB) are incorrect. While high-throughput biochemical screening techniques have provided valuable functional information for limited sets of SG proteins, the biochemical functions for most SG proteins are still unknown or uncertain. Therefore, computational methods for the reliable prediction of protein function from structure can add tremendous value to the existing SG data. In this article, we show how computational methods may be used to predict the function of SG proteins, using examples from the six-hairpin glycosidase (6-HG) and the concanavalin A-like lectin/glucanase (CAL/G) superfamilies. Using a set of predicted functional residues, obtained from computed electrostatic and chemical properties for each protein structure, it is shown that these superfamilies may be sorted into functional families according to biochemical function. Within these superfamilies, a total of 18 SG proteins were analyzed according to their predicted, local functional sites: 13 from the 6-HG superfamily, five from the CAL/G superfamily. Within the 6-HG superfamily, an uncharacterized protein BACOVA_03626 from Bacteroides ovatus (PDB 3ON6) and a hypothetical protein BT3781 from Bacteroides thetaiotaomicron (PDB 2P0V) are shown to have very strong active site matches with exo-α-1,6-mannosidases, thus likely possessing this function. Also in this superfamily, it is shown that protein BH0842, a putative glycoside hydrolase from Bacillus halodurans (PDB 2RDY), has a predicted active site that matches well with a known α-L-galactosidase. In the CAL/G superfamily, an uncharacterized glycosyl hydrolase family 16 protein from Mycobacterium smegmatis (PDB 3RQ0) is shown to have local structural

  18. Surface display of heterologous proteins in Bacillus thuringiensis using a peptidoglycan hydrolase anchor

    PubMed Central

    Shao, Xiaohu; Jiang, Mengtian; Yu, Ziniu; Cai, Hao; Li, Lin

    2009-01-01

    Background Previous studies have revealed that the lysin motif (LysM) domains of bacterial cell wall-degrading enzymes are able to bind to peptidoglycan moieties of the cell wall. This suggests an approach for a cell surface display system in Gram-positive bacteria using a LysM-containing protein as the anchoring motif. In this study, we developed a new surface display system in B. thuringiensis using a LysM-containing peptidoglycan hydrolase, endo-β-N-acetylglucosaminidase (Mbg), as the anchor protein. Results Homology searching in the B. thuringiensis YBT-1520 genome revealed a putative peptidoglycan hydrolase gene. The encoded protein, Mbg, exhibited substantial cell-wall binding capacity. The deduced amino acid sequence of Mbg was structurally distinguished as an N-terminal domain with two tandemly aligned LysMs and a C-terminal catalytic domain. A GFP-fusion protein was expressed and used to verify the surface localization by Western blot, flow cytometry, protease accessibility, SDS sensitivity, immunofluorescence, and electron microscopy assays. Low-level constitutive expression of Mbg was elevated by introducing a sporulation-independent promoter of cry3Aa. Truncated Mbg domains with separate N-terminus (Mbgn), C-terminus (Mbgc), LysM1, or LysM2 were further compared for their cell-wall displaying efficiencies. The Mbgn moiety contributed to cell-wall anchoring, while LysM1 was the active domain. Two tandemly repeated Mbgns exhibited the highest display activity, while the activity of three repeated Mbgns was decreased. A heterologous bacterial multicopper oxidase (WlacD) was successfully displayed onto the surface of B. thuringiensis target cells using the optimum (Mbgn)2 anchor, without radically altering its catalytic activity. Conclusion Mbg can be a functional anchor protein to target different heterologous proteins onto the surface of B. thuringiensis cells. Since the LysM domain appears to be universal in Gram-positive bacteria, the strategy

  19. Leishmania donovani Nucleoside Hydrolase Terminal Domains in Cross-Protective Immunotherapy Against Leishmania amazonensis Murine Infection

    PubMed Central

    Nico, Dirlei; Gomes, Daniele Crespo; Palatnik-de-Sousa, Iam; Morrot, Alexandre; Palatnik, Marcos; Palatnik-de-Sousa, Clarisa Beatriz

    2014-01-01

    Nucleoside hydrolases of the Leishmania genus are vital enzymes for the replication of the DNA and conserved phylogenetic markers of the parasites. Leishmania donovani nucleoside hydrolase (NH36) induced a main CD4+ T cell driven protective response against L. chagasi infection in mice which is directed against its C-terminal domain. In this study, we used the three recombinant domains of NH36: N-terminal domain (F1, amino acids 1–103), central domain (F2 aminoacids 104–198), and C-terminal domain (F3 amino acids 199–314) in combination with saponin and assayed their immunotherapeutic effect on Balb/c mice previously infected with L. amazonensis. We identified that the F1 and F3 peptides determined strong cross-immunotherapeutic effects, reducing the size of footpad lesions to 48 and 64%, and the parasite load in footpads to 82.6 and 81%, respectively. The F3 peptide induced the strongest anti-NH36 antibody response and intradermal response (IDR) against L. amazonenis and a high secretion of IFN-γ and TNF-α with reduced levels of IL-10. The F1 vaccine, induced similar increases of IgG2b antibodies and IFN-γ and TNF-α levels, but no IDR and no reduction of IL-10. The multiparameter flow cytometry analysis was used to assess the immune response after immunotherapy and disclosed that the degree of the immunotherapeutic effect is predicted by the frequencies of the CD4+ and CD8+ T cells producing IL-2 or TNF-α or both. Total frequencies and frequencies of double-cytokine CD4 T cell producers were enhanced by F1 and F3 vaccines. Collectively, our multifunctional analysis disclosed that immunotherapeutic protection improved as the CD4 responses progressed from 1+ to 2+, in the case of the F1 and F3 vaccines, and as the CD8 responses changed qualitatively from 1+ to 3+, mainly in the case of the F1 vaccine, providing new correlates of immunotherapeutic protection against cutaneous leishmaniasis in mice based on T-helper TH1 and CD8+ mediated immune responses

  20. Evaluation of epichlorohydrin (ECH) genotoxicity. Microsomal epoxide hydrolase-dependent deactivation of ECH mutagenicity in Schizosaccharomyces pombe in vitro.

    PubMed

    Rossi, A M; Migliore, L; Loprieno, N; Romano, M; Salmona, M

    1983-04-01

    The mutagenic effect of epichlorohydrin (ECH) on the yeast Schizosaccharomyces pombe was studied in vitro in the presence of mouse-liver S9 mix and microsomal and cytosolic fractions. The incubations were always performed in the absence of NADPH-generating systems. S9 mix and microsomes from phenobarbital-pretreated mice significantly reduced ECH mutagenicity, whereas the cytosol did not result in any deactivating effect. The various protein contents of the subcellular fractions were not involved in any scavenger effect as regards ECH mutagenic activity. Moreover, the addition of reduced glutathione to the incubation mixtures indicated that it did not play an important role, either per se or through the enzyme(s) glutathione-S-epoxide transferase(s), in preventing ECH genotoxicity. Our results suggest that microsomal epoxide hydrolase(s) represents the major step in the detoxifying pathway of ECH. These observations were supported by measurements of the specific epoxide hydrolase activity in the various fractions on the same substrate. PMID:6835236

  1. Stimulation of lignocellulosic biomass hydrolysis by proteins of glycoside hydrolase family 61: structure and function of a large, enigmatic family.

    PubMed

    Harris, Paul V; Welner, Ditte; McFarland, K C; Re, Edward; Navarro Poulsen, Jens-Christian; Brown, Kimberly; Salbo, Rune; Ding, Hanshu; Vlasenko, Elena; Merino, Sandy; Xu, Feng; Cherry, Joel; Larsen, Sine; Lo Leggio, Leila

    2010-04-20

    Currently, the relatively high cost of enzymes such as glycoside hydrolases that catalyze cellulose hydrolysis represents a barrier to commercialization of a biorefinery capable of producing renewable transportable fuels such as ethanol from abundant lignocellulosic biomass. Among the many families of glycoside hydrolases that catalyze cellulose and hemicellulose hydrolysis, few are more enigmatic than family 61 (GH61), originally classified based on measurement of very weak endo-1,4-beta-d-glucanase activity in one family member. Here we show that certain GH61 proteins lack measurable hydrolytic activity by themselves but in the presence of various divalent metal ions can significantly reduce the total protein loading required to hydrolyze lignocellulosic biomass. We also solved the structure of one highly active GH61 protein and find that it is devoid of conserved, closely juxtaposed acidic side chains that could serve as general proton donor and nucleophile/base in a canonical hydrolytic reaction, and we conclude that the GH61 proteins are unlikely to be glycoside hydrolases. Structure-based mutagenesis shows the importance of several conserved residues for GH61 function. By incorporating the gene for one GH61 protein into a commercial Trichoderma reesei strain producing high levels of cellulolytic enzymes, we are able to reduce by 2-fold the total protein loading (and hence the cost) required to hydrolyze lignocellulosic biomass. PMID:20230050

  2. Characterization of the First Fungal Glycosyl Hydrolase Family 19 Chitinase (NbchiA) from Nosema bombycis (Nb).

    PubMed

    Han, Bing; Zhou, Kang; Li, Zhihong; Sun, Bin; Ni, Qi; Meng, Xianzhi; Pan, Guoqing; Li, Chunfeng; Long, Mengxian; Li, Tian; Zhou, Congzhao; Li, Weifang; Zhou, Zeyang

    2016-01-01

    Chitinases (EC 3.2.1.14), as one kind of glycosyl hydrolase, hydrolyze the β-(1,4) linkages of chitin. According to the sequence similarity, chitinases can be divided into glycoside hydrolase family 18 and family 19. Here, a chitinase from Nosema bombycis (NbchiA) was cloned and purified by metal affinity chromatography and molecular exclusion chromatography. Sequence analysis indicated that NbchiA belongs to glycoside hydrolase family 19 class IV chitinase. The optimal pH and temperature of NbchiA are 7.0 and 40 °C, respectively. This purified chitinase showed high activity toward soluble substrates such as ethylene glycol chitin and soluble chitosan. The degradation of chitin oligosaccharides (GlcNAc)(2-5) detected by high-performance liquid chromatography showed that NbchiA hydrolyzed mainly the second glycosidic linkage from the reducing end of (GlcNAc)(3-5). On the basis of structure-based multiple-sequence alignment, Glu51 and Glu60 are believed to be the key catalytic residues. The site-directed mutation analysis revealed that the enzymatic activity was decreased upon mutation of Glu60, whereas mutation of Glu51 totally abolished the enzymatic activity. This is the first report of a GH19 chitinase in fungi and in Microsporidia. PMID:26108336

  3. Signal transduction in the carnivorous plant Sarracenia purpurea. Regulation of secretory hydrolase expression during development and in response to resources.

    PubMed Central

    Gallie, D R; Chang, S C

    1997-01-01

    Carnivory in plants has developed as an evolutionary adaptation to nutrient-poor environments. A significant investment of the resources of a carnivorous plant is committed to producing the traps, attractants, and digestive enzymes needed for the carnivory. The cost:benefit ratio of carnivory can be improved by either maximizing the prey capture rate or by reducing the metabolic commitment toward carnivory. Using the pitcher plant Sarracenia purpurea, we have investigated whether the expression of the hydrolytic enzymes needed for digestion is regulated in response to the presence of prey. Expression of protease, RNase, nuclease, and phosphatase activities could be induced in the fluid of nonactive traps by the addition of nucleic acids, protein, or reduced nitrogen, suggesting that hydrolase expression is induced upon perception of the appropriate chemical signal. Hydrolase expression was also developmentally controlled since expression commenced upon opening of a trap, increased for several days, and in the absence of prey largely ceased within 2 weeks. Nevertheless, the traps remained competent to induce expression in response to the appropriate signals. These data suggest that in young traps hydrolase expression is developmentally regulated, which is later replaced by a signal transduction mechanism, and they demonstrate the ability of a carnivorous species to respond to the availability of resources. PMID:9414556

  4. Ingestion of the epoxide hydrolase inhibitor AUDA modulates immune responses of the mosquito, Culex quinquefasciatus during blood feeding.

    PubMed

    Xu, Jiawen; Morisseau, Christophe; Yang, Jun; Lee, Kin Sing Stephen; Kamita, Shizuo G; Hammock, Bruce D

    2016-09-01

    Epoxide hydrolases (EHs) are enzymes that play roles in metabolizing xenobiotic epoxides from the environment, and in regulating lipid signaling molecules, such as juvenile hormones in insects and epoxy fatty acids in mammals. In this study we fed mosquitoes with an epoxide hydrolase inhibitor AUDA during artificial blood feeding, and we found the inhibitor increased the concentration of epoxy fatty acids in the midgut of female mosquitoes. We also observed ingestion of AUDA triggered early expression of defensin A, cecropin A and cecropin B2 at 6 h after blood feeding. The expression of cecropin B1 and gambicin were not changed more than two fold compared to controls. The changes in gene expression were transient possibly because more than 99% of the inhibitor was metabolized or excreted at 42 h after being ingested. The ingestion of AUDA also affected the growth of bacteria colonizing in the midgut, but did not affect mosquito longevity, fecundity and fertility in our laboratory conditions. When spiked into the blood, EpOMEs and DiHOMEs were as effective as the inhibitor AUDA in reducing the bacterial load in the midgut, while EETs rescued the effects of AUDA. Our data suggest that epoxy fatty acids from host blood are immune response regulators metabolized by epoxide hydrolases in the midgut of female mosquitoes, inhibition of which causes transient changes in immune responses, and affects growth of microbes in the midgut. PMID:27369469

  5. Inhibition of xenobiotic-degrading hydrolases by organophosphinates. Annual progress report No. 1 Jul 82-1 Jul 83

    SciTech Connect

    Brown, T.M.; Zimmerman, J.K.; Bryson, P.K.; Grothusen, J.R.

    1983-07-01

    Organophosphinate pretreatment agents for chemical warfare defense inhibited carboxylester hydrolase from porcine liver and from rabbit liver. Recovery of rabbit liver monomeric carboxylester hydrolase to at least 30% of its initial activity was observed 48 hr. after inhibition by certain 4-nitrophenyl alkyl(phenyl)phosphinates and analogues. When ranked according to the initial rates at which their phosphinylated enzymes recovered, they were methyl(phenyl)>methyl(2-thienyl)>methyl(2-furyl)>ethyl(phenyl)>di-2-thienyl>diphenyl. Recovery was less than 20% in 96 hr. following inhibition by methyl(naphthyl),di-2-furyl, isopropyl(phenyl), dichloromethyl(phenyl), and bis chloromethyl substituted analogues. High performance liquid chromatography on silica using 10% to 20% 2-propanol in hexane as mobile phase resulted in satisfactory chromatograms for all except the most polar phosphinates. This method was useful in determining purity and decomposition of the compounds. Arylester hydrolase was purified 30-fold from rabbit serum by a sequence of polyethylene glycol fractionation, ion exchange chromatography, ammonium sulfate fractionation, molecular exclusion chromatography and pseudo-affinity chromatography. The partially purified enzyme was activated by 2.5 mM divalent calcium.

  6. Identification and Characterization of a New Alkaline SGNH Hydrolase from a Thermophilic Bacterium Bacillus sp. K91.

    PubMed

    Yu, Tingting; Ding, Junmei; Zheng, Qingxia; Han, Nanyu; Yu, Jialin; Yang, Yunjuan; Li, Junjun; Mu, Yuelin; Wu, Qian; Huang, Zunxi

    2016-04-28

    est19 is a gene from Bacillus sp. K91 that encodes a new esterase. A comparison of the amino acid sequence showed that Est19 has typical Ser-Gly-Asn-His (SGNH) family motifs and could be grouped into the SGNH hydrolase family. The Est19 protein was functionally cloned, and expressed and purified from Escherichia coli BL21(DE3). The enzyme activity was optimal at 60°C and pH 9.0, and displayed esterase activity towards esters with short-chain acyl esters (C₂-C₆). A structural model of Est19 was constructed using phospholipase A1 from Streptomyces albidoflavus NA297 as a template. The structure showed an α/β-hydrolase fold and indicated the presence of the typical catalytic triad Ser49-Asp227-His230, which were further investigated by site-directed mutagenesis. To the best of our knowledge, Est19 is a new member of the SGNH hydrolase family identified from thermophiles, which may be applicable in the industrial production of semisynthetic β-lactam antibiotics after modification. PMID:26699742

  7. Molecular cloning, sequence analysis, and characterization of a new cell wall hydrolase, CwlL, of Bacillus licheniformis.

    PubMed

    Oda, Y; Nakayama, R; Kuroda, A; Sekiguchi, J

    1993-11-01

    We have cloned a DNA fragment containing the gene for a cell wall hydrolase from Bacillus licheniformis FD0120 into Escherichia coli. Sequencing of the fragment showed the presence of an open reading frame (ORF; designated as cwlL), which is different from the B. licheniformis cell wall hydrolase gene cwlM, and encodes a polypeptide of 360 amino acids with a molecular mass of 38,994. The enzyme purified from the E. coli clone is an N-acetylmuramoyl-L-alanine amidase, which has a M(r) value of 41 kDa as determined by SDS-polyacrylamide gel electrophoresis, and is able to digest B. licheniformis, B. subtilis and Micrococcus luteus cell walls. The nucleotide and deduced amino acid sequences of cwlL are very similar to those of ORF3 in the putative operon xpaL1-xpaL2-ORF3 in B. licheniformis MC14. Moreover, the amino acid sequence homology of CwlL with the B. subtilis amidase CwlA indicates two evolutionarily distinguishable regions in CwlL. The sequence homology of CwlL with other cell wall hydrolases and the regulation of cwlL are discussed. PMID:7902527

  8. Structural Basis of the Novel S. pneumoniae Virulence Factor, GHIP, a Glycosyl Hydrolase 25 Participating in Host-Cell Invasion

    PubMed Central

    Niu, Siqiang; Luo, Miao; Tang, Jian; Zhou, Hua; Zhang, Yangli; Min, Xun; Cai, Xuefei; Zhang, Wenlu; Xu, Wenchu; Li, Defeng; Ding, Jingjin; Hu, Yonglin; Wang, Dacheng; Huang, Ailong

    2013-01-01

    Pathogenic bacteria produce a wide variety of virulence factors that are considered to be potential antibiotic targets. In this study, we report the crystal structure of a novel S. pneumoniae virulence factor, GHIP, which is a streptococcus-specific glycosyl hydrolase. This novel structure exhibits an α/β-barrel fold that slightly differs from other characterized hydrolases. The GHIP active site, located at the negatively charged groove in the barrel, is very similar to the active site in known peptidoglycan hydrolases. Functionally, GHIP exhibited weak enzymatic activity to hydrolyze the PNP-(GlcNAc)5 peptidoglycan by the general acid/base catalytic mechanism. Animal experiments demonstrated a marked attenuation of S. pneumoniae-mediated virulence in mice infected by ΔGHIP-deficient strains, suggesting that GHIP functions as a novel S. pneumoniae virulence factor. Furthermore, GHIP participates in allowing S. pneumoniae to colonize the nasopharynx and invade host epithelial cells. Taken together, these findings suggest that GHIP can potentially serve as an antibiotic target to effectively treat streptococcus-mediated infection. PMID:23874703

  9. Cocoa pod husk, a new source of hydrolase enzymes for preparation of cross-linked enzyme aggregate.

    PubMed

    Yusof, Faridah; Khanahmadi, Soofia; Amid, Azura; Mahmod, Safa Senan

    2016-01-01

    Cocoa pod husk (CPH) is a by-product of cocoa production obtained after removing the beans from the fruit. The analysis of CPH has shown that it contains high amounts of protein. This study is aimed to utilize this protein source in hydrolase enzyme production. In this study, seven hydrolase enzymes (amylase, fructosyltransferase, mannanase, glucosidase, glucanase, lipase and protease) were screened from CPH for the first time for feasible industrial production. Among these hydrolases, lipase was chosen for the next steps of experiments as it has a lot of applications in different industries. The extraction of high active lipase from CPH has been done under optimum conditions. The condition that was optimum for the three major factors was achieved using Face centered central composite design (FCCCD) with response surface methodology (RSM) to obtain the highest enzyme activity of crude lipase from CPH. The optimum condition of extraction is used for preparation of cross-linked enzyme aggregate (CLEA). For the production of immobilized biocatalyst, the technique of CLEA is considered as an effective technique for its industrially attractive advantages. Referring to the results of OFAT, CLEA-lipase was prepared in the best condition at the presence of 30 mM ammonium sulphate, 70 mM glutaraldehyde with 0.23 mM Bovine serum albumin as an additive. Immobilization effectively improved the stability of lipase against various organic solvents. PMID:26904389

  10. First Glycoside Hydrolase Family 2 Enzymes from Thermus antranikianii and Thermus brockianus with β-Glucosidase Activity

    PubMed Central

    Schröder, Carola; Blank, Saskia; Antranikian, Garabed

    2015-01-01

    Two glycoside hydrolase encoding genes (tagh2 and tbgh2) were identified from different Thermus species using functional screening. Based on amino acid similarities, the enzymes were predicted to belong to glycoside hydrolase (GH) family 2. Surprisingly, both enzymes (TaGH2 and TbGH2) showed twofold higher activities for the hydrolysis of nitrophenol-linked β-D-glucopyranoside than of -galactopyranoside. Specific activities of 3,966 U/mg for TaGH2 and 660 U/mg for TbGH2 were observed. In accordance, Km values for both enzymes were significantly lower when β-D-glucopyranoside was used as substrate. Furthermore, TaGH2 was able to hydrolyze cellobiose. TaGH2 and TbGH2 exhibited highest activity at 95 and 90°C at pH 6.5. Both enzymes were extremely thermostable and showed thermal activation up to 250% relative activity at temperatures of 50 and 60°C. Especially, TaGH2 displayed high tolerance toward numerous metal ions (Cu2+, Co2+, Zn2+), which are known as glycoside hydrolase inhibitors. In this study, the first thermoactive GH family 2 enzymes with β-glucosidase activity have been identified and characterized. The hydrolysis of cellobiose is a unique property of TaGH2 when compared to other enzymes of GH family 2. Our work contributes to a broader knowledge of substrate specificities in GH family 2. PMID:26090361

  11. Recessive loss of function of the neuronal ubiquitin hydrolase UCHL1 leads to early-onset progressive neurodegeneration

    PubMed Central

    Bilguvar, Kaya; Tyagi, Navneet K.; Ozkara, Cigdem; Tuysuz, Beyhan; Bakircioglu, Mehmet; Choi, Murim; Delil, Sakir; Caglayan, Ahmet O.; Baranoski, Jacob F.; Erturk, Ozdem; Yalcinkaya, Cengiz; Karacorlu, Murat; Dincer, Alp; Johnson, Michele H.; Mane, Shrikant; Chandra, Sreeganga S.; Louvi, Angeliki; Boggon, Titus J.; Lifton, Richard P.; Horwich, Arthur L.; Gunel, Murat

    2013-01-01

    Ubiquitin C-terminal hydrolase-L1 (UCHL1), a neuron-specific de-ubiquitinating enzyme, is one of the most abundant proteins in the brain. We describe three siblings from a consanguineous union with a previously unreported early-onset progressive neurodegenerative syndrome featuring childhood onset blindness, cerebellar ataxia, nystagmus, dorsal column dysfuction, and spasticity with upper motor neuron dysfunction. Through homozygosity mapping of the affected individuals followed by whole-exome sequencing of the index case, we identified a previously undescribed homozygous missense mutation within the ubiquitin binding domain of UCHL1 (UCHL1GLU7ALA), shared by all affected subjects. As demonstrated by isothermal titration calorimetry, purified UCHL1GLU7ALA, compared with WT, exhibited at least sevenfold reduced affinity for ubiquitin. In vitro, the mutation led to a near complete loss of UCHL1 hydrolase activity. The GLU7ALA variant is predicted to interfere with the substrate binding by restricting the proper positioning of the substrate for tunneling underneath the cross-over loop spanning the catalytic cleft of UCHL1. This interference with substrate binding, combined with near complete loss of hydrolase activity, resulted in a >100-fold reduction in the efficiency of UCHL1GLU7ALA relative to WT. These findings demonstrate a broad requirement of UCHL1 in the maintenance of the nervous system. PMID:23359680

  12. Recessive loss of function of the neuronal ubiquitin hydrolase UCHL1 leads to early-onset progressive neurodegeneration.

    PubMed

    Bilguvar, Kaya; Tyagi, Navneet K; Ozkara, Cigdem; Tuysuz, Beyhan; Bakircioglu, Mehmet; Choi, Murim; Delil, Sakir; Caglayan, Ahmet O; Baranoski, Jacob F; Erturk, Ozdem; Yalcinkaya, Cengiz; Karacorlu, Murat; Dincer, Alp; Johnson, Michele H; Mane, Shrikant; Chandra, Sreeganga S; Louvi, Angeliki; Boggon, Titus J; Lifton, Richard P; Horwich, Arthur L; Gunel, Murat

    2013-02-26

    Ubiquitin C-terminal hydrolase-L1 (UCHL1), a neuron-specific de-ubiquitinating enzyme, is one of the most abundant proteins in the brain. We describe three siblings from a consanguineous union with a previously unreported early-onset progressive neurodegenerative syndrome featuring childhood onset blindness, cerebellar ataxia, nystagmus, dorsal column dysfuction, and spasticity with upper motor neuron dysfunction. Through homozygosity mapping of the affected individuals followed by whole-exome sequencing of the index case, we identified a previously undescribed homozygous missense mutation within the ubiquitin binding domain of UCHL1 (UCHL1(GLU7ALA)), shared by all affected subjects. As demonstrated by isothermal titration calorimetry, purified UCHL1(GLU7ALA), compared with WT, exhibited at least sevenfold reduced affinity for ubiquitin. In vitro, the mutation led to a near complete loss of UCHL1 hydrolase activity. The GLU7ALA variant is predicted to interfere with the substrate binding by restricting the proper positioning of the substrate for tunneling underneath the cross-over loop spanning the catalytic cleft of UCHL1. This interference with substrate binding, combined with near complete loss of hydrolase activity, resulted in a >100-fold reduction in the efficiency of UCHL1(GLU7ALA) relative to WT. These findings demonstrate a broad requirement of UCHL1 in the maintenance of the nervous system. PMID:23359680

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

    PubMed Central

    Wang, Meng; Lai, Guo-Li; Nie, Yong; Geng, Shuang; Liu, Liming; Zhu, Baoli; Shi, Zhongping; Wu, Xiao-Lei

    2015-01-01

    In biofuel production from lignocellulose, low thermostability and product inhibition strongly restrict the enzyme activities and production process. Application of multiple thermostable glycoside hydrolases, forming an enzyme “cocktail”, can result in a synergistic action and therefore improve production efficiency and reduce operational costs. Therefore, increasing enzyme thermostabilities and compatibility are important for the biofuel industry. In this study, we reported the screening, cloning and biochemical characterization of four novel thermostable lignocellulose hydrolases from a metagenomic library of a long-term dry thermophilic methanogenic digester community, which were highly compatible with optimal conditions and specific activities. The optimal temperatures of the four enzymes, β-xylosidase, xylanase, β-glucosidase, and cellulase ranged from 60 to 75°C, and over 80% residual activities were observed after 2 h incubation at 50°C. Mixtures of these hydrolases retained high residual synergistic activities after incubation with cellulose, xylan, and steam-exploded corncob at 50°C for 72 h. In addition, about 55% dry weight of steam-exploded corncob was hydrolyzed to glucose and xylose by the synergistic action of the four enzymes at 50°C for 48 h. This work suggested that since different enzymes from a same ecosystem could be more compatible, screening enzymes from a long-term enriching community could be a favorable strategy. PMID:26052323

  14. S-Inosyl-l-Homocysteine Hydrolase, a Novel Enzyme Involved in S-Adenosyl-l-Methionine Recycling

    PubMed Central

    Miller, Danielle; Xu, Huimin

    2015-01-01

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

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

    SciTech Connect

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

    2015-09-15

    ABSTRACT

    Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. These enzymes all have γ-d-Glu-A2pm (A2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structure consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.

    IMPORTANCEPeptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural

  16. Insights into substrate specificity of NlpC/P60 cell wall hydrolases containing bacterial SH3 domains

    DOE PAGESBeta

    Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.; Patin, Delphine; Farr, Carol L.; Grant, Joanna C.; Chiu, Hsiu -Ju; Jaroszewski, Lukasz; Knuth, Mark W.; Godzik, Adam; et al

    2015-09-15

    Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. In addition, these enzymes all have γ-d-Glu-A2pm (A2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structure consisting ofmore » two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.Peptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show

  17. Insights into substrate specificity of NlpC/P60 cell wall hydrolases containing bacterial SH3 domains

    SciTech Connect

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

    2015-09-15

    Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. In addition, these enzymes all have γ-d-Glu-A2pm (A2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structure consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.

    Peptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60

  18. The Molecular Basis for Dual Fatty Acid Amide Hydrolase (FAAH)/Cyclooxygenase (COX) Inhibition.

    PubMed

    Palermo, Giulia; Favia, Angelo D; Convertino, Marino; De Vivo, Marco

    2016-06-20

    The design of multitarget-directed ligands is a promising strategy for discovering innovative drugs. Here, we report a mechanistic study that clarifies key aspects of the dual inhibition of the fatty acid amide hydrolase (FAAH) and the cyclooxygenase (COX) enzymes by a new multitarget-directed ligand named ARN2508 (2-[3-fluoro-4-[3-(hexylcarbamoyloxy)phenyl]phenyl]propanoic acid). This potent dual inhibitor combines, in a single scaffold, the pharmacophoric elements often needed to block FAAH and COX, that is, a carbamate moiety and the 2-arylpropionic acid functionality, respectively. Molecular modeling and molecular dynamics simulations suggest that ARN2508 uses a noncovalent mechanism of inhibition to block COXs, while inhibiting FAAH via the acetylation of the catalytic Ser241, in line with previous experimental evidence for covalent FAAH inhibition. This study proposes the molecular basis for the dual FAAH/COX inhibition by this novel hybrid scaffold, stimulating further experimental studies and offering new insights for the rational design of novel anti-inflammatory agents that simultaneously act on FAAH and COX. PMID:26593700

  19. Balancing the stability and the catalytic specificities of OP hydrolases with enhanced V-agent activities.

    PubMed

    Reeves, T E; Wales, M E; Grimsley, J K; Li, P; Cerasoli, D M; Wild, J R

    2008-06-01

    Rational site-directed mutagenesis and biophysical analyses have been used to explore the thermodynamic stability and catalytic capabilities of organophosphorus hydrolase (OPH) and its genetically modified variants. There are clear trade-offs in the stability of modifications that enhance catalytic activities. For example, the H254R/H257L variant has higher turnover numbers for the chemical warfare agents VX (144 versus 14 s(-1) for the native enzyme (wild type) and VR (Russian VX, 465 versus 12 s(-1) for wild type). These increases are accompanied by a loss in stability in which the total Gibb's free energy for unfolding is 19.6 kcal/mol, which is 5.7 kcal/mol less than that of the wild-type enzyme. X-ray crystallographic studies support biophysical data that suggest amino acid residues near the active site contribute to the chemical and thermal stability through hydrophobic and cation-pi interactions. The cation-pi interactions appear to contribute an additional 7 kcal/mol to the overall global stability of the enzyme. Using rational design, it has been possible to make amino acid changes in this region that restored the stability, yet maintained effective V-agent activities, with turnover numbers of 68 and 36 s(-1) for VX and VR, respectively. This study describes the first rationally designed, stability/activity balance for an OPH enzyme with a legitimate V-agent activity, and its crystal structure. PMID:18434422

  20. EHPred: an SVM-based method for epoxide hydrolases recognition and classification.

    PubMed

    Jia, Jia; Yang, Liang; Zhang, Zi-Zhang

    2006-01-01

    A two-layer method based on support vector machines (SVMs) has been developed to distinguish epoxide hydrolases (EHs) from other enzymes and to classify its subfamilies using its primary protein sequences. SVM classifiers were built using three different feature vectors extracted from the primary sequence of EHs: the amino acid composition (AAC), the dipeptide composition (DPC), and the pseudo-amino acid composition (PAAC). Validated by 5-fold cross tests, the first layer SVM classifier can differentiate EHs and non-EHs with an accuracy of 94.2% and has a Matthew's correlation coefficient (MCC) of 0.84. Using 2-fold cross validation, PAAC-based second layer SVM can further classify EH subfamilies with an overall accuracy of 90.7% and MCC of 0.87 as compared to AAC (80.0%) and DPC (84.9%). A program called EHPred has also been developed to assist readers to recognize EHs and to classify their subfamilies using primary protein sequences with greater accuracy. PMID:16365918

  1. Liver-specific transgenic expression of cholesteryl ester hydrolase reduces atherosclerosis in Ldlr-/- mice.

    PubMed

    Bie, Jinghua; Wang, Jing; Yuan, Quan; Kakiyama, Genta; Ghosh, Siddhartha S; Ghosh, Shobha

    2014-04-01

    The liver plays a central role in the final elimination of cholesterol from the body either as bile acids or as free cholesterol (FC), and lipoprotein-derived cholesterol is the major source of total biliary cholesterol. HDL is the major lipoprotein responsible for removal and transport of cholesterol, mainly as cholesteryl esters (CEs), from the peripheral tissues to the liver. While HDL-FC is rapidly secreted into bile, the fate of HDL-CE remains unclear. We have earlier demonstrated the role of human CE hydrolase (CEH, CES1) in hepatic hydrolysis of HDL-CE and increasing bile acid synthesis, a process dependent on scavenger receptor BI expression. In the present study, we examined the hypothesis that by enhancing the elimination of HDL-CE into bile/feces, liver-specific transgenic expression of CEH will be anti-atherogenic. Increased CEH expression in the liver significantly increased the flux of HDL-CE to bile acids. In the LDLR(-/-) background, this enhanced elimination of cholesterol led to attenuation of diet-induced atherosclerosis with a consistent increase in fecal sterol secretion primarily as bile acids. Taken together with the observed reduction in atherosclerosis by increasing macrophage CEH-mediated cholesterol efflux, these studies establish CEH as an important regulator in enhancing cholesterol elimination and also as an anti-atherogenic target. PMID:24563511

  2. An insect farnesyl phosphatase homologous to the N-terminal domain of soluble epoxide hydrolase

    PubMed Central

    Cao, Li; Zhang, Ping; Grant, David F.

    2009-01-01

    In insects, farnesyl pyrophosphate (FPP) is converted to juvenile hormone (JH) via a conserved pathway consisting of isoprenoid derived metabolites. The first step of this pathway is presumed to be hydrolysis of FPP to farnesol in the ring gland. Based on alignment of putative phosphatases from D. melanogaster with the phosphatase domain of soluble epoxide hydrolase, Phos2680 and Phos15739 with conserved phosphatase motifs were identified, cloned and purified. Both D. melanogaster phosphatases hydrolyzed para-nitrophenyl phosphate, however, Phos15739 also hydrolyzed FPP with a Kcat/Km of 2.1 X 105 M−1s−1. RT-PCR analysis revealed that Phos15739 was expressed in the ring gland and its expression was correlated with JHIII titer during development of D. melanogaster. N-acetyl-S-geranylgeranyl-L-cysteine was found to be a potent inhibitor of Phos15739 with an IC50 value of 4.4 μM. Thus, our data identify Phos15739 as a FPP phosphatase that likely catalyzes the hydrolysis of FPP to farnesol in D. melanogaster. PMID:19168029

  3. Eukaryote to gut bacteria transfer of a glycoside hydrolase gene essential for starch breakdown in plants

    PubMed Central

    Arias, Maria Cecilia; Danchin, Étienne G.J.; Coutinho, Pedro; Henrissat, Bernard; Ball, Steven

    2012-01-01

    Lateral gene transfer (LGT) between bacteria constitutes a strong force in prokaryote evolution, transforming the hierarchical tree of life into a network of relationships between species. In contrast, only a few cases of LGT from eukaryotes to prokaryotes have been reported so far. The distal animal intestine is predominantly a bacterial ecosystem, supplying the host with energy from dietary polysaccharides through carbohydrate-active enzymes absent from its genome. It has been suggested that LGT is particularly important for the human microbiota evolution. Here we show evidence for the first eukaryotic gene identified in multiple gut bacterial genomes. We found in the genome sequence of several gut bacteria, a typically eukaryotic glycoside-hydrolase necessary for starch breakdown in plants. The distribution of this gene is patchy in gut bacteria with presence otherwise detected only in a few environmental bacteria. We speculate that the transfer of this gene to gut bacteria occurred by a sequence of two key LGT events; first, an original eukaryotic gene was transferred probably from Archaeplastida to environmental bacteria specialized in plant polysaccharides degradation and second, the gene was transferred from the environmental bacteria to gut microbes. PMID:22934241

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

    PubMed

    Ogawa, Shintaro; Kunugi, Hiroshi

    2015-01-01

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

  5. Conformational Disorganization within the Active Site of a Recently Evolved Organophosphate Hydrolase Limits Its Catalytic Efficiency.

    PubMed

    Mabbitt, Peter D; Correy, Galen J; Meirelles, Tamara; Fraser, Nicholas J; Coote, Michelle L; Jackson, Colin J

    2016-03-01

    The evolution of new enzymatic activity is rarely observed outside of the laboratory. In the agricultural pest Lucilia cuprina, a naturally occurring mutation (Gly137Asp) in α-esterase 7 (LcαE7) results in acquisition of organophosphate hydrolase activity and confers resistance to organophosphate insecticides. Here, we present an X-ray crystal structure of LcαE7:Gly137Asp that, along with kinetic data, suggests that Asp137 acts as a general base in the new catalytic mechanism. Unexpectedly, the conformation of Asp137 observed in the crystal structure obstructs the active site and is not catalytically productive. Molecular dynamics simulations reveal that alternative, catalytically competent conformers of Asp137 are sampled on the nanosecond time scale, although these states are less populated. Thus, although the mutation introduces the new reactive group responsible for organophosphate detoxification, the catalytic efficiency appears to be limited by conformational disorganization: the frequent sampling of low-energy nonproductive states. This result is consistent with a model of molecular evolution in which initial function-changing mutations can result in enzymes that display only a fraction of their catalytic potential due to conformational disorganization. PMID:26881849

  6. Superfamily-wide portrait of serine hydrolase inhibition achieved by library-versus-library screening.

    PubMed

    Bachovchin, Daniel A; Ji, Tianyang; Li, Weiwei; Simon, Gabriel M; Blankman, Jacqueline L; Adibekian, Alexander; Hoover, Heather; Niessen, Sherry; Cravatt, Benjamin F

    2010-12-01

    Serine hydrolases (SHs) are one of the largest and most diverse enzyme classes in mammals. They play fundamental roles in virtually all physiological processes and are targeted by drugs to treat diseases such as diabetes, obesity, and neurodegenerative disorders. Despite this, we lack biological understanding for most of the 110+ predicted mammalian metabolic SHs, in large part because of a dearth of assays to assess their biochemical activities and a lack of selective inhibitors to probe their function in living systems. We show here that the vast majority (> 80%) of mammalian metabolic SHs can be labeled in proteomes by a single, active site-directed fluorophosphonate probe. We exploit this universal activity-based assay in a library-versus-library format to screen 70+ SHs against 140+ structurally diverse carbamates. Lead inhibitors were discovered for ∼40% of the screened enzymes, including many poorly characterized SHs. Global profiles identified carbamate inhibitors that discriminate among highly sequence-related SHs and, conversely, enzymes that share inhibitor sensitivity profiles despite lacking sequence homology. These findings indicate that sequence relatedness is not a strong predictor of shared pharmacology within the SH superfamily. Finally, we show that lead carbamate inhibitors can be optimized into pharmacological probes that inactivate individual SHs with high specificity in vivo. PMID:21084632

  7. Superfamily-wide portrait of serine hydrolase inhibition achieved by library-versus-library screening

    PubMed Central

    Bachovchin, Daniel A.; Ji, Tianyang; Li, Weiwei; Simon, Gabriel M.; Blankman, Jacqueline L.; Adibekian, Alexander; Hoover, Heather; Niessen, Sherry; Cravatt, Benjamin F.

    2010-01-01

    Serine hydrolases (SHs) are one of the largest and most diverse enzyme classes in mammals. They play fundamental roles in virtually all physiological processes and are targeted by drugs to treat diseases such as diabetes, obesity, and neurodegenerative disorders. Despite this, we lack biological understanding for most of the 110+ predicted mammalian metabolic SHs, in large part because of a dearth of assays to assess their biochemical activities and a lack of selective inhibitors to probe their function in living systems. We show here that the vast majority (> 80%) of mammalian metabolic SHs can be labeled in proteomes by a single, active site-directed fluorophosphonate probe. We exploit this universal activity-based assay in a library-versus-library format to screen 70+ SHs against 140+ structurally diverse carbamates. Lead inhibitors were discovered for ∼40% of the screened enzymes, including many poorly characterized SHs. Global profiles identified carbamate inhibitors that discriminate among highly sequence-related SHs and, conversely, enzymes that share inhibitor sensitivity profiles despite lacking sequence homology. These findings indicate that sequence relatedness is not a strong predictor of shared pharmacology within the SH superfamily. Finally, we show that lead carbamate inhibitors can be optimized into pharmacological probes that inactivate individual SHs with high specificity in vivo. PMID:21084632

  8. Mechanistic Insights into the Rate-Limiting Step in Purine-Specific Nucleoside Hydrolase.

    PubMed

    Chen, Nanhao; Zhao, Yuan; Lu, Jianing; Wu, Ruibo; Cao, Zexing

    2015-07-14

    A full enzymatic catalysis cycle in the inosine-adenosine-guanosine specific nucleoside hydrolase (IAG-NH) was assumed to be comprised of four steps: substrate binding, chemical reaction, base release, and ribose release. Nevertheless, the mechanistic details for the rate-limiting step of the entire enzymatic reaction are still unknown, even though the ribose release was likely to be the most difficult stage. Based on state-of-the-art quantum mechanics and molecular mechanics (QM/MM) molecular dynamics (MD) simulations, the ribose release process can be divided into two steps: "ribose dissociation" and "ribose release". The "ribose dissociation" includes "cleavage" and "exchange" stages, in which a metastable 6-fold intermediate will recover to an 8-fold coordination shell of Ca(2+) as observed in apo- IAG-NH. Extensive random acceleration molecular dynamics and MD simulations have been employed to verify plausible release channels, and the estimated barrier for the rate-determining step of the entire reaction is 13.0 kcal/mol, which is comparable to the experimental value of 16.7 kcal/mol. Moreover, the gating mechanism arising from loop1 and loop2, as well as key residues around the active pocket, has been found to play an important role in manipulating the ribose release. PMID:26575755

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

    PubMed Central

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

    2016-01-01

    Glycoside hydrolases (GHs) play fundamental roles in the decomposition of lignocellulosic biomaterials. Here, we report the full-length structure of a cellulase from Bacillus licheniformis (BlCel5B), a member of the GH5 subfamily 4 that is entirely dependent on its two ancillary modules (Ig-like module and CBM46) for catalytic activity. Using X-ray crystallography, small-angle X-ray scattering and molecular dynamics simulations, we propose that the C-terminal CBM46 caps the distal N-terminal catalytic domain (CD) to establish a fully functional active site via a combination of large-scale multidomain conformational selection and induced-fit mechanisms. The Ig-like module is pivoting the packing and unpacking motions of CBM46 relative to CD in the assembly of the binding subsite. This is the first example of a multidomain GH relying on large amplitude motions of the CBM46 for assembly of the catalytically competent form of the enzyme. PMID:27032335

  10. Immunohistochemical distribution of the cannabinoid receptor 1 and fatty acid amide hydrolase in the dog claustrum.

    PubMed

    Pirone, Andrea; Cantile, Carlo; Miragliotta, Vincenzo; Lenzi, Carla; Giannessi, Elisabetta; Cozzi, Bruno

    2016-07-01

    Cannabinoid receptor 1 (CB1R) and fatty acid amide hydrolase (FAAH) are part of the endocannabinoid system (ECB) which exerts a neuromodulatory activity on different brain functions and plays a key role in neurogenesis. Although many studies have reported FAAH and CB1R expression in the brain of different animal species, to the best of our knowledge they have never been described in the canine claustrum. Claustrum samples, obtained from necropsy of four neurologically normal dogs, were formalin fixed for paraffin embedding. Sections were either stained for morpho-histological analysis or immunostained for CB1R and FAAH. Analysis of adjacent sections incubated with the two antisera showed a complementary labeling pattern in the claustrum, with CB1R antibody staining fibers while anti-FAAH antibody stained cell bodies and the proximal portion of dendrites; this particular anatomical relationship suggests a retrograde endocannabinoid action via CB1R. CB1R and FAAH complementary immunostaining and their cellular localization reported here provide the first anatomical evidence for existence of the ECB in the dog claustrum. PMID:26907575

  11. Molecular and biochemical characterization of juvenile hormone epoxide hydrolase from the silkworm, Bombyx mori.

    PubMed

    Zhang, Qi-Rui; Xu, Wei-Hua; Chen, Fu-Sheng; Li, Sheng

    2005-02-01

    One major route of insect juvenile hormone (JH) degradation is epoxide hydration by JH epoxide hydrolase (JHEH). A full-length cDNA (1536 bp) encoding a microsomal JHEH was isolated from the silkworm, Bombyx mori. Bommo-JHEH cDNA contains an open reading frame encoding a 461-amino acid protein (52 kDa), which reveals a high degree of similarity to the previously reported insect JHEHs. The residues Tyr298, Tyr373, and the HGWP motif corresponding to the oxyanion hole of JHEHs and the residues Asp227, His430, and Glu403 in the catalytic triad are well conserved in Bommo-JHEH. Bommo-JHEH was highly expressed in the fat body, where its mRNA expression pattern was in contrast to the pattern of hemolymph levels of JH during the larval development, suggesting that Bommo-JHEH plays an important role in JH degradation. Recombinant Bommo-JHEH (52 kDa) expressed in Sf9 insect cells was membrane-bound and had a high level of enzyme activity (300-fold over the control activity). This Bommo-JHEH study provides a better understanding of how JH levels are regulated in the domesticated silkworm. PMID:15681225

  12. Destructuring plant biomass: focus on fungal and extremophilic cell wall hydrolases.

    PubMed

    Guerriero, Gea; Hausman, Jean-Francois; Strauss, Joseph; Ertan, Haluk; Siddiqui, Khawar Sohail

    2015-05-01

    The use of plant biomass as feedstock for biomaterial and biofuel production is relevant in the current bio-based economy scenario of valorizing renewable resources. Fungi, which degrade complex and recalcitrant plant polymers, secrete different enzymes that hydrolyze plant cell wall polysaccharides. The present review discusses the current research trends on fungal, as well as extremophilic cell wall hydrolases that can withstand extreme physico-chemical conditions required in efficient industrial processes. Secretomes of fungi from the phyla Ascomycota, Basidiomycota, Zygomycota and Neocallimastigomycota are presented along with metabolic cues (nutrient sensing, coordination of carbon and nitrogen metabolism) affecting their composition. We conclude the review by suggesting further research avenues focused on the one hand on a comprehensive analysis of the physiology and epigenetics underlying cell wall degrading enzyme production in fungi and on the other hand on the analysis of proteins with unknown function and metagenomics of extremophilic consortia. The current advances in consolidated bioprocessing, altered secretory pathways and creation of designer plants are also examined. Furthermore, recent developments in enhancing the activity, stability and reusability of enzymes based on synergistic, proximity and entropic effects, fusion enzymes, structure-guided recombination between homologous enzymes and magnetic enzymes are considered with a view to improving saccharification. PMID:25804821

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

    PubMed

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

    2015-01-01

    In order to optimize the use of agaves for commercial applications, an understanding of fructan metabolism in these species at the molecular and genetic level is essential. Based on transcriptome data, this report describes the identification and molecular characterization of cDNAs and deduced amino acid sequences for genes encoding fructosyltransferases, invertases and fructan exohydrolases (FEH) (enzymes belonging to plant glycoside hydrolase family 32) from four different agave species (A. tequilana, A. deserti, A. victoriae-reginae, and A. striata). Conserved amino acid sequences and a hypervariable domain allowed classification of distinct isoforms for each enzyme type. Notably however neither 1-FFT nor 6-SFT encoding cDNAs were identified. In silico analysis revealed that distinct isoforms for certain enzymes found in a single species, showed different levels and tissue specific patterns of expression whereas in other cases expression patterns were conserved both within the species and between different species. Relatively high levels of in silico expression for specific isoforms of both invertases and fructosyltransferases were observed in floral tissues in comparison to vegetative tissues such as leaves and stems and this pattern was confirmed by Quantitative Real Time PCR using RNA obtained from floral and leaf tissue of A. tequilana. Thin layer chromatography confirmed the presence of fructans with degree of polymerization (DP) greater than DP three in both immature buds and fully opened flowers also obtained from A. tequilana. PMID:26300895

  14. Fatty-acid amide hydrolase polymorphisms and post-traumatic stress disorder after penetrating brain injury

    PubMed Central

    Pardini, M; Krueger, F; Koenigs, M; Raymont, V; Hodgkinson, C; Zoubak, S; Goldman, D; Grafman, J

    2012-01-01

    The past few years have seen an increase in the clinical awareness of post-traumatic stress disorder (PTSD), one of the most disabling and least understood behavioral disorders. Although the biological bases of PTSD are poorly understood, fatty-acid amide hydrolase (FAAH) activity has been linked with arousability and aversive-memories extinction, that is, two key features of PTSD. In this study, we investigated the association between the FAAH genetic polymorphisms and PTSD development and maintenance. We assessed PTSD frequency in a group of male Vietnam war veterans who suffered combat-related penetrating traumatic brain injury, that is, a relatively homogeneous population regarding the nature of the events that led to PTSD. We showed that rs2295633, a single-nucleotide polymorphism of FAAH, was significantly associated with PTSD diagnosis in subjects without lesions in the ventromedial prefrontal cortex. Moreover, the presence of the C allele was associated with more severe re-experiencing of trauma and more negative reported childhood experiences. In conclusion, our data suggest that FAAH has an important role in PTSD through modulation of aversive memories and point to both a novel therapeutic target and a possible risk marker for this condition. PMID:22832737

  15. Regulation of homocysteine metabolism by Mycobacterium tuberculosis S-adenosylhomocysteine hydrolase

    PubMed Central

    Singhal, Anshika; Arora, Gunjan; Sajid, Andaleeb; Maji, Abhijit; Bhat, Ajay; Virmani, Richa; Upadhyay, Sandeep; Nandicoori, Vinay K.; Sengupta, Shantanu; Singh, Yogendra

    2013-01-01

    Mycobacterium tuberculosis modulates expression of various metabolism-related genes to adapt in the adverse host environment. The gene coding for M. tuberculosis S-adenosylhomocysteine hydrolase (Mtb-SahH) is essential for optimal growth and the protein product is involved in intermediary metabolism. However, the relevance of SahH in mycobacterial physiology is unknown. In this study, we analyze the role of Mtb-SahH in regulating homocysteine concentration in surrogate host Mycobacterium smegmatis. Mtb-SahH catalyzes reversible hydrolysis of S-adenosylhomocysteine to homocysteine and adenosine and we demonstrate that the conserved His363 residue is critical for bi-directional catalysis. Mtb-SahH is regulated by serine/threonine phosphorylation of multiple residues by M. tuberculosis PknB. Major phosphorylation events occur at contiguous residues Thr219, Thr220 and Thr221, which make pivotal contacts with cofactor NAD+. Consequently, phosphorylation negatively modulates affinity of enzyme towards NAD+ as well as SAH-synthesis. Thr219, Thr220 and Thr221 are essential for enzyme activity, and therefore, responsible for SahH-mediated regulation of homocysteine. PMID:23877358

  16. Two-way traffic of glycoside hydrolase family 18 processive chitinases on crystalline chitin

    NASA Astrophysics Data System (ADS)

    Igarashi, Kiyohiko; Uchihashi, Takayuki; Uchiyama, Taku; Sugimoto, Hayuki; Wada, Masahisa; Suzuki, Kazushi; Sakuda, Shohei; Ando, Toshio; Watanabe, Takeshi; Samejima, Masahiro

    2014-06-01

    Processivity refers to the ability of synthesizing, modifying and degrading enzymes to catalyse multiple successive cycles of reaction with polymeric substrates without disengaging from the substrates. Since biomass polysaccharides, such as chitin and cellulose, often form recalcitrant crystalline regions, their degradation is highly dependent on the processivity of degrading enzymes. Here we employ high-speed atomic force microscopy to directly visualize the movement of two processive glycoside hydrolase family 18 chitinases (ChiA and ChiB) from the chitinolytic bacterium Serratia marcescens on crystalline β-chitin. The half-life of processive movement and the velocity of ChiA are larger than those of ChiB, suggesting that asymmetric subsite architecture determines both the direction and the magnitude of processive degradation of crystalline polysaccharides. The directions of processive movements of ChiA and ChiB are observed to be opposite. The molecular mechanism of the two-way traffic is discussed, including a comparison with the processive cellobiohydrolases of the cellulolytic system.

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

    PubMed Central

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

    2014-01-01

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

  18. Altering the substrate specificity of methyl parathion hydrolase with directed evolution.

    PubMed

    Ng, Tee-Kheang; Gahan, Lawrence R; Schenk, Gerhard; Ollis, David L

    2015-05-01

    Many organophosphates (OPs) are used as pesticides in agriculture. They pose a severe health hazard due to their inhibitory effect on acetylcholinesterase. Therefore, detoxification of water and soil contaminated by OPs is important. Metalloenzymes such as methyl parathion hydrolase (MPH) from Pseudomonas sp. WBC-3 hold great promise as bioremediators as they are able to hydrolyze a wide range of OPs. MPH is highly efficient towards methyl parathion (1 × 10(6) s(-1) M(-1)), but its activity towards other OPs is more modest. Thus, site saturation mutagenesis (SSM) and DNA shuffling were performed to find mutants with improved activities on ethyl paraxon (6.1 × 10(3) s(-1) M(-1)). SSM was performed on nine residues lining the active site. Several mutants with modest activity enhancement towards ethyl paraoxon were isolated and used as templates for DNA shuffling. Ultimately, 14 multiple-site mutants with enhanced activity were isolated. One mutant, R2F3, exhibited a nearly 100-fold increase in the kcat/Km value for ethyl paraoxon (5.9 × 10(5) s(-1) M(-1)). These studies highlight the 'plasticity' of the MPH active site that facilitates the fine-tuning of its active site towards specific substrates with only minor changes required. MPH is thus an ideal candidate for the development of an enzyme-based bioremediation system. PMID:25797441

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

    PubMed

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

    2012-07-27

    In the advancement of green syntheses and sustainable reactions, enzymatic biocatalysis offers extremely high reaction rates and selectivity that goes far beyond the reach of chemical catalysts; however, these enzymes suffer from typical environmental constraints, e.g. operational temperature, pH and tolerance to oxidative environments. A common hydrolase enzyme, diisopropylfluorophosphatase (DFPase, EC 3.1.8.2), has demonstrated a pronounced efficacy for the hydrolysis of a variety of substrates for potential toxin remediation, but suffers from the aforementioned limitations. As a means to enhance DFPase's stability in oxidative environments, enzymatic covalent immobilization within the polymeric matrix of poly(propylene sulfide) (PPS) nanoparticles was performed. By modifying the enzyme's exposed lysine residues via thiolation, DFPase is utilized as a comonomer/crosslinker in a mild emulsion polymerization. The resultant polymeric polysulfide shell acts as a 'sacrificial barrier' by first oxidizing to polysulfoxides and polysulfones, rendering DFPase in an active state. DFPase-PPS nanoparticles thus retain activity upon exposure to as high as 50 parts per million (ppm) of hypochlorous acid (HOCl), while native DFPase is observed as inactive at 500 parts per billion (ppb). This trend is also confirmed by enzyme-generated (chloroperoxidase (CPO), EC 1.11.1.10) reactive oxygen species (ROS) including both HOCl (3 ppm) and ClO(2) (100 ppm). PMID:22743846

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

    PubMed Central

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

    2015-01-01

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

  1. Soluble Epoxide Hydrolase as a Potential Key Factor for Human Prenatal Development.

    PubMed

    Cizkova, Katerina; Rajdova, Aneta; Ehrmann, Jiri

    2016-01-01

    Soluble epoxide hydrolase (sEH) converts highly active epoxyeicosatrienoic acids (EETs) generated by cytochrome P450 (CYP) epoxygenases from arachidonic acid to less active dihydroxyeicosatrienoic acids. Because of the role of EETs in processes potentially relevant to the development of organisms, EETs could be suggested as potential morphogens. Unfortunately, only little is known about sEH expression during human intrauterine development (IUD). We investigated the spatio-temporal expression pattern of sEH in human embryonic/foetal intestines, liver and kidney from the 6th to the 20th week of IUD by two-step immunohistochemistry. sEH was expressed during the whole tested period of prenatal development and its level of expression remained more or less the same during the estimated period of IUD. Distribution of CYP epoxygenases and sEH in the intestinal epithelium and the nephrogenic zone of the kidney suggests an influence of EETs on cell proliferation and differentiation and, consequently, on the development of intestines and kidney. Thus, alterations in the strict spatio-temporal pattern of expression of CYP epoxygenases and/or sEH during human prenatal development by xenobiotics could have a harmful impact for developing organisms. PMID:27144772

  2. Crystal structures of glycoside hydrolase family 3 β-glucosidase 1 from Aspergillus aculeatus.

    PubMed

    Suzuki, Kentaro; Sumitani, Jun-ichi; Nam, Young-Woo; Nishimaki, Toru; Tani, Shuji; Wakagi, Takayoshi; Kawaguchi, Takashi; Fushinobu, Shinya

    2013-06-01

    GH3 (glycoside hydrolase family 3) BGLs (β-glucosidases) from filamentous fungi have been widely and commercially used for the supplementation of cellulases. AaBGL1 (Aspergillus aculeatus BGL1) belongs to the GH3 and shows high activity towards cellooligosaccharides up to high degree of polymerization. In the present study we determined the crystal structure of AaBGL1. In addition to the substrate-free structure, the structures of complexes with glucose and various inhibitors were determined. The structure of AaBGL1 is highly glycosylated with 88 monosaccharides (18 N-glycan chains) in the dimer. The largest N-glycan chain comprises ten monosaccharides and is one of the largest glycans ever observed in protein crystal structures. A prominent insertion region exists in a fibronectin type III domain, and this region extends to cover a wide surface area of the enzyme. The subsite +1 of AaBGL1 is highly hydrophobic. Three aromatic residues are present at subsite +1 and are located in short loop regions that are uniquely present in this enzyme. There is a long cleft extending from subsite +1, which appears to be suitable for binding long cellooligosaccharides. The crystal structures of AaBGL1 from the present study provide an important structural basis for the technical improvement of enzymatic cellulosic biomass conversion. PMID:23537284

  3. Colorectal polyp type and the association with charred meat consumption, smoking, and microsomal epoxide hydrolase polymorphisms.

    PubMed

    Burnett-Hartman, Andrea N; Newcomb, Polly A; Mandelson, Margaret T; Adams, Scott V; Wernli, Karen J; Shadman, Mazyar; Wurscher, Michelle A; Makar, Karen W

    2011-01-01

    We determined the association between charred meat consumption, cigarette smoking, microsomal epoxide hydrolase (mEH) polymorphisms (rs1051740 and rs2234922), and colorectal adenomas and hyperplastic polyps (HPs) and explored gene-environment interactions. Men and women with colorectal adenomas (n = 519), HPs (n = 691), or concurrently with both types of polyps (n = 227) and polyp-free controls (n = 772) receiving a colonoscopy from December 2004 to September 2007 were recruited. Participants completed telephone interviews and provided buccal cell samples; genotyping of mEH was completed using Taqman assays. We conducted polytomous regression and calculated odd ratios (OR) and 95% confidence intervals. Interactions were evaluated using Wald chi-square tests. Consumption of >3 servings of charred meat per week was associated with distal HPs (OR = 2.0, 1.2-3.4) but not adenomas nor either type of proximal polyp. Heavy cigarette smoking (≥ 22 pack-years) was associated with an increased risk for colorectal adenomas (OR = 1.7, 95% CI: 1.2-2.4), HPs (OR = 2.4, 95% CI: 1.7-3.3), and both types (OR = 2.8, 95% CI: 1.8-4.3) with the strongest association for distal polyps. There was no association between mEH genotype and colorectal polyps, nor were any statistically significant gene-environment interactions identified. Future investigation of BaP exposure and colorectal neoplasia should analyze whether associations are dependent upon anatomic location. PMID:21598178

  4. Induction of epoxide hydrolase, glucuronosyl transferase, and sulfotransferase by phenethyl isothiocyanate in male Wistar albino rats.

    PubMed

    Abdull Razis, Ahmad Faizal; Mohd Noor, Noramaliza; Konsue, Nattaya

    2014-01-01

    Phenethyl isothiocyanate (PEITC) is an isothiocyanate found in watercress as the glucosinolate (gluconasturtiin). The isothiocyanate is converted from the glucosinolate by intestinal microflora or when contacted with myrosinase during the chopping and mastication of the vegetable. PEITC manifested protection against chemically-induced cancers in various tissues. A potential mechanism of chemoprevention is by modulating the metabolism of carcinogens so as to promote deactivation. The principal objective of this study was to investigate in rats the effect of PEITC on carcinogen-metabolising enzyme systems such as sulfotransferase (SULT), N-acetyltransferase (NAT), glucuronosyl transferase (UDP), and epoxide hydrolase (EH) following exposure to low doses that simulate human dietary intake. Rats were fed for 2 weeks diets supplemented with PEITC at 0.06 µmol/g (low dose, i.e., dietary intake), 0.6 µmol/g (medium dose), and 6.0 µmol/g (high dose), and the enzymes were monitored in rat liver. At the Low dose, no induction of the SULT, NAT, and EH was noted, whereas UDP level was elevated. At the Medium dose, only SULT level was increased, whereas at the High dose marked increase in EH level was observed. It is concluded that PEITC modulates carcinogen-metabolising enzyme systems at doses reflecting human intake thus elucidating the mechanism of its chemoprevention. PMID:24592387

  5. Human Carboxymethylenebutenolidase as a Bioactivating Hydrolase of Olmesartan Medoxomil in Liver and Intestine

    PubMed Central

    Ishizuka, Tomoko; Fujimori, Izumi; Kato, Mitsunori; Noji-Sakikawa, Chisa; Saito, Motoko; Yoshigae, Yasushi; Kubota, Kazuishi; Kurihara, Atsushi; Izumi, Takashi; Ikeda, Toshihiko; Okazaki, Osamu

    2010-01-01

    Olmesartan medoxomil (OM) is a prodrug type angiotensin II type 1 receptor antagonist widely prescribed as an antihypertensive agent. Herein, we describe the identification and characterization of the OM bioactivating enzyme that hydrolyzes the prodrug and converts to its pharmacologically active metabolite olmesartan in human liver and intestine. The protein was purified from human liver cytosol by successive column chromatography and was identified by mass spectrometry to be a carboxymethylenebutenolidase (CMBL) homolog. Human CMBL, whose endogenous function has still not been reported, is a human homolog of Pseudomonas dienelactone hydrolase involved in the bacterial halocatechol degradation pathway. The ubiquitous expression of human CMBL gene transcript in various tissues was observed. The recombinant human CMBL expressed in mammalian cells was clearly shown to activate OM. By comparing the enzyme kinetics and chemical inhibition properties between the recombinant protein and human tissue preparations, CMBL was demonstrated to be the primary OM bioactivating enzyme in the liver and intestine. The recombinant CMBL also converted other prodrugs having the same ester structure as OM, faropenem medoxomil and lenampicillin, to their active metabolites. CMBL exhibited a unique sensitivity to chemical inhibitors, thus, being distinguishable from other known esterases. Site-directed mutagenesis on the putative active residue Cys132 of the recombinant CMBL caused a drastic reduction of the OM-hydrolyzing activity. We report for the first time that CMBL serves as a key enzyme in the bioactivation of OM, hydrolyzing the ester bond of the prodrug type xenobiotics. PMID:20177059

  6. Human carboxymethylenebutenolidase as a bioactivating hydrolase of olmesartan medoxomil in liver and intestine.

    PubMed

    Ishizuka, Tomoko; Fujimori, Izumi; Kato, Mitsunori; Noji-Sakikawa, Chisa; Saito, Motoko; Yoshigae, Yasushi; Kubota, Kazuishi; Kurihara, Atsushi; Izumi, Takashi; Ikeda, Toshihiko; Okazaki, Osamu

    2010-04-16

    Olmesartan medoxomil (OM) is a prodrug type angiotensin II type 1 receptor antagonist widely prescribed as an antihypertensive agent. Herein, we describe the identification and characterization of the OM bioactivating enzyme that hydrolyzes the prodrug and converts to its pharmacologically active metabolite olmesartan in human liver and intestine. The protein was purified from human liver cytosol by successive column chromatography and was identified by mass spectrometry to be a carboxymethylenebutenolidase (CMBL) homolog. Human CMBL, whose endogenous function has still not been reported, is a human homolog of Pseudomonas dienelactone hydrolase involved in the bacterial halocatechol degradation pathway. The ubiquitous expression of human CMBL gene transcript in various tissues was observed. The recombinant human CMBL expressed in mammalian cells was clearly shown to activate OM. By comparing the enzyme kinetics and chemical inhibition properties between the recombinant protein and human tissue preparations, CMBL was demonstrated to be the primary OM bioactivating enzyme in the liver and intestine. The recombinant CMBL also converted other prodrugs having the same ester structure as OM, faropenem medoxomil and lenampicillin, to their active metabolites. CMBL exhibited a unique sensitivity to chemical inhibitors, thus, being distinguishable from other known esterases. Site-directed mutagenesis on the putative active residue Cys(132) of the recombinant CMBL caused a drastic reduction of the OM-hydrolyzing activity. We report for the first time that CMBL serves as a key enzyme in the bioactivation of OM, hydrolyzing the ester bond of the prodrug type xenobiotics. PMID:20177059

  7. Development of a High Throughput Platform for Screening Glycoside Hydrolases Based on Oxime-NIMS

    PubMed Central

    Deng, Kai; Guenther, Joel M.; Gao, Jian; Bowen, Benjamin P.; Tran, Huu; Reyes-Ortiz, Vimalier; Cheng, Xiaoliang; Sathitsuksanoh, Noppadon; Heins, Richard; Takasuka, Taichi E.; Bergeman, Lai F.; Geertz-Hansen, Henrik; Deutsch, Samuel; Loqué, Dominique; Sale, Kenneth L.; Simmons, Blake A.; Adams, Paul D.; Singh, Anup K.; Fox, Brian G.; Northen, Trent R.

    2015-01-01

    Cost-effective hydrolysis of biomass into sugars for biofuel production requires high-performance low-cost glycoside hydrolase (GH) cocktails that are active under demanding process conditions. Improving the performance of GH cocktails depends on knowledge of many critical parameters, including individual enzyme stabilities, optimal reaction conditions, kinetics, and specificity of reaction. With this information, rate- and/or yield-limiting reactions can be potentially improved through substitution, synergistic complementation, or protein engineering. Given the wide range of substrates and methods used for GH characterization, it is difficult to compare results across a myriad of approaches to identify high performance and synergistic combinations of enzymes. Here, we describe a platform for systematic screening of GH activities using automatic biomass handling, bioconjugate chemistry, robotic liquid handling, and nanostructure-initiator mass spectrometry (NIMS). Twelve well-characterized substrates spanning the types of glycosidic linkages found in plant cell walls are included in the experimental workflow. To test the application of this platform and substrate panel, we studied the reactivity of three engineered cellulases and their synergy of combination across a range of reaction conditions and enzyme concentrations. We anticipate that large-scale screening using the standardized platform and substrates will generate critical datasets to enable direct comparison of enzyme activities for cocktail design. PMID:26528471

  8. Characterization of a highly thermostable glycoside hydrolase family 10 xylanase from Malbranchea cinnamomea.

    PubMed

    Fan, Guangsen; Yang, Shaoqing; Yan, Qiaojuan; Guo, Yu; Li, Yanxiao; Jiang, Zhengqiang

    2014-09-01

    A thermostable xylanase (McXyn10) from the thermophilic fungus Malbranchea cinnamomea strain S168 was purified and biochemically characterized. The enzyme was purified to homogeneity with a molecular mass of 43.5 kDa on SDS-PAGE. The optimal pH and temperature of the purified enzyme were pH 6.5 and 80°C, respectively. The enzyme showed a broad range of pH stability (pH 4.0-10.5), and was stable up to 70°C with a thermal denaturing half life of 76.0 min. The enzyme exhibited strict specificity for various xylans as substrates, but displayed no activity toward other tested polysaccharides. McXyn10 hydrolyzed birchwood xylan, beechwood xylan and oat-spelt xylan, yielded mainly xylobiose, xylotriose and xylooligosaccharides with degree of polymerization (DP) above 5, while yielded xylobiose from xylotriose and xylotetraose. The xylanase gene was further cloned. It had an open reading frame of 1191 bp with two introns. The deduced amino acid sequence of the gene showed highest identity (58%) with a glycoside hydrolase family 10 xylanase from Aureobasidium pullulans. PMID:25058752

  9. Multi-functional glycoside hydrolase: Blon_0625 from Bifidobacterium longum subsp. infantis ATCC 15697.

    PubMed

    Matsumoto, Takuya; Shimada, Shota; Hata, Yuto; Tanaka, Tsutomu; Kondo, Akihiko

    2015-01-01

    We here describe a unique β-D-glucosidase (BGL; Blon_0625) derived from Bifidobacterium longum subsp. infantis ATCC 15697. The Blon_0625 gene was expressed by recombinant Escherichia coli. Purified recombinant Blon_0625 retains hydrolyzing activity against both p-nitrophenyl-β-D-glucopyranoside (pNPG; 17.3±0.24Umg(-1)) and p-nitrophenyl-β-D-xylopyranoside (pNPX; 16.7±0.32Umg(-1)) at pH 6.0, 30°C. To best of our knowledge, no previously described BGL retains the same level of both pNPGase and pNPXase activity. Furthermore, Blon_0625 also retains the activity against 4-nitrophenyl-α-l-arabinofranoside (pNPAf; 5.6±0.09Umg(-1)). In addition, the results of the degradation of phosphoric acid swollen cellulose (PASC) or xylan using endoglucanase from Thermobifida fusca YX (Tfu_0901) or xylanase from Kitasatospora setae KM-6054 (KSE_59480) show that Blon_0625 acts as a BGL and as a β-D-xylosidase (XYL) for hydrolyzing oligosaccharides. These results clearly indicate that Blon_0625 is a multi-functional glycoside hydrolase which retains the activity of BGL, XYL, and also α-l-arabinofuranosidase. Therefore, the utilization of multi-functional Blon_0625 may contribute to facilitating the efficient degradation of lignocellulosic materials and help enhance bioconversion processes. PMID:25435500

  10. Direct production of ethanol from neoagarobiose using recombinant yeast that secretes α-neoagarooligosaccharide hydrolase.

    PubMed

    Syazni; Yanagisawa, Mitsunori; Kasuu, Masahiro; Ariga, Osamu; Nakasaki, Kiyohiko

    2016-04-01

    An α-neoagarooligosaccharide hydrolase, AgaNash, was purified from Cellvibrio sp. OA-2007, which utilizes agarose as a substrate. The agaNash gene, which encodes AgaNash, was obtained by comparing the N-terminal amino acid sequence of AgaNash with that deduced from the nucleotide sequence of the full-length OA-2007 genome. The agaNash gene combined with the Saccharomyces cerevisiae signal peptide α-mating factor was transformed into the YPH499 strain of S. cerevisiae to generate YPH499/pTEF-MF-agaNash, and the recombinant yeast was confirmed to produce AgaNash, though it was mainly retained within the recombinant cell. To enhance AgaNash secretion from the cell, the signal peptide was replaced with a combination of the signal peptide and a threonine- and serine-rich tract (T-S region) of the S. diastaticus STA1 gene. The new recombinant yeast, YPH499/pTEF-STA1SP-agaNash, was demonstrated to secrete AgaNash and hydrolyze neoagarobiose with an efficiency of as high as 84%, thereby producing galactose, which is a fermentable sugar for the yeast, and ethanol, at concentrations of up to 1.8 g/L, directly from neoagarobiose. PMID:26920485

  11. Discovery and molecular basis of potent noncovalent inhibitors of fatty acid amide hydrolase (FAAH)

    PubMed Central

    Min, Xiaoshan; Thibault, Stephen T.; Porter, Amy C.; Gustin, Darin J.; Carlson, Timothy J.; Xu, Haoda; Lindstrom, Michelle; Xu, Guifen; Uyeda, Craig; Ma, Zhihua; Li, Yihong; Kayser, Frank; Walker, Nigel P. C.; Wang, Zhulun

    2011-01-01

    Fatty acid amide hydrolase (FAAH), an amidase-signature family member, is an integral membrane enzyme that degrades lipid amides including the endogenous cannabinoid anandamide and the sleep-inducing molecule oleamide. Both genetic knock out and pharmacological administration of FAAH inhibitors in rodent models result in analgesic, anxiolytic, and antiinflammatory phenotypes. Targeting FAAH activity, therefore, presents a promising new therapeutic strategy for the treatment of pain and other neurological-related or inflammatory disorders. Nearly all FAAH inhibitors known to date attain their binding potency through a reversible or irreversible covalent modification of the nucleophile Ser241 in the unusual Ser-Ser-Lys catalytic triad. Here, we report the discovery and mechanism of action of a series of ketobenzimidazoles as unique and potent noncovalent FAAH inhibitors. Compound 2, a representative of these ketobenzimidazoles, was designed from a series of ureas that were identified from high-throughput screening. While urea compound 1 is characterized as an irreversible covalent inhibitor, the cocrystal structure of FAAH complexed with compound 2 reveals that these ketobenzimidazoles, though containing a carbonyl moiety, do not covalently modify Ser241. These inhibitors achieve potent inhibition of FAAH activity primarily from shape complementarity to the active site and through numerous hydrophobic interactions. These noncovalent compounds exhibit excellent selectivity and good pharmacokinetic properties. The discovery of this distinctive class of inhibitors opens a new avenue for modulating FAAH activity through nonmechanism-based inhibition. PMID:21502526

  12. Effects of a Soluble Epoxide Hydrolase Inhibitor on Lipopolysaccharide-Induced Acute Lung Injury in Mice

    PubMed Central

    Yang, Liu-Qing; Ma, Yong-Bo

    2016-01-01

    Objectives Inflammation plays a key role in the pathogenesis of acute lung injury (ALI). Soluble epoxide hydrolase (sEH) is suggested as a vital pharmacologic target for inflammation. In this study, we determined whether a sEH inhibitor, AUDA, exerts lung protection in lipopolysaccharide (LPS)-induced ALI in mice. Methods Male BALB/c mice were randomized to receive AUDA or vehicle intraperitoneal injection 4 h after LPS or phosphate buffered saline (PBS) intratracheal instillation. Samples were harvested 24 h post LPS or PBS administration. Results AUDA administration decreased the pulmonary levels of monocyte chemoattractant protein (MCP)-1 and tumor necrosis factor (TNF)-α. Improvement of oxygenation and lung edema were observed in AUDA treated group. AUDA significantly inhibited sEH activity, and elevated epoxyeicosatrienoic acids (EETs) levels in lung tissues. Moreover, LPS induced the activation of nuclear factor (NF)-κB was markedly dampened in AUDA treated group. Conclusion Administration of AUDA after the onset of LPS-induced ALI increased pulmonary levels of EETs, and ameliorated lung injury. sEH is a potential pharmacologic target for ALI. PMID:27490848

  13. An efficient thermostable organophosphate hydrolase and its application in pesticide decontamination.

    PubMed

    Del Giudice, Immacolata; Coppolecchia, Rossella; Merone, Luigia; Porzio, Elena; Carusone, Teresa Maria; Mandrich, Luigi; Worek, Franz; Manco, Giuseppe

    2016-04-01

    In vitro evolution of enzymes represents a powerful device to evolve new or to improve weak enzymatic functions. In the present work a semi-rational engineering approach has been used to design an efficient and thermostable organophosphate hydrolase, starting from a lactonase scaffold (SsoPox from Sulfolobus solfataricus). In particular, by in vitro evolution of the SsoPox ancillary promiscuous activity, the triple mutant C258L/I261F/W263A has been obtained which, retaining its inherent stability, showed an enhancement of its hydrolytic activity on paraoxon up to 300-fold, achieving absolute values of catalytic efficiency up to 10(5) M(-1) s(-1). The kinetics and structural determinants of this enhanced activity were thoroughly investigated and, in order to evaluate its potential biotechnological applications, the mutant was tested in formulations of different solvents (methanol or ethanol) or detergents (SDS or a commercial soap) for the cleaning of pesticide-contaminated surfaces. PMID:26416557

  14. Crystallization and preliminary X-ray diffraction analysis of tetrathionate hydrolase from Acidithiobacillus ferrooxidans

    PubMed Central

    Kanao, Tadayoshi; Kosaka, Megumi; Yoshida, Kyoya; Nakayama, Hisayuki; Tamada, Taro; Kuroki, Ryota; Yamada, Hidenori; Takada, Jun; Kamimura, Kazuo

    2013-01-01

    Tetrathionate hydrolase (4THase) from the iron- and sulfur-oxidizing bacterium Acidithiobacillus ferrooxidans catalyses the disproportionate hydrolysis of tetrathionate to elemental sulfur, thiosulfate and sulfate. The gene encoding 4THase (Af-tth) was expressed as inclusion bodies in recombinant Escherichia coli. Recombinant Af-Tth was activated by refolding under acidic conditions and was then purified to homogeneity. The recombinant protein was crystallized in 20 mM glycine buffer pH 10 containing 50 mM sodium chloride and 33%(v/v) PEG 1000 using the hanging-drop vapour-diffusion method. The crystal was a hexagonal cylinder with dimensions of 0.2 × 0.05 × 0.05 mm. X-ray crystallographic analysis showed that the crystal diffracted to 2.15 Å resolution and belongs to space group P31 or P32, with unit-cell parameters a = b = 92.1, c = 232.6 Å. PMID:23722856

  15. Identification and Characterization of a Periplasmic Aminoacyl-phosphatidylglycerol Hydrolase Responsible for Pseudomonas aeruginosa Lipid Homeostasis*

    PubMed Central

    Arendt, Wiebke; Groenewold, Maike K.; Hebecker, Stefanie; Dickschat, Jeroen S.; Moser, Jürgen

    2013-01-01

    Specific aminoacylation of the phospholipid phosphatidylglycerol (PG) with alanine (or with lysine) was shown to render various organisms less susceptible to antimicrobial agents and environmental stresses. In this study, we make use of the opportunistic pathogen Pseudomonas aeruginosa to decode ORF PA0919-dependent lipid homeostasis. Analysis of the polar lipid content of the deletion mutant ΔPA0919 indicated significantly enlarged levels of alanyl-PG. The resulting phenotype manifested an increased susceptibility to several antimicrobial compounds when compared with the wild type. A pH-dependent PA0919 promoter located within the upstream gene PA0920 was identified. Localization experiments demonstrated that the PA0919 protein is anchored to the periplasmic surface of the inner bacterial membrane. The recombinant overproduction of wild type and several site-directed mutant proteins in the periplasm of Escherichia coli facilitated a detailed in vitro analysis of the enzymatic PA0919 function. A series of artificial substrates (p-nitrophenyl esters of various amino acids/aliphatic acids) indicated enzymatic hydrolysis of the alanine, glycine, or lysine moiety of the respective ester substrates. Our final in vitro activity assay in the presence of radioactively labeled alanyl-PG then revealed hydrolysis of the aminoacyl linkage, resulting in the formation of alanine and PG. Consequently, PA0919 was termed alanyl-PG hydrolase. The elucidated enzymatic activity implies a new regulatory circuit for the appropriate tuning of cellular alanyl-PG concentrations. PMID:23792962

  16. Rational Design of Potent and Selective Inhibitors of an Epoxide Hydrolase Virulence Factor from Pseudomonas aeruginosa.

    PubMed

    Kitamura, Seiya; Hvorecny, Kelli L; Niu, Jun; Hammock, Bruce D; Madden, Dean R; Morisseau, Christophe

    2016-05-26

    The virulence factor cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif) is secreted by Pseudomonas aeruginosa and is the founding member of a distinct class of epoxide hydrolases (EHs) that triggers the catalysis-dependent degradation of the CFTR. We describe here the development of a series of potent and selective Cif inhibitors by structure-based drug design. Initial screening revealed 1a (KB2115), a thyroid hormone analog, as a lead compound with low micromolar potency. Structural requirements for potency were systematically probed, and interactions between Cif and 1a were characterized by X-ray crystallography. On the basis of these data, new compounds were designed to yield additional hydrogen bonding with residues of the Cif active site. From this effort, three compounds were identified that are 10-fold more potent toward Cif than our first-generation inhibitors and have no detectable thyroid hormone-like activity. These inhibitors will be useful tools to study the pathological role of Cif and have the potential for clinical application. PMID:27120257

  17. Synthesis of Phenoxyacyl-Ethanolamides and Their Effects on Fatty Acid Amide Hydrolase Activity*

    PubMed Central

    Faure, Lionel; Nagarajan, Subbiah; Hwang, Hyeondo; Montgomery, Christa L.; Khan, Bibi Rafeiza; John, George; Koulen, Peter; Blancaflor, Elison B.; Chapman, Kent D.

    2014-01-01

    N-Acylethanolamines (NAEs) are involved in numerous biological activities in plant and animal systems. The metabolism of these lipids by fatty acid amide hydrolase (FAAH) is a key regulatory point in NAE signaling activity. Several active site-directed inhibitors of FAAH have been identified, but few compounds have been described that enhance FAAH activity. Here we synthesized two sets of phenoxyacyl-ethanolamides from natural products, 3-n-pentadecylphenolethanolamide and cardanolethanolamide, with structural similarity to NAEs and characterized their effects on the hydrolytic activity of FAAH. Both compounds increased the apparent Vmax of recombinant FAAH proteins from both plant (Arabidopsis) and mammalian (Rattus) sources. These NAE-like compounds appeared to act by reducing the negative feedback regulation of FAAH activity by free ethanolamine. Both compounds added to seedlings relieved, in part, the negative growth effects of exogenous NAE12:0. Cardanolethanolamide reduced neuronal viability and exacerbated oxidative stress-mediated cell death in primary cultured neurons at nanomolar concentrations. This was reversed by FAAH inhibitors or exogenous NAE substrate. Collectively, our data suggest that these phenoxyacyl-ethanolamides act to enhance the activity of FAAH and may stimulate the turnover of NAEs in vivo. Hence, these compounds might be useful pharmacological tools for manipulating FAAH-mediated regulation of NAE signaling in plants or animals. PMID:24558037

  18. Synthesis of phenoxyacyl-ethanolamides and their effects on fatty acid amide hydrolase activity.

    PubMed

    Faure, Lionel; Nagarajan, Subbiah; Hwang, Hyeondo; Montgomery, Christa L; Khan, Bibi Rafeiza; John, George; Koulen, Peter; Blancaflor, Elison B; Chapman, Kent D

    2014-03-28

    N-Acylethanolamines (NAEs) are involved in numerous biological activities in plant and animal systems. The metabolism of these lipids by fatty acid amide hydrolase (FAAH) is a key regulatory point in NAE signaling activity. Several active site-directed inhibitors of FAAH have been identified, but few compounds have been described that enhance FAAH activity. Here we synthesized two sets of phenoxyacyl-ethanolamides from natural products, 3-n-pentadecylphenolethanolamide and cardanolethanolamide, with structural similarity to NAEs and characterized their effects on the hydrolytic activity of FAAH. Both compounds increased the apparent Vmax of recombinant FAAH proteins from both plant (Arabidopsis) and mammalian (Rattus) sources. These NAE-like compounds appeared to act by reducing the negative feedback regulation of FAAH activity by free ethanolamine. Both compounds added to seedlings relieved, in part, the negative growth effects of exogenous NAE12:0. Cardanolethanolamide reduced neuronal viability and exacerbated oxidative stress-mediated cell death in primary cultured neurons at nanomolar concentrations. This was reversed by FAAH inhibitors or exogenous NAE substrate. Collectively, our data suggest that these phenoxyacyl-ethanolamides act to enhance the activity of FAAH and may stimulate the turnover of NAEs in vivo. Hence, these compounds might be useful pharmacological tools for manipulating FAAH-mediated regulation of NAE signaling in plants or animals. PMID:24558037

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

    NASA Astrophysics Data System (ADS)

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

    2012-07-01

    In the advancement of green syntheses and sustainable reactions, enzymatic biocatalysis offers extremely high reaction rates and selectivity that goes far beyond the reach of chemical catalysts; however, these enzymes suffer from typical environmental constraints, e.g. operational temperature, pH and tolerance to oxidative environments. A common hydrolase enzyme, diisopropylfluorophosphatase (DFPase, EC 3.1.8.2), has demonstrated a pronounced efficacy for the hydrolysis of a variety of substrates for potential toxin remediation, but suffers from the aforementioned limitations. As a means to enhance DFPase’s stability in oxidative environments, enzymatic covalent immobilization within the polymeric matrix of poly(propylene sulfide) (PPS) nanoparticles was performed. By modifying the enzyme’s exposed lysine residues via thiolation, DFPase is utilized as a comonomer/crosslinker in a mild emulsion polymerization. The resultant polymeric polysulfide shell acts as a ‘sacrificial barrier’ by first oxidizing to polysulfoxides and polysulfones, rendering DFPase in an active state. DFPase-PPS nanoparticles thus retain activity upon exposure to as high as 50 parts per million (ppm) of hypochlorous acid (HOCl), while native DFPase is observed as inactive at 500 parts per billion (ppb). This trend is also confirmed by enzyme-generated (chloroperoxidase (CPO), EC 1.11.1.10) reactive oxygen species (ROS) including both HOCl (3 ppm) and ClO2 (100 ppm).

  20. Rapid purification of cytosolic epoxide hydrolase from normal and clofibrate-treated animals by affinity chromatography.

    PubMed Central

    Prestwich, G D; Hammock, B D

    1985-01-01

    Epoxide hydrolase from liver cytosol (cEH) of both normal and clofibrate-treated mice can be bioselectively adsorbed onto an affinity column prepared from epoxy-activated Sepharose and 7-methoxycitronellyl thiol. The free ligand is a modest inhibitor of cEH (I50, approximately equal to 3 X 10(-4) M) and lacks the epoxide function necessary for it to be turned over as a substrate. This study demonstrates that a methoxy group can be used to mimic an oxirane in a vertebrate system. Bioselective elution of cEH can be accomplished with several chalcone oxides, which are selective potent inhibitors (I50, 1-50 X 10(-7) M), and activity can be recovered by dialysis. This procedure thus enhances the purification by offering independent opportunities for selective binding and selective elution. Conservatively, a 40%-80% recovery of partially inhibited enzyme activity can be achieved in 4-48 hr with a 30- to 90-fold purification. The purified cEH from clofibrate-induced animals was essentially homogeneous by NaDodSO4/PAGE and had an apparent subunit molecular weight of 58,000. The cEHs from normal and clofibrate-induced animals appeared identical by NaDodSO4/PAGE. Since the cEH activity in normal and clofibrate-treated animals is due to the same enzyme, the increase in cEH activity caused by selected hypolipidemic agents appears to be true induction. Images PMID:3856846

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

    PubMed

    Kumar, Vikash

    2010-05-01

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

  2. ASTROGLIOSIS AND BEHAVIORAL CHANGES IN MICE LACKING THE NEUTRAL CYSTEINE PROTEASE BLEOMYCIN HYDROLASE

    PubMed Central

    Montoya, S.E.; Thiels, E.; Card, J.P.; Lazo, J.S.

    2007-01-01

    Bleomycin hydrolase is a multifaceted neutral cysteine protease with a suggested role in antigen presentation, homocysteine-thiolactone metabolism, and Alzheimer’s disease pathogenesis. Deletion of the protease in mice results in increased neonatal mortality and dermatopathology. Immunohistochemical and behavioral studies of BLMH knockout mice were undertaken to further evaluate the role of the protease in the brain. No gross abnormalities in the central nervous system were observed upon preliminary histological examination of B6.129Blmhtm1Geh/J null animals. However, glial fibrillary acid protein immunohistochemistry revealed a global reactive astrogliosis in the aged null animals, indicative of undefined brain pathology. The role of BLMH in the brain was further explored by characterizing the behavioral phenotype of hybrid [129S6-Blmhtm1Geh/J X B6.129 Blmhtm1Geh/J]F1 null and littermate controls using multiple behavioral paradigms. In the water maze, deletion of BLMH resulted in poorer performance during water maze probe trials without detectable effect of the mutation on sensorimotor function. In addition, no age-dependent decline in discriminative performance on probe trials was observed in null animals. These data suggest a physiological non-redundant function for BLMH in the central nervous system. PMID:17391860

  3. Effect of soluble epoxide hydrolase polymorphism on substrate and inhibitor selectivity and dimer formation[S

    PubMed Central

    Morisseau, Christophe; Wecksler, Aaron T.; Deng, Catherine; Dong, Hua; Yang, Jun; Lee, Kin Sing S.; Kodani, Sean D.; Hammock, Bruce D.

    2014-01-01

    Epoxy FAs (EpFAs) are important lipid mediators that are mainly metabolized by soluble epoxide hydrolase (sEH). Thus, sEH inhibition is a promising therapeutic target to treat numerous ailments. Several sEH polymorphisms result in amino acid substitutions and alter enzyme activity. K55R and R287Q are associated with inflammatory, cardiovascular, and metabolic diseases. R287Q seems to affect sEH activity through reducing formation of a catalytically active dimer. Thus, understanding how these SNPs affect the selectivity of sEH for substrates and inhibitors is of potential clinical importance. We investigated the selectivity of four sEH SNPs toward a series of EpFAs and inhibitors. We found that the SNPs alter the catalytic activity of the enzyme but do not alter the relative substrate and inhibitor selectivity. We also determined their dimer/monomer constants (KD/M). The WT sEH formed a very tight dimer, with a KD/M in the low picomolar range. Only R287Q resulted in a large change of the KD/M. However, human tissue concentrations of sEH suggest that it is always in its dimer form independently of the SNP. These results suggest that the different biologies associated with K55R and R287Q are not explained by alteration in dimer formation or substrate selectivity. PMID:24771868

  4. Soluble epoxide hydrolase inhibition is antinociceptive in a mouse model of diabetic neuropathy

    PubMed Central

    Wagner, Karen; Yang, Jun; Inceoglu, Bora; Hammock, Bruce D.

    2014-01-01

    Neuropathic pain is currently an insufficiently treated clinical condition. There remains a critical need for efficacious therapies without severe side effects to treat the uniquely persistent and tonic pain of neuropathy. Inhibitors of the soluble epoxide hydrolase (sEH) enzyme which stabilize endogenous epoxy fatty acids have demonstrated antihyperalgesia in clinical chronic inflammatory pain and modeled neuropathic pain. Recently, the conditioned place preference (CPP) assay has been used to evaluate the tonic nature of neuropathy in several animal models. The current experiments use the CPP assay alongside withdrawal thresholds to investigate the antihyperalgesic efficacy of sEH inhibitors in a murine model of diabetic neuropathy. Here, the sEH inhibitor t-TUCB at 10 mg/kg induced a robust place preference in diabetic neuropathic mice representative of pain relief. Importantly, this effect was absent both in control mice and in sEH knockout mice at the same dose indicating t-TUCB is not positively reinforcing or rewarding. When compared to gabapentin, t-TUCB elicited a similar degree of withdrawal threshold improvement without the same degree of spontaneous locomotion decline in neuropathic mice. Overall, these experiments show that inhibiting the sEH attenuates chronic pain and offers an alternative to current side-effect limited therapies to meet this clinical need. PMID:24924124

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

    SciTech Connect

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

    2009-03-24

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

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

    PubMed Central

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

    2015-01-01

    In order to optimize the use of agaves for commercial applications, an understanding of fructan metabolism in these species at the molecular and genetic level is essential. Based on transcriptome data, this report describes the identification and molecular characterization of cDNAs and deduced amino acid sequences for genes encoding fructosyltransferases, invertases and fructan exohydrolases (FEH) (enzymes belonging to plant glycoside hydrolase family 32) from four different agave species (A. tequilana, A. deserti, A. victoriae-reginae, and A. striata). Conserved amino acid sequences and a hypervariable domain allowed classification of distinct isoforms for each enzyme type. Notably however neither 1-FFT nor 6-SFT encoding cDNAs were identified. In silico analysis revealed that distinct isoforms for certain enzymes found in a single species, showed different levels and tissue specific patterns of expression whereas in other cases expression patterns were conserved both within the species and between different species. Relatively high levels of in silico expression for specific isoforms of both invertases and fructosyltransferases were observed in floral tissues in comparison to vegetative tissues such as leaves and stems and this pattern was confirmed by Quantitative Real Time PCR using RNA obtained from floral and leaf tissue of A. tequilana. Thin layer chromatography confirmed the presence of fructans with degree of polymerization (DP) greater than DP three in both immature buds and fully opened flowers also obtained from A. tequilana. PMID:26300895

  7. Bacillus thuringiensis peptidoglycan hydrolase SleB171 involved in daughter cell separation during cell division.

    PubMed

    Li, Hua; Hu, Penggao; Zhao, Xiuyun; Yu, Ziniu; Li, Lin

    2016-04-01

    Whole-genome analyses have revealed a putative cell wall hydrolase gene (sleB171) that constitutes an operon with two other genes (ypeBandyhcN) of unknown function inBacillus thuringiensisBMB171. The putative SleB171 protein consists of 259 amino acids and has a molecular weight of 28.3 kDa. Gene disruption ofsleB171in the BMB171 genome causes the formation of long cell chains during the vegetative growth phase and delays spore formation and spore release, although it has no significant effect on cell growth and the ultimate release of the spores. The inseparable vegetative cells were nearly restored through the complementation ofsleB171expression. Real-time quantitative polymerase chain reaction analysis revealed thatsleB171is mainly active in the vegetative growth phase, with a maximum activity at the early stationary growth phase. Western blot analysis also confirmed thatsleB171is preferentially expressed during the vegetative growth phase. These results demonstrated that SleB171 plays an essential role in the daughter cell separation during cell division. PMID:26922318

  8. Urea carboxylase and allophanate hydrolase are components of a multifunctional protein in yeast.

    PubMed

    Sumrada, R A; Cooper, T G

    1982-08-10

    Saccharomyces cerevisiae can use urea as sole nitrogen source by degrading it in two steps (urea carboxylase and allophanate hydrolase) to ammonia and carbon dioxide. We previously demonstrated that: 1) the enzymatic functions required for degradation are encoded in two tightly linked genetic loci and 2) pleiotropic mutations each resulting in the loss of both activities are found in both loci. These and other observations led to the hypothesis that urea degradation might be catalyzed by a multifunctional polypeptide. Waheed and Castric (1977) J. Biol. Chem. 252, 1628-1632), on the other hand, purified urea amidolyase from Candida utilis and reported it to be a tetramer composed of nonidentical 70- and 170-kilodalton subunits. To resolve the differing views of urea amidolyase structure, we purified the protein using rapid methods designed to avoid proteolytic cleavage. Application of these methods resulted in the isolation of a single, inducible and repressible, 204-kilodalton species. We observed no evidence for the existence of nonidentical subunits. A similar inducible, high molecular weight species was also detected in C. utilis. These biochemical results support our earlier hypothesis that urea degradation is carried out in yeast by an inducible and repressible protein composed of identical, multifunctional subunits. PMID:6124544

  9. Coacervate Core Micelles for the Dispersion and Stabilization of Organophosphate Hydrolase in Organic Solvents

    NASA Astrophysics Data System (ADS)

    Mills, Carolyn; Obermeyer, Allie; Dong, Xuehui; Olsen, Bradley D.

    Bulk organophosphate (OP) nerve agents are difficult to decontaminate on site and dangerous to transport. The organophosphate hydrolase (OPH) enzyme is an efficient catalyst for hydrolyzing, and thus decontaminating, these compounds, but suffers from poor stability in the hydrophobic bulk OP environment. Here, we exploit the complex coacervation phase separation phenomenon to form complex coacervate core micelles (C3Ms) that can protect this OPH enzyme under these conditions. Stable C3Ms form when mixing a charged-neutral block copolymer methyl-quaternized poly(4-vinylpyridine)-block-poly(oligo(ethylene glycol) methacrylate) (Qp4vp- b-POEGMA), a homopolymer poly(acrylic acid) (PAA), and OPH under a certain conditions. The C3Ms are then transferred into two organic solvents, ethanol and dimethyl methylphosphonate (DMMP), which is a good simulant for the physical properties of the OP compounds. The C3Ms retain their nanostructures in the organic solvents. The activity test of OPH indicates that the C3Ms successfully protect OPH activity in organic solvents.

  10. Distribution and evolution of glycoside hydrolase family 45 cellulases in nematodes and fungi

    PubMed Central

    2014-01-01

    Background Horizontal gene transfer (HGT) has been suggested as the mechanism by which various plant parasitic nematode species have obtained genes important in parasitism. In particular, cellulase genes have been acquired by plant parasitic nematodes that allow them to digest plant cell walls. Unlike the typical glycoside hydrolase (GH) family 5 cellulase genes which are found in several nematode species from the order Tylenchida, members of the GH45 cellulase have only been identified in a cluster including the families Parasitaphelenchidae (with the pinewood nematode Bursaphelenchus xylophilus) and Aphelenchoididae, and their origins remain unknown. Results In order to investigate the distribution and evolution of GH45 cellulase genes in nematodes and fungi we performed a wide ranging screen for novel putative GH45 sequences. This revealed that the sequences are widespread mainly in Ascomycetous fungi and have so far been found in a single major nematode lineage. Close relationships between the sequences from nematodes and fungi were found through our phylogenetic analyses. An intron position is shared by sequences from Bursaphelenchus nematodes and several Ascomycetous fungal species. Conclusions The close phylogenetic relationships and conserved gene structure between the sequences from nematodes and fungi strongly supports the hypothesis that nematode GH45 cellulase genes were acquired via HGT from fungi. The rapid duplication and turnover of these genes within Bursaphelenchus genomes demonstrate that useful sequences acquired via HGT can become established in the genomes of recipient organisms and may open novel niches for these organisms to exploit. PMID:24690293

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

    PubMed

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

    2016-08-01

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

  12. A Novel α/β-Hydrolase Gene IbMas Enhances Salt Tolerance in Transgenic Sweetpotato

    PubMed Central

    Song, Xuejin; He, Shaozhen; Liu, Qingchang

    2014-01-01

    Salt stress is one of the major environmental stresses in agriculture worldwide and affects crop productivity and quality. The development of crops with elevated levels of salt tolerance is therefore highly desirable. In the present study, a novel maspardin gene, named IbMas, was isolated from salt-tolerant sweetpotato (Ipomoea batatas (L.) Lam.) line ND98. IbMas contains maspardin domain and belongs to α/β-hydrolase superfamily. Expression of IbMas was up-regulated in sweetpotato under salt stress and ABA treatment. The IbMas-overexpressing sweetpotato (cv. Shangshu 19) plants exhibited significantly higher salt tolerance compared with the wild-type. Proline content was significantly increased, whereas malonaldehyde content was significantly decreased in the transgenic plants. The activities of superoxide dismutase (SOD) and photosynthesis were significantly enhanced in the transgenic plants. H2O2 was also found to be significantly less accumulated in the transgenic plants than in the wild-type. Overexpression of IbMas up-regulated the salt stress responsive genes, including pyrroline-5-carboxylate synthase, pyrroline-5-carboxylate reductase, SOD, psbA and phosphoribulokinase genes, under salt stress. These findings suggest that overexpression of IbMas enhances salt tolerance of the transgenic sweetpotato plants by regulating osmotic balance, protecting membrane integrity and photosynthesis and increasing reactive oxygen species scavenging capacity. PMID:25501819

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

    PubMed

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

    2016-02-18

    The transition from replication to non-replication underlies much of Mycobacterium tuberculosis (Mtb) pathogenesis, as non- or slowly replicating Mtb are responsible for persistence and poor treatment outcomes. Therapeutic targeting of non-replicating populations is a priority for tuberculosis treatment, but few drug targets in non-replicating Mtb are currently known. Here, we directly measured the activity of the highly diverse and druggable serine hydrolases (SHs) during active replication and non-replication using activity-based proteomics. We predict SH activity for 78 proteins, including 27 proteins with unknown function, and identify 37 SHs that remain active in the absence of replication, providing a set of candidate persistence targets. Non-replication was associated with major shifts in SH activity. These activity changes were largely independent of SH abundance, indicating extensive post-translational regulation of SHs. By probing a large cross-section of druggable Mtb enzyme space during replication and non-replication, we identify new SHs and suggest new persistence targets. PMID:26853625

  14. Hydrolases in Polymer Chemistry: Part III: Synthesis and Limited Surface Hydrolysis of Polyesters and Other Polymers

    NASA Astrophysics Data System (ADS)

    Guebitz, Georg M.

    Limited enzymatic surface hydrolysis of polyamides, polyethyleneterphthalates (PET) and polyacrylonitriles has been demonstrated to be a powerful and yet mild strategy for directly improving polymer surface properties (e.g., hydrophilicity) or activating materials for further processing. Recently, mechanistic details on enzymatic surface hydrolysis have become available, especially for the functionalisation of PET, which has been investigated in most detail. Generally, enzymes show a strong preference for amorphous regions of polymers. Consequently, during hydrolysis, the degree of crystallinity increases according to FTIR and DSC analysis. MALDI-TOF analysis has shown that PET hydrolases (i.e. cutinases and lipases) cleave the polymer endo-wise, in contrast to alkaline hydrolysis. As a result, an increase in the amount of carboxyl and hydroxyl groups has been found upon enzymatic hydrolysis, according to X-ray photoelectron spectroscopy and various derivatisation and titration methods recently adapted for this purpose. These mechanistic data, combined with advances in structural and molecular biology, help to explain the considerably different activities of closely related enzymes (e.g. cutinases) on polymers.

  15. A novel member of glycoside hydrolase family 30 subfamily 8 with altered substrate specificity.

    PubMed

    St John, Franz J; Dietrich, Diane; Crooks, Casey; Pozharski, Edwin; González, Javier M; Bales, Elizabeth; Smith, Kennon; Hurlbert, Jason C

    2014-11-01

    Endoxylanases classified into glycoside hydrolase family 30 subfamily 8 (GH30-8) are known to hydrolyze the hemicellulosic polysaccharide glucuronoxylan (GX) but not arabinoxylan or neutral xylooligosaccharides. This is owing to the specificity of these enzymes for the α-1,2-linked glucuronate (GA) appendage of GX. Limit hydrolysis of this substrate produces a series of aldouronates each containing a single GA substituted on the xylose penultimate to the reducing terminus. In this work, the structural and biochemical characterization of xylanase 30A from Clostridium papyrosolvens (CpXyn30A) is presented. This xylanase possesses a high degree of amino-acid identity to the canonical GH30-8 enzymes, but lacks the hallmark β8-α8 loop region which in part defines the function of this GH30 subfamily and its role in GA recognition. CpXyn30A is shown to have a similarly low activity on all xylan substrates, while hydrolysis of xylohexaose revealed a competing transglycosylation reaction. These findings are directly compared with the model GH30-8 enzyme from Bacillus subtilis, XynC. Despite its high sequence identity to the GH30-8 enzymes, CpXyn30A does not have any apparent specificity for the GA appendage. These findings confirm that the typically conserved β8-α8 loop region of these enzymes influences xylan substrate specificity but not necessarily β-1,4-xylanase function. PMID:25372685

  16. α/β-Hydrolase domain-6-accessible monoacylglycerol controls glucose-stimulated insulin secretion.

    PubMed

    Zhao, Shangang; Mugabo, Yves; Iglesias, Jose; Xie, Li; Delghingaro-Augusto, Viviane; Lussier, Roxane; Peyot, Marie-Line; Joly, Erik; Taïb, Bouchra; Davis, Matthew A; Brown, J Mark; Abousalham, Abdelkarim; Gaisano, Herbert; Madiraju, S R Murthy; Prentki, Marc

    2014-06-01

    Glucose metabolism in pancreatic β cells stimulates insulin granule exocytosis, and this process requires generation of a lipid signal. However, the signals involved in lipid amplification of glucose-stimulated insulin secretion (GSIS) are unknown. Here we show that in β cells, glucose stimulates production of lipolysis-derived long-chain saturated monoacylglycerols, which further increase upon inhibition of the membrane-bound monoacylglycerol lipase α/β-Hydrolase Domain-6 (ABHD6). ABHD6 expression in β cells is inversely proportional to GSIS. Exogenous monoacylglycerols stimulate β cell insulin secretion and restore GSIS suppressed by the pan-lipase inhibitor orlistat. Whole-body and β-cell-specific ABHD6-KO mice exhibit enhanced GSIS, and their islets show elevated monoacylglycerol production and insulin secretion in response to glucose. Inhibition of ABHD6 in diabetic mice restores GSIS and improves glucose tolerance. Monoacylglycerol binds and activates the vesicle priming protein Munc13-1, thereby inducing insulin exocytosis. We propose saturated monoacylglycerol as a signal for GSIS and ABHD6 as a negative modulator of insulin secretion. PMID:24814481

  17. Abnormal Hypermethylation at Imprinting Control Regions in Patients with S-Adenosylhomocysteine Hydrolase (AHCY) Deficiency

    PubMed Central

    Motzek, Antje; Knežević, Jelena; Switzeny, Olivier J.; Cooper, Alexis; Barić, Ivo; Beluzić, Robert; Strauss, Kevin A.; Puffenberger, Erik G.; Vugrek, Oliver; Zechner, Ulrich

    2016-01-01

    S-adenosylhomocysteine hydrolase (AHCY) deficiency is a rare autosomal recessive disorder in methionine metabolism caused by mutations in the AHCY gene. Main characteristics are psychomotor delay including delayed myelination and myopathy (hypotonia, absent tendon reflexes etc.) from birth, mostly associated with hypermethioninaemia, elevated serum creatine kinase levels and increased genome wide DNA methylation. The prime function of AHCY is to hydrolyse and efficiently remove S-adenosylhomocysteine, the by-product of transmethylation reactions and one of the most potent methyltransferase inhibitors. In this study, we set out to more specifically characterize DNA methylation changes in blood samples from patients with AHCY deficiency. Global DNA methylation was increased in two of three analysed patients. In addition, we analysed the DNA methylation levels at differentially methylated regions (DMRs) of six imprinted genes (MEST, SNRPN, LIT1, H19, GTL2 and PEG3) as well as Alu and LINE1 repetitive elements in seven patients. Three patients showed a hypermethylation in up to five imprinted gene DMRs. Abnormal methylation in Alu and LINE1 repetitive elements was not observed. We conclude that DNA hypermethylation seems to be a frequent but not a constant feature associated with AHCY deficiency that affects different genomic regions to different degrees. Thus AHCY deficiency may represent an ideal model disease for studying the molecular origins and biological consequences of DNA hypermethylation due to impaired cellular methylation status. PMID:26974671

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

    PubMed Central

    Hickey, Raymond D.; Mao, Shennen A.; Glorioso, Jaime; Lillegard, Joseph B.; Fisher, James E.; Amiot, Bruce; Rinaldo, Piero; Harding, Cary O.; Marler, Ronald; Finegold, Milton J.; Grompe, Markus; Nyberg, Scott L.

    2014-01-01

    Hereditary tyrosinemia type I (HT1) is caused by deficiency in fumarylacetoacetate hydrolase (FAH), an enzyme that catalyzes the last step of tyrosine metabolism. The most severe form of the disease presents acutely during infancy, and is characterized by severe liver involvement, most commonly resulting in death if untreated. Generation of FAH+/− pigs was previously accomplished by adeno-associated virus-mediated gene knockout in fibroblasts and somatic cell nuclear transfer. Subsequently, these animals were outbred and crossed to produce the first FAH−/− pigs. FAH-deficiency produced a lethal defect in utero that was corrected by administration of 2-(2-nitro-4-trifluoromethylbenzyol)-1,3 cyclohexanedione (NTBC) throughout pregnancy. Animals on NTBC were phenotypically normal at birth; however, animals were euthanized approximately four weeks after withdrawal of NTBC due to clinical decline and physical examination findings of severe liver injury and encephalopthy consistent with acute liver failure. Biochemical and histological analyses, characterized by diffuse and severe hepatocellular damage, confirmed the diagnosis of severe liver injury. FAH−/− pigs provide the first genetically engineered large animal model of a metabolic liver disorder. Future applications of FAH−/− pigs include discovery research as a large animal model of HT1 and spontaneous acute liver failure, and preclinical testing of efficacy of liver cell therapies, including transplantation of hepatocytes, liver stem cells, and pluripotent stem cell-derived hepatocytes. PMID:24879068

  19. Structure–Activity Relationships of α-Keto Oxazole Inhibitors of Fatty Acid Amide Hydrolase

    PubMed Central

    Hardouin, Christophe; Kelso, Michael J.; Romero, F. Anthony; Rayl, Thomas J.; Leung, Donmienne; Hwang, Inkyu; Cravatt, Benjamin F.; Boger, Dale L.

    2008-01-01

    A systematic study of the structure–activity relationships (SAR) of 2b (OL-135), a potent inhibitor of fatty acid amide hydrolase (FAAH), is detailed targeting the C2 acyl side chain. A series of aryl replacements or substituents for the terminal phenyl group provided effective inhibitors (e.g., 5c, aryl = 1-napthyl, Ki = 2.6 nM) with 5hh (aryl = 3-Cl-Ph, Ki = 900 pM) being 5-fold more potent than 2b. Conformationally-restricted C2 side chains were examined and many provided exceptionally potent inhibitors of which 11j (ethylbiphenyl side chain) was established to be a 750 pM inhibitor. A systematic series of heteroatoms (O, NMe, S), electron-withdrawing groups (SO, SO2), and amides positioned within and hydroxyl substitutions on the linking side chain were investigated which typically led to a loss in potency. The most tolerant positions provided effective inhibitors (12p, 6-position S, Ki = 3 nM or 13d, 2-position OH, Ki = 8 nM) comparable in potency to 2b. Proteomic-wide screening of selected inhibitors from the systematic series of >100 candidates prepared revealed that they are selective for FAAH over all other mammalian serine proteases. PMID:17559203

  20. Peptide hydrolase activities in seedlings and hormone-treated cotyledons of pumpkin (Cucurbita pepo).

    PubMed

    Weidhase, R A; Parthier, B

    1983-01-01

    Enzymes hydrolyzing Gly-Ala-, Met-Met- and Pro-4-phenylazo-phenylamides, and N-benzoyl-L-arginine-4-nitroanilide have been identified in germinating seeds and cotyledons of pumpkin (Cucurbita pepo). The enzyme activities per cotyledon increase markedly during the germination process, but the proportion of enhancement depends on the type of enzyme species. The increase in enzyme activities is due to de novo synthesis as shown by cycloheximide treatment and is influenced by phytohormones (cytokinins and abscissic acid). In isolated cotyledons exogenous cytokinin (benzyladenine) obviously can replace the effect of the embryo as the source of endogenous hormone. Abscissic acid counteracts the cytokinin effect. It is suggested that aminopeptidases have a biological function in reserve protein degradation of the cotyledons during seed germination. Our results do not support the assumption that the embryonic axis of the growing seedling serves as a "sink" of proteolytic products resulting in an activation of peptide hydrolases in the cotyledons, but rather de novo synthesis of these enzymes seems to be controlled by substances (phytohormones) originating from the embryo. PMID:6360167

  1. Comparative gene identification 58/α/β hydrolase domain 5 lacks lysophosphatidic acid acyltransferase activity

    PubMed Central

    McMahon, Derek; Dinh, Anna; Kurz, Daniel; Shah, Dharika; Han, Gil-Soo; Carman, George M.; Brasaemle, Dawn L.

    2014-01-01

    Mutations in the gene encoding comparative gene identification 58 (CGI-58)/α/β hydrolase domain 5 (ABHD5) cause Chanarin-Dorfman syndrome, characterized by excessive triacylglycerol storage in cells and tissues. CGI-58 has been identified as a coactivator of adipose TG lipase (ATGL) and a lysophosphatidic acid acyltransferase (LPAAT). We developed a molecular model of CGI-58 structure and then mutated predicted active site residues and performed LPAAT activity assays of recombinant WT and mutated CGI-58. When mutations of predicted catalytic residues failed to reduce LPAAT activity, we determined that LPAAT activity was due to a bacterial contaminant of affinity purification procedures, plsC, the sole LPAAT in Escherichia coli. Purification protocols were optimized to reduce plsC contamination, in turn reducing LPAAT activity. When CGI-58 was expressed in SM2-1(DE3) cells that lack plsC, lysates lacked LPAAT activity. Additionally, mouse CGI-58 expressed in bacteria as a glutathione-S-transferase fusion protein and human CGI-58 expressed in yeast lacked LPAAT activity. Previously reported lipid binding activity of CGI-58 was revisited using protein-lipid overlays. Recombinant CGI-58 failed to bind lysophosphatidic acid, but interestingly, bound phosphatidylinositol 3-phosphate [PI(3)P] and phosphatidylinositol 5-phosphate [PI(5)P]. Prebinding CGI-58 with PI(3)P or PI(5)P did not alter its coactivation of ATGL in vitro. In summary, purified recombinant CGI-58 that is functional as an ATGL coactivator lacks LPAAT activity. PMID:24879803

  2. Cloning, Expression and Characterization of a Glycoside Hydrolase Family 39 Xylosidase from Bacillus Halodurans C-125

    NASA Astrophysics Data System (ADS)

    Wagschal, Kurt; Franqui-Espiet, Diana; Lee, Charles C.; Robertson, George H.; Wong, Dominic W. S.

    The gene encoding a glycoside hydrolase family 39 xylosidase (BH1068) from the alkaliphile Bacillus halodurans strain C-125 was cloned with a C-terminal His-tag, and the recombinant gene product termed BH1068(His)6 was expressed in Escherichia coli. Of the artificial substrates tested, BH1068(His)6 hydrolyzed nitrophenyl derivatives of β-d-xylopyranose, α-l-arabinofuranose, and α-l-arabinopyranose. Deviation from Michaelis-Menten kinetics at higher substrate concentrations indicative of transglycosylation was observed, and k cat and K m values were measured at both low and high substrate concentrations to illuminate the relative propensities to proceed along this alternate reaction pathway. The pH maximum was 6.5, and under the conditions tested, maximal activity was at 47°C, and thermal instability occurred above 45°C. BH1068(His)6 was inactive on arabinan, hydrolyzed xylooligosaccharides, and released only xylose from oat, wheat, rye, beech, and birch arabinoxylan, and thus, can be classified as a xylosidase with respect to natural substrate specificity. The enzyme was not inhibited by up to 200 mM xylose. The oligomerization state was tetrameric under the size-exclusion chromatography conditions employed.

  3. ROLE OF SOLUBLE EPOXIDE HYDROLASE IN AGE-RELATED VASCULAR COGNITIVE DECLINE

    PubMed Central

    Nelson, Jonathan W.; Young, Jennifer M.; Borkar, Rohan; Woltjer, Randy L.; Quinn, Joseph F.; Silbert, Lisa C.; Grafe, Marjorie R.; Alkayed, Nabil J.

    2014-01-01

    P450 eicosanoids are important regulators of the cerebral microcirculation, but their role in cerebral small vessel disease is unclear. We tested the hypothesis that vascular cognitive impairment (VCI) is linked to reduced cerebral microvascular eicosanoid signaling. We analyzed human brain tissue from individuals formerly enrolled in the Oregon Brain Aging Study, who had a history of cognitive impairment histopathological evidence of microvascular disease. VCI subjects had significantly higher lesion burden both on premortem MRI and postmortem histopathology compared to age- and sex-matched controls. Mass spectrometry-based eicosanoid analysis revealed that 14,15-dihydroxyeicosatrienoic acid (DHET) was elevated in cortical brain tissue from VCI subjects. Immunoreactivity of soluble epoxide hydrolase (sEH), the enzyme responsible for 14,15-DHET formation, was localized to cerebral microvascular endothelium, and was enhanced in microvessels of affected tissue. Finally, we evaluated the genotype frequency of two functional single nucleotide polymorphisms of sEH gene EPHX2 in VCI and control groups. Our findings support a role for sEH and a potential benefit from sEH inhibitors in age-related VCI. PMID:25277097

  4. Microencapsulated bile salt hydrolase producing Lactobacillus reuteri for oral targeted delivery in the gastrointestinal tract.

    PubMed

    Martoni, Christopher; Bhathena, Jasmine; Urbanska, Aleksandra Malgorzata; Prakash, Satya

    2008-11-01

    This is the first study of its kind to screen probiotic lactic acid bacteria for the purpose of microencapsulating a highly bile salt hydrolase (BSH)-active strain. A Lactobacillus reuteri strain and a Bifidobacterium longum strain were isolated as the highest BSH producers among the candidates. Microcapsules were prepared with a diameter of 619 +/- 31 mum and a cell load of 5 x 10(9) cfu/ml. Post de Man, Rogosa, and Sharpe broth-acid challenge, L. reuteri microcapsules metabolized glyco- and tauro-conjugated bile salts at rates of 10.16 +/- 0.46 and 1.85 +/- 0.33 micromol/g microcapsule per hour, respectively, over the first 2 h. Microencapsulated B. longum had minimal BSH activity and were significantly (P < 0.05) more susceptible to acid challenge. Further testing of L. reuteri microcapsules in a simulated human gastrointestinal (GI) model showed an improved rate, with 49.4 +/- 6.21% of glyco-conjugates depleted after 60 min and complete deconjugation after 4 h. Microcapsules protected the encased cells in the simulated stomach maintaining L. reuteri viability above 10(9), 10(8), and 10(6) cfu/ml after 2 h at pH 3.0, 2.5, and 2.0, respectively. Results show excellent potential for this highly BSH-active microencapsulation system in vitro, highlighted by improved viability and substrate utilization in simulated GI transit. PMID:18719901

  5. Soluble Epoxide Hydrolase: Sex Differences and Role in Endothelial Cell Survival

    PubMed Central

    Gupta, Nandita C.; Davis, Catherine M.; Nelson, Jonathan W.; Young, Jennifer M.; Alkayed, Nabil J.

    2012-01-01

    OBJECTIVE Sex differences in cerebral ischemic injury are in part due to differences in cerebrovascular perfusion. We determined if brain microvascular endothelial cells (ECs) isolated from female (F) brain are more resistant to ischemic injury compared to male (M) ECs, and if the difference is due to lower expression of soluble epoxide hydrolase (sEH) and higher levels of vasoprotective epoxyeicosatrienoic acids (EETs). We also determined if protection by EETs is linked to inhibition of Rho-kinase (ROCK). METHODS EC ischemic damage was measured after oxygen-glucose deprivation (OGD) using propidium iodide (PI) and cleaved caspase-3 labeling. Expression of sEH was determined by quantitative PCR and immunocytochemistry, EETs levels by liquid chromatography-tandem mass spectrometry, and ROCK activity by ELISA. RESULTS EC damage was higher in M vs. F ECs, which correlated with higher sEH mRNA, stronger immunoreactivity and lower EETs compared to F ECs. Inhibition of sEH abolished the sex difference in EC damage. ROCK activity was higher in M vs. F ECs after OGD, and sex differences in EC damage and ROCK activity were abolished by 14,15-EET and ROCK inhibition. CONCLUSION Sex differences in ischemic brain injury are in part due to differences in EETs-mediated inhibition of EC ROCK activation after ischemia. PMID:22723436

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

    PubMed Central

    Ogawa, Shintaro; Kunugi, Hiroshi

    2015-01-01

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

  7. Diversity of glycosyl hydrolases from cellulose-depleting communities enriched from casts of two earthworm species.

    PubMed

    Beloqui, Ana; Nechitaylo, Taras Y; López-Cortés, Nieves; Ghazi, Azam; Guazzaroni, María-Eugenia; Polaina, Julio; Strittmatter, Axel W; Reva, Oleg; Waliczek, Agnes; Yakimov, Michail M; Golyshina, Olga V; Ferrer, Manuel; Golyshin, Peter N

    2010-09-01

    The guts and casts of earthworms contain microbial assemblages that process large amounts of organic polymeric substrates from plant litter and soil; however, the enzymatic potential of these microbial communities remains largely unexplored. In the present work, we retrieved carbohydrate-modifying enzymes through the activity screening of metagenomic fosmid libraries from cellulose-depleting microbial communities established with the fresh casts of two earthworm species, Aporrectodea caliginosa and Lumbricus terrestris, as inocula. Eight glycosyl hydrolases (GHs) from the A. caliginosa-derived community were multidomain endo-beta-glucanases, beta-glucosidases, beta-cellobiohydrolases, beta-galactosidase, and beta-xylosidases of known GH families. In contrast, two GHs derived from the L. terrestris microbiome had no similarity to any known GHs and represented two novel families of beta-galactosidases/alpha-arabinopyranosidases. Members of these families were annotated in public databases as conserved hypothetical proteins, with one being structurally related to isomerases/dehydratases. This study provides insight into their biochemistry, domain structures, and active-site architecture. The two communities were similar in bacterial composition but significantly different with regard to their eukaryotic inhabitants. Further sequence analysis of fosmids and plasmids bearing the GH-encoding genes, along with oligonucleotide usage pattern analysis, suggested that those apparently originated from Gammaproteobacteria (pseudomonads and Cellvibrio-like organisms), Betaproteobacteria (Comamonadaceae), and Alphaproteobacteria (Rhizobiales). PMID:20622123

  8. Isolation and characterization of novel multifunctional recombinant family 26 glycoside hydrolase from Mehsani buffalo rumen metagenome.

    PubMed

    Patel, Avani B; Patel, Amrutlal K; Shah, Mihir P; Parikh, Ishan K; Joshi, Chaitanya G

    2016-01-01

    Rumen microbiota harbor a diverse set of carbohydrate-active enzymes (CAZymes), which play a crucial role in the degradation of a complex plant polysaccharide thereby providing metabolic energy to the host animals. Earlier, we reported CAZYme analysis from the buffalo rumen metagenome by high throughput shotgun sequencing. Among the various CAZymes, glycoside hydrolase family 26 (GH26) enzymes have a number of industrial applications including in paper, oil, biofuel, food, feed, pharmaceutical, coffee, and detergent industries. Here, we report isolation and characterization of GH26 enzyme from the buffalo rumen metagenome. A novel GH26 gene composed of 1,119 base pairs was successfully amplified using the gene-specific primers inferred based on the contig generated from metagenome sequence assembly and cloned in a pET32a (+) expression vector as an N-terminal histidine tag fusion protein. A novel GH26 protein from an unknown rumen microorganism shared a maximum of 68% identity with the Prevotella ruminicola 23 encoded carbohydrate esterase family 7 and 46% with Bacteroides sp. 2_1_33B encoded mannan endo-1, 4-β-mannosidase. The recombinant GH26-histidine tag fusion protein was expressed in Escherichia coli and purified using Ni-NTA affinity chromatography. The purified enzyme displayed multifunctional activities against various carbohydrate substrates including locust bean gum, beechwood xylan, pectin, and carboxymethyl cellulose suggesting mannanase, xylanase, pectin esterase, and endoglucanase activities, respectively. PMID:25644118

  9. Loss of a Biofilm-Inhibiting Glycosyl Hydrolase during the Emergence of Yersinia pestis▿

    PubMed Central

    Erickson, David L.; Jarrett, Clayton O.; Callison, Julie A.; Fischer, Elizabeth R.; Hinnebusch, B. Joseph

    2008-01-01

    Yersinia pestis, the bacterial agent of plague, forms a biofilm in the foregut of its flea vector to produce a transmissible infection. The closely related Yersinia pseudotuberculosis, from which Y. pestis recently evolved, can colonize the flea midgut but does not form a biofilm in the foregut. Y. pestis biofilm in the flea and in vitro is dependent on an extracellular matrix synthesized by products of the hms genes; identical genes are present in Y. pseudotuberculosis. The Yersinia Hms proteins contain functional domains present in Escherichia coli and Staphylococcus proteins known to synthesize a poly-β-1,6-N-acetyl-d-glucosamine biofilm matrix. In this study, we show that the extracellular matrices (ECM) of Y. pestis and staphylococcal biofilms are antigenically related, indicating a similar biochemical structure. We also characterized a glycosyl hydrolase (NghA) of Y. pseudotuberculosis that cleaved β-linked N-acetylglucosamine residues and reduced biofilm formation by staphylococci and Y. pestis in vitro. The Y. pestis nghA ortholog is a pseudogene, and overexpression of functional nghA reduced ECM surface accumulation and inhibited the ability of Y. pestis to produce biofilm in the flea foregut. Mutational loss of this glycosidase activity in Y. pestis may have contributed to the recent evolution of flea-borne transmission. PMID:18931111

  10. Sequence, Structure, and Evolution of Cellulases in Glycoside Hydrolase Family 48*

    PubMed Central

    Sukharnikov, Leonid O.; Alahuhta, Markus; Brunecky, Roman; Upadhyay, Amit; Himmel, Michael E.; Lunin, Vladimir V.; Zhulin, Igor B.

    2012-01-01

    Currently, the cost of cellulase enzymes remains a key economic impediment to commercialization of biofuels (1). Enzymes from glycoside hydrolase family 48 (GH48) are a critical component of numerous natural lignocellulose-degrading systems. Although computational mining of large genomic data sets is a promising new approach for identifying novel cellulolytic activities, current computational methods are unable to distinguish between cellulases and enzymes with different substrate specificities that belong to the same protein family. We show that by using a robust computational approach supported by experimental studies, cellulases and non-cellulases can be effectively identified within a given protein family. Phylogenetic analysis of GH48 showed non-monophyletic distribution, an indication of horizontal gene transfer. Enzymatic function of GH48 proteins coded by horizontally transferred genes was verified experimentally, which confirmed that these proteins are cellulases. Computational and structural studies of GH48 enzymes identified structural elements that define cellulases and can be used to computationally distinguish them from non-cellulases. We propose that the structural element that can be used for in silico discrimination between cellulases and non-cellulases belonging to GH48 is an ω-loop located on the surface of the molecule and characterized by highly conserved rare amino acids. These markers were used to screen metagenomics data for “true” cellulases. PMID:23055526

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

    SciTech Connect

    Abbott,D.; Boraston, A.

    2007-01-01

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

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

    PubMed

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

    2015-06-01

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

  13. Heterogeneous expression and biological function of ubiquitin carboxy-terminal hydrolase-L1 in osteosarcoma.

    PubMed

    Zheng, Shuier; Qiao, Guanglei; Min, Daliu; Zhang, Zhichang; Lin, Feng; Yang, Qingcheng; Feng, Tao; Tang, Lina; Sun, Yuanjue; Zhao, Hui; Li, Hongtao; Yu, Wenxi; Yang, Yumei; Shen, Zan; Yao, Yang

    2015-04-01

    Ubiquitin carboxyl terminal hydrolase 1 (UCHL1), a member of the UCH class of DUBs, has been reported as either an oncogene or a tumor suppressor. However, the molecular mechanism underlying the biological function of UCHL1 in osteosarcoma is still unclear. This study was aimed at elucidating the roles of UCHL1 in regulating the biological behavior of osteosarcoma cells. In this study, we found that UCHL1 was elevated in osteosarcoma compared with normal bone tissue. Moreover, UCHL1 expression level was correlated with tumor maximum diameter, high rate of lung metastases and short survival time. Then, we found that knockdown of UCHL1 in osteosarcoma cell MG63 inhibited cell proliferation and significantly increased cell population in the G1 phase. Several cyclins promoting G1/S phase transition were reduced after UCHL1 knockdown, including cell cycle regulator cyclin D1, cyclin E1 and CDK6. Moreover, inhibition of UCHL1 in MG63 cells dramatically induced cell apoptosis. We also found that down-regulation of UCHL1 in MG63 significantly inhibited cell invasion. Then, we found that there was a positive correlation between UCHL1 expression level and the Akt and ERK phosphorylation status. Finally, in vivo data showed that knockdown of UCHL1 inhibited osteosarcoma growth in nude mice. These results indicate that UCHL1 could work as an oncogene and may serve as a promising therapeutic strategy for osteosarcoma. PMID:25578779

  14. Bioprospecting metagenomics of a microbial community on cotton degradation: Mining for new glycoside hydrolases.

    PubMed

    Zhang, Guoxiu; Liu, Pei; Zhang, Lei; Wei, Wei; Wang, Xuedong; Wei, Dongzhi; Wang, Wei

    2016-09-20

    Glycoside hydrolases (GHases) of higher performance are immediately needed for efficient degradation of plant biomass into fermentable sugars in industrial processes. The current study represents functional characterization of the enzymatic repertoire involved in crude cotton biomass degradation. Physical contact between cells and substrate is necessary for efficient hydrolysis of cellulose. Cytophagales, which plays a major role in cotton biomass decomposition, was identified as a prevalent community member by 16S rRNA analysis. From the metagenome data, 2058 GHase homologs were identified, of which sixteen were successfully expressed in E. coli. Four enzymes showed activities on p-nitrophenyl-β-d-xylopyranoside, four showed activities on p-nitrophenyl-β-d-glucopyranoside, two had activities against p-nitrophenyl-β-d-glucuronide, one showed activity on laminarin, three had activities against p-nitrophenyl-N-acetyl-β-d-glucosaminide, one had activity towards carboxymethyl cellulose, and one towards p-nitrophenyl-β-d-mannopyranoside. Metagenomics provides a good resource for mining novel biomass degrading enzymes. The sixteen GHases that were cloned may have potential application for biomass conversion and bioproduct production. Functional characterization of the enzymatic repertoire in cotton biomass degradation and analysis of the GHases provide insight into the composition and interaction of enzymes and pathways of plant biomass degradation. PMID:27460447

  15. Induction of Epoxide Hydrolase, Glucuronosyl Transferase, and Sulfotransferase by Phenethyl Isothiocyanate in Male Wistar Albino Rats

    PubMed Central

    Mohd Noor, Noramaliza; Konsue, Nattaya

    2014-01-01

    Phenethyl isothiocyanate (PEITC) is an isothiocyanate found in watercress as the glucosinolate (gluconasturtiin). The isothiocyanate is converted from the glucosinolate by intestinal microflora or when contacted with myrosinase during the chopping and mastication of the vegetable. PEITC manifested protection against chemically-induced cancers in various tissues. A potential mechanism of chemoprevention is by modulating the metabolism of carcinogens so as to promote deactivation. The principal objective of this study was to investigate in rats the effect of PEITC on carcinogen-metabolising enzyme systems such as sulfotransferase (SULT), N-acetyltransferase (NAT), glucuronosyl transferase (UDP), and epoxide hydrolase (EH) following exposure to low doses that simulate human dietary intake. Rats were fed for 2 weeks diets supplemented with PEITC at 0.06 µmol/g (low dose, i.e., dietary intake), 0.6 µmol/g (medium dose), and 6.0 µmol/g (high dose), and the enzymes were monitored in rat liver. At the Low dose, no induction of the SULT, NAT, and EH was noted, whereas UDP level was elevated. At the Medium dose, only SULT level was increased, whereas at the High dose marked increase in EH level was observed. It is concluded that PEITC modulates carcinogen-metabolising enzyme systems at doses reflecting human intake thus elucidating the mechanism of its chemoprevention. PMID:24592387

  16. Crystal structure of Homo sapiens PTD012 reveals a zinc-containing hydrolase fold.

    PubMed

    Manjasetty, Babu A; Büssow, Konrad; Fieber-Erdmann, Martin; Roske, Yvette; Gobom, Johan; Scheich, Christoph; Götz, Frank; Niesen, Frank H; Heinemann, Udo

    2006-04-01

    The human protein PTD012 is the longer product of an alternatively spliced gene and was described to be localized in the nucleus. The X-ray structure analysis at 1.7 A resolution of PTD012 through SAD phasing reveals a monomeric protein and a novel fold. The shorter splice form was also studied and appears to be unfolded and non-functional. The structure of PTD012 displays an alphabetabetaalpha four-layer topology. A metal ion residing between the central beta-sheets is partially coordinated by three histidine residues. X-ray absorption near-edge structure (XANES) analysis identifies the PTD012-bound ion as Zn(2+). Tetrahedral coordination of the ion is completed by the carboxylate oxygen atom of an acetate molecule taken up from the crystallization buffer. The binding of Zn(2+) to PTD012 is reminiscent of zinc-containing enzymes such as carboxypeptidase, carbonic anhydrase, and beta-lactamase. Biochemical assays failed to demonstrate any of these enzyme activities in PTD012. However, PTD012 exhibits ester hydrolase activity on the substrate p-nitrophenyl acetate. PMID:16522806

  17. Destructuring plant biomass: Focus on fungal and extremophilic cell wall hydrolases

    PubMed Central

    Guerriero, Gea; Hausman, Jean-Francois; Strauss, Joseph; Ertan, Haluk; Siddiqui, Khawar Sohail

    2016-01-01

    The use of plant biomass as feedstock for biomaterial and biofuel production is relevant in the current bio-based economy scenario of valorizing renewable resources. Fungi, which degrade complex and recalcitrant plant polymers, secrete different enzymes that hydrolyze plant cell wall polysaccharides. The present review discusses the current research trends on fungal, as well as extremophilic cell wall hydrolases that can withstand extreme physico-chemical conditions required in efficient industrial processes. Secretomes of fungi from the phyla Ascomycota, Basidiomycota, Zygomycota and Neocalli-mastigomycota are presented along with metabolic cues (nutrient sensing, coordination of carbon and nitrogen metabolism) affecting their composition. We conclude the review by suggesting further research avenues focused on the one hand on a comprehensive analysis of the physiology and epigenetics underlying cell wall degrading enzyme production in fungi and on the other hand on the analysis of proteins with unknown function and metagenomics of extremophilic consortia. The current advances in consolidated bioprocessing, altered secretory pathways and creation of designer plants are also examined. Furthermore, recent developments in enhancing the activity, stability and reusability of enzymes based on synergistic, proximity and entropic effects, fusion enzymes, structure-guided recombination between homologous enzymes and magnetic enzymes are considered with a view to improving saccharification. PMID:25804821

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

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

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

  19. Substrate recognition by glycoside hydrolase family 74 xyloglucanase from the basidiomycete Phanerochaete chrysosporium.

    PubMed

    Ishida, Takuya; Yaoi, Katsuro; Hiyoshi, Ayako; Igarashi, Kiyohiko; Samejima, Masahiro

    2007-11-01

    The basidiomycete Phanerochaete chrysosporium produces xyloglucanase Xgh74B, which has the glycoside hydrolase (GH) family 74 catalytic domain and family 1 carbohydrate-binding module, in cellulose-grown culture. The recombinant enzyme, which was heterologously expressed in the yeast Pichia pastoris, had high hydrolytic activity toward xyloglucan from tamarind seed (TXG), whereas other beta-1,4-glucans examined were poor substrates for the enzyme. The existence of the carbohydrate-binding module significantly affects adsorption of the enzyme on crystalline cellulose, but has no effect on the hydrolysis of xyloglucan, indicating that the domain may contribute to the localization of the enzyme. HPLC and MALDI-TOF MS analyses of the hydrolytic products of TXG clearly indicated that Xgh74B hydrolyzes the glycosidic bonds of unbranched glucose residues, like other GH family 74 xyloglucanases. However, viscometric analysis suggested that Xgh74B hydrolyzes TXG in a different manner from other known GH family 74 xyloglucanases. Gel permeation chromatography showed that Xgh74B initially produced oligosaccharides of degree of polymerization (DP) 16-18, and these oligosaccharides were then slowly hydrolyzed to final products of DP 7-9. In addition, the ratio of oligosaccharides of DP 7-9 versus those of DP 16-18 was dependent upon the pH of the reaction mixture, indicating that the affinity of Xgh74B for the oligosaccharides of DP 16-18 is affected by the ionic environment at the active site. PMID:17922847

  20. YZGD from Paenibacillus thiaminolyticus, a pyridoxal phosphatase of the HAD (haloacid dehalogenase) superfamily and a versatile member of the Nudix (nucleoside diphosphate x) hydrolase superfamily.

    PubMed

    Tirrell, Isaac M; Wall, Jennifer L; Daley, Christopher J; Denial, Sarah J; Tennis, Frances G; Galens, Kevin G; O'Handley, Suzanne F

    2006-03-15

    YZGD from Paenibacillus thiaminolyticus is a novel bifunctional enzyme with both PLPase (pyridoxal phosphatase) and Nudix (nucleoside diphosphate x) hydrolase activities. The PLPase activity is catalysed by the HAD (haloacid dehalogenase) superfamily motif of the enzyme, and the Nudix hydrolase activity is catalysed by the conserved Nudix signature sequence within a separate portion of the enzyme, as confirmed by site-directed mutagenesis. YZGD's phosphatase activity is very specific, with pyridoxal phosphate being the only natural substrate, while YZGD's Nudix activity is just the opposite, with YZGD being the most versatile Nudix hydrolase characterized to date. YZGD's Nudix substrates include the CDP-alcohols (CDP-ethanol, CDP-choline and CDP-glycerol), the ADP-coenzymes (NADH, NAD and FAD), ADP-sugars, TDP-glucose and, to a lesser extent, UDP- and GDP-sugars. Regardless of the Nudix substrate, one of the products is always a nucleoside monophosphate, suggesting a role in nucleotide salvage. Both the PLPase and Nudix hydrolase activities require a bivalent metal cation, but while PLPase activity is supported by Co2+, Mg2+, Zn2+ and Mn2+, the Nudix hydrolase activity is Mn2+-specific. YZGD's phosphatase activity is optimal at an acidic pH (pH 5), while YZGD's Nudix activities are optimal at an alkaline pH (pH 8.5). YZGD is the first enzyme reported to be a member of both the HAD and Nudix hydrolase superfamilies, the first PLPase to be recognized as a member of the HAD superfamily and the first Nudix hydrolase capable of hydrolysing ADP-x, CDP-x and TDP-x substrates with comparable substrate specificity. PMID:16336194

  1. YZGD from Paenibacillus thiaminolyticus, a pyridoxal phosphatase of the HAD (haloacid dehalogenase) superfamily and a versatile member of the Nudix (nucleoside diphosphate x) hydrolase superfamily

    PubMed Central

    Tirrell, Isaac M.; Wall, Jennifer L.; Daley, Christopher J.; Denial, Sarah J.; Tennis, Frances G.; Galens, Kevin G.; O'Handley, Suzanne F.

    2005-01-01

    YZGD from Paenibacillus thiaminolyticus is a novel bifunctional enzyme with both PLPase (pyridoxal phosphatase) and Nudix (nucleoside diphosphate x) hydrolase activities. The PLPase activity is catalysed by the HAD (haloacid dehalogenase) superfamily motif of the enzyme, and the Nudix hydrolase activity is catalysed by the conserved Nudix signature sequence within a separate portion of the enzyme, as confirmed by site-directed mutagenesis. YZGD's phosphatase activity is very specific, with pyridoxal phosphate being the only natural substrate, while YZGD's Nudix activity is just the opposite, with YZGD being the most versatile Nudix hydrolase characterized to date. YZGD's Nudix substrates include the CDP-alcohols (CDP-ethanol, CDP-choline and CDP-glycerol), the ADP-coenzymes (NADH, NAD and FAD), ADP-sugars, TDP-glucose and, to a lesser extent, UDP- and GDP-sugars. Regardless of the Nudix substrate, one of the products is always a nucleoside monophosphate, suggesting a role in nucleotide salvage. Both the PLPase and Nudix hydrolase activities require a bivalent metal cation, but while PLPase activity is supported by Co2+, Mg2+, Zn2+ and Mn2+, the Nudix hydrolase activity is Mn2+-specific. YZGD's phosphatase activity is optimal at an acidic pH (pH 5), while YZGD's Nudix activities are optimal at an alkaline pH (pH 8.5). YZGD is the first enzyme reported to be a member of both the HAD and Nudix hydrolase superfamilies, the first PLPase to be recognized as a member of the HAD superfamily and the first Nudix hydrolase capable of hydrolysing ADP-x, CDP-x and TDP-x substrates with comparable substrate specificity. PMID:16336194

  2. Relationship between plasma lipids and palmitoyl-CoA hydrolase and synthetase activities with peroxisomal proliferation in rats treated with fibrates.

    PubMed Central

    Alegret, M.; Ferrando, R.; Vázquez, M.; Adzet, T.; Merlos, M.; Laguna, J. C.

    1994-01-01

    1. The time-course of the effect of clofibrate (CFB), bezafibrate (BFB) and gemfibrozil (GFB) on lipid plasma levels and palmitoyl-CoA hydrolase and synthetase activities, as well as the correlations with the peroxisomal proliferation phenomenon have been studied in male Sprague-Dawley rats. 2. The administration of the three drugs caused a significant reduction in body weight gain, accompanied with a paradoxical increase in food intake in groups treated with BFB and GFB. 3. Drug treatment produced gross hepatomegaly and increase in peroxisomal beta-oxidation, and these parameters were strongly correlated. The order of potency was BFB > CFB > or = GFB. 4. Both plasma cholesterol (BFB approximately CFB > GFB) and triglyceride (BFB approximately GFB > CFB) levels were reduced in treated animals. There was an inverse correlation between these parameters and peroxisomal beta-oxidation, although the peroxisomal proliferation seemed to explain only a small part of the hypolipidemic effect observed. 5. Cytosolic and microsomal (but not mitochondrial) palmitoyl-CoA hydrolase activities were increased by the three drugs (BFB > CFB > GFB), probably by inducing the hydrolase I isoform, which is insensitive to inhibition by fibrates in vitro. The increased hydrolase activities were directly and strongly correlated with peroxisomal beta-oxidation. 6. Palmitoyl-CoA synthetase activity was also increased by the treatment with fibrates (BFB > CFB > GFB), probably as a consequence of the enhancement of hydrolase activities.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7915611

  3. Effects of synthetic alkamides on Arabidopsis fatty acid amide hydrolase activity and plant development.

    PubMed

    Faure, Lionel; Cavazos, Ronaldo; Khan, Bibi Rafeiza; Petros, Robby A; Koulen, Peter; Blancaflor, Elison B; Chapman, Kent D

    2015-02-01

    Alkamides and N-acylethanolamines (NAEs) are bioactive, amide-linked lipids that influence plant development. Alkamides are restricted to several families of higher plants and some fungi, whereas NAEs are widespread signaling molecules in both plants and animals. Fatty acid amide hydrolase (FAAH) has been described as a key contributor to NAE hydrolysis; however, no enzyme has been associated with alkamide degradation in plants. Herein reported is synthesis of 12 compounds structurally similar to a naturally occurring alkamide (N-isobutyl-(2E,6Z,8E)decatrienamide or affinin) with different acyl compositions more similar to plant NAEs and various amino alkyl head groups. These "hybrid" synthetic alkamides were tested for activity toward recombinant Arabidopsis FAAH and for their effects on plant development (i.e., cotyledon expansion and primary root length). A substantial increase in FAAH activity was discovered toward NAEs in vitro in the presence of some of these synthetic alkamides, such as N-ethyllauroylamide (4). This "enhancement" effect was found to be due, at least in part, to relief from product inhibition of FAAH by ethanolamine, and not due to an alteration in the oligomerization state of the FAAH enzyme. For several of these alkamides, an inhibition of seedling growth was observed with greater results in FAAH knockouts and less in FAAH over-expressing plants, suggesting that these alkamides could be hydrolyzed by FAAH in planta. The tight regulation of NAE levels in vivo appears to be important for proper seedling establishment, and as such, some of these synthetic alkamides may be useful pharmacological tools to manipulate the effects of NAEs in situ. PMID:25491532

  4. Discovery of bile salt hydrolase inhibitors using an efficient high-throughput screening system.

    PubMed

    Smith, Katie; Zeng, Ximin; Lin, Jun

    2014-01-01

    The global trend of restricting the use of antibiotic growth promoters (AGP) in animal production necessitates the need to develop valid alternatives to maintain productivity and sustainability of food animals. Previous studies suggest inhibition of bile salt hydrolase (BSH), an intestinal bacteria-produced enzyme that exerts negative impact on host fat digestion and utilization, is a promising approach to promote animal growth performance. To achieve the long term goal of developing novel alternatives to AGPs, in this study, a rapid and convenient high-throughput screening (HTS) system was developed and successfully used for identification of BSH inhibitors. With the aid of a high-purity BSH from a chicken Lactobacillus salivarius strain, we optimized various screening conditions (e.g. BSH concentration, reaction buffer pH, incubation temperature and length, substrate type and concentration) and establish a precipitation-based screening approach to identify BSH inhibitors using 96-well or 384-well microplates. A pilot HTS was performed using a small compound library comprised of 2,240 biologically active and structurally diverse compounds. Among the 107 hits, several promising and potent BSH inhibitors (e.g. riboflavin and phenethyl caffeate) were selected and validated by standard BSH activity assay. Interestingly, the HTS also identified a panel of antibiotics as BSH inhibitor; in particular, various tetracycline antibiotics and roxarsone, the widely used AGP, have been demonstrated to display potent inhibitory effect on BSH. Together, this study developed an efficient HTS system and identified several BSH inhibitors with potential as alternatives to AGP. In addition, the findings from this study also suggest a new mode of action of AGP for promoting animal growth. PMID:24454844

  5. Identifying glycoside hydrolase family 18 genes in the mycoparasitic fungal species Clonostachys rosea.

    PubMed

    Tzelepis, Georgios; Dubey, Mukesh; Jensen, Dan Funck; Karlsson, Magnus

    2015-07-01

    Clonostachysrosea is a mycoparasitic fungal species that is an efficient biocontrol agent against many plant diseases. During mycoparasitic interactions, one of the most crucial steps is the hydrolysis of the prey's fungal cell wall, which mainly consists of glucans, glycoproteins and chitin. Chitinases are hydrolytic enzymes responsible for chitin degradation and it is suggested that they play an important role in fungal-fungal interactions. Fungal chitinases belong exclusively to the glycoside hydrolase (GH) family 18.These GH18 proteins are categorized into three distinct phylogenetic groups (A, B and C), subdivided into several subgroups. In this study, we identified 14 GH18 genes in the C. rosea genome, which is remarkably low compared with the high numbers found in mycoparasitic Trichoderma species. Phylogenetic analysis revealed that C. rosea contains eight genes in group A, two genes in group B, two genes in group C, one gene encoding a putative ENGase (endo-β-N-acetylglucosaminidase) and the ech37 gene, which is of bacterial origin. Gene expression analysis showed that only two genes had higher transcription levels during fungal-fungal interactions, while eight out of 14 GH18 genes were triggered by chitin. Furthermore, deletion of the C group chiC2 gene decreased the growth inhibitory activity of C. rosea culture filtrates against Botrytis cinerea and Rhizoctonia solani, although the biocontrol ability of C. rosea against B. cinerea was not affected. In addition, a potential role of the CHIC2 chitinase in the sporulation process was revealed. These results provide new information about the role of GH18 proteins in mycoparasitic interactions. PMID:25881898

  6. Crystal Structures of a Glycoside Hydrolase Family 20 Lacto-N-biosidase from Bifidobacterium bifidum *

    PubMed Central

    Ito, Tasuku; Katayama, Takane; Hattie, Mitchell; Sakurama, Haruko; Wada, Jun; Suzuki, Ryuichiro; Ashida, Hisashi; Wakagi, Takayoshi; Yamamoto, Kenji; Stubbs, Keith A.; Fushinobu, Shinya

    2013-01-01

    Human milk oligosaccharides contain a large variety of oligosaccharides, of which lacto-N-biose I (Gal-β1,3-GlcNAc; LNB) predominates as a major core structure. A unique metabolic pathway specific for LNB has recently been identified in the human commensal bifidobacteria. Several strains of infant gut-associated bifidobacteria possess lacto-N-biosidase, a membrane-anchored extracellular enzyme, that liberates LNB from the nonreducing end of human milk oligosaccharides and plays a key role in the metabolic pathway of these compounds. Lacto-N-biosidase belongs to the glycoside hydrolase family 20, and its reaction proceeds via a substrate-assisted catalytic mechanism. Several crystal structures of GH20 β-N-acetylhexosaminidases, which release monosaccharide GlcNAc from its substrate, have been determined, but to date, a structure of lacto-N-biosidase is unknown. Here, we have determined the first three-dimensional structures of lacto-N-biosidase from Bifidobacterium bifidum JCM1254 in complex with LNB and LNB-thiazoline (Gal-β1,3-GlcNAc-thiazoline) at 1.8-Å resolution. Lacto-N-biosidase consists of three domains, and the C-terminal domain has a unique β-trefoil-like fold. Compared with other β-N-acetylhexosaminidases, lacto-N-biosidase has a wide substrate-binding pocket with a −2 subsite specific for β-1,3-linked Gal, and the residues responsible for Gal recognition were identified. The bound ligands are recognized by extensive hydrogen bonds at all of their hydroxyls consistent with the enzyme's strict substrate specificity for the LNB moiety. The GlcNAc sugar ring of LNB is in a distorted conformation near 4E, whereas that of LNB-thiazoline is in a 4C1 conformation. A possible conformational pathway for the lacto-N-biosidase reaction is discussed. PMID:23479733

  7. High level expression of organophosphorus hydrolase in Pichia pastoris by multicopy ophcM assembly.

    PubMed

    Shen, Wei; Shu, Min; Ma, Lixin; Ni, Hong; Yan, Hong

    2016-03-01

    The residues of organophosphorus pesticides bring serious impact on the environmental safety and people's health. Biodegradation of organophosphorus pesticides is recognized as an ideal method. An organophosphorus hydrolase (OPHCM) from Pseudomonas pseudoalcaligenes was synthesized and expressed in Pichia pastoris. The yield reached approximately 470 mg/l after a 6-d induction in shake flasks. To improve the enzyme production, we describe a novel approach to express OPHCM efficiently with a biobrick assembly method in vitro. Four recombinant plasmids containing 1-4 copies of ophcM-expressing cassettes were constructed and transformed into P. pastoris. Increasing the copy number of ophcM gene enhanced the expression level of OPHCM. The maximum yield and specific activity in P. pastoris harboring two-copy tandem ophcM-expressing cassettes reached 610 mg/l after a 6-d induction in shake flasks and 7.8 g/l in high-density fermentation with specific activity of 13.7 U/mg. The optimum pH and temperature of the recombinant OPHCM activity were 11.0 and 50 °C, respectively. In addition, the enzyme activity of recombinant OPHCM enhanced 57.6% and 30.1% in the presence of 1 mM Cd(2+) and 5% glycerol, respectively. The high expression and good properties of recombinant OPHCM provide an effective solution to solve the pollution of organophosphorus pesticides in the environment. Moreover, the approach for generating multicopy gene expressing vectors here will benefit the study for enhancing the expression level of genes of interest. PMID:26611611

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

    PubMed Central

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

    2016-01-01

    Proton pump inhibitors such as omeprazole (OME) reduce the severity of gastrointestinal (GI) ulcers induced by nonsteroidal anti-inflammatory drugs (NSAIDs) but can also increase the chance of dysbiosis. The aim of this study was to test the hypothesis that preventive use of a soluble epoxide hydrolase inhibitor (sEHI) such as TPPU can decrease NSAID-induced ulcers by increasing anti-inflammatory epoxyeicosatrienoic acids (EETs). Dose- [10, 30, and 100 mg/kg, by mouth (PO)] and time-dependent (6 and 18 hours) ulcerative effects of diclofenac sodium (DCF, an NSAID) were studied in the small intestine of Swiss Webster mice. Dose-dependent effects of TPPU (0.001–0.1 mg/kg per day for 7 days, in drinking water) were evaluated in DCF-induced intestinal toxicity and compared with OME (20 mg/kg, PO). In addition, the effect of treatment was studied on levels of Hb in blood, EETs in plasma, inflammatory markers such as myeloperoxidase (MPO) in intestinal tissue homogenates, and tissue necrosis factor-α (TNF-α) in serum. DCF dose dependently induced ulcers that were associated with both a significant (P < 0.05) loss of Hb and an increase in the level of MPO and TNF-α, with severity of ulceration highest at 18 hours. Pretreatment with TPPU dose dependently prevented ulcer formation by DCF, increased the levels of epoxy fatty acids, including EETs, and TPPU’s efficacy was comparable to OME. TPPU significantly (P < 0.05) reversed the effect of DCF on the level of Hb, MPO, and TNF-α. Thus sEHI might be useful in the management of NSAID-induced ulcers. PMID:26989141

  9. Synergism of Glycoside Hydrolase Secretomes from Two Thermophilic Bacteria Cocultivated on Lignocellulose

    PubMed Central

    Zhang, Kundi; Chen, Xiaohua; Schwarz, Wolfgang H.

    2014-01-01

    Two cellulolytic thermophilic bacterial strains, CS-3-2 and CS-4-4, were isolated from decayed cornstalk by the addition of growth-supporting factors to the medium. According to 16S rRNA gene-sequencing results, these strains belonged to the genus Clostridium and showed 98.87% and 98.86% identity with Clostridium stercorarium subsp. leptospartum ATCC 35414T and Clostridium cellulosi AS 1.1777T, respectively. The endoglucanase and exoglucanase activities of strain CS-4-4 were approximately 3 to 5 times those of strain CS-3-2, whereas the β-glucosidase activity of strain CS-3-2 was 18 times higher than that of strain CS-4-4. The xylanase activity of strain CS-3-2 was 9 times that of strain CS-4-4, whereas the β-xylosidase activity of strain CS-4-4 was 27 times that of strain CS-3-2. The enzyme activities in spent cultures following cocultivation of the two strains with cornstalk as the substrate were much greater than those in pure cultures or an artificial mixture of samples, indicating synergism of glycoside hydrolase secretomes between the two strains. Quantitative measurement of the two strains in the cocultivation system indicated that strain CS-3-2 grew robustly during the initial stages, whereas strain CS-4-4 dominated the system in the late-exponential phase. Liquid chromatography-tandem mass spectrometry analysis of protein bands appearing in the native zymograms showed that ORF3880 and ORF3883 from strain CS-4-4 played key roles in the lignocellulose degradation process. Both these open reading frames (ORFs) exhibited endoglucanase and xylanase activities, but ORF3880 showed tighter adhesion to insoluble substrates at 4, 25, and 60°C owing to its five carbohydrate-binding modules (CBMs). PMID:24532065

  10. Crystal Structure and Mutational Analysis of Isomalto-dextranase, a Member of Glycoside Hydrolase Family 27.

    PubMed

    Okazawa, Yuka; Miyazaki, Takatsugu; Yokoi, Gaku; Ishizaki, Yuichi; Nishikawa, Atsushi; Tonozuka, Takashi

    2015-10-23

    Arthrobacter globiformis T6 isomalto-dextranase (AgIMD) is an enzyme that liberates isomaltose from the non-reducing end of a polymer of glucose, dextran. AgIMD is classified as a member of the glycoside hydrolase family (GH) 27, which comprises mainly α-galactosidases and α-N-acetylgalactosaminidases, whereas AgIMD does not show α-galactosidase or α-N-acetylgalactosaminidase activities. Here, we determined the crystal structure of AgIMD. AgIMD consists of the following three domains: A, C, and D. Domains A and C are identified as a (β/α)8-barrel catalytic domain and an antiparallel β-structure, respectively, both of which are commonly found in GH27 enzymes. However, domain A of AgIMD has subdomain B, loop-1, and loop-2, all of which are not found in GH27 human α-galactosidase. AgIMD in a complex with trisaccharide panose shows that Asp-207, a residue in loop-1, is involved in subsite +1. Kinetic parameters of the wild-type and mutant enzymes for the small synthetic saccharide p-nitrophenyl α-isomaltoside and the polysaccharide dextran were compared, showing that Asp-207 is important for the catalysis of dextran. Domain D is classified as carbohydrate-binding module (CBM) 35, and an isomaltose molecule is seen in this domain in the AgIMD-isomaltose complex. Domain D is highly homologous to CBM35 domains found in GH31 and GH66 enzymes. The results here indicate that some features found in GH13, -31, and -66 enzymes, such as subdomain B, residues at the subsite +1, and the CBM35 domain, are also observed in the GH27 enzyme AgIMD and thus provide insights into the evolutionary relationships among GH13, -27, -31, -36, and -66 enzymes. PMID:26330557

  11. Insights into Exo- and Endoglucanase Activities of Family 6 Glycoside Hydrolases from Podospora anserina

    PubMed Central

    Poidevin, Laetitia; Feliu, Julia; Doan, Annick; Berrin, Jean-Guy; Bey, Mathieu; Coutinho, Pedro M.; Henrissat, Bernard; Record, Eric

    2013-01-01

    The ascomycete Podospora anserina is a coprophilous fungus that grows at late stages on droppings of herbivores. Its genome encodes a large diversity of carbohydrate-active enzymes. Among them, four genes encode glycoside hydrolases from family 6 (GH6), the members of which comprise putative endoglucanases and exoglucanases, some of them exerting important functions for biomass degradation in fungi. Therefore, this family was selected for functional analysis. Three of the enzymes, P. anserina Cel6A (PaCel6A), PaCel6B, and PaCel6C, were functionally expressed in the yeast Pichia pastoris. All three GH6 enzymes hydrolyzed crystalline and amorphous cellulose but were inactive on hydroxyethyl cellulose, mannan, galactomannan, xyloglucan, arabinoxylan, arabinan, xylan, and pectin. PaCel6A had a catalytic efficiency on cellotetraose comparable to that of Trichoderma reesei Cel6A (TrCel6A), but PaCel6B and PaCel6C were clearly less efficient. PaCel6A was the enzyme with the highest stability at 45°C, while PaCel6C was the least stable enzyme, losing more than 50% of its activity after incubation at temperatures above 30°C for 24 h. In contrast to TrCel6A, all three studied P. anserina GH6 cellulases were stable over a wide range of pHs and conserved high activity at pH values of up to 9. Each enzyme displayed a distinct substrate and product profile, highlighting different modes of action, with PaCel6A being the enzyme most similar to TrCel6A. PaCel6B was the only enzyme with higher specific activity on carboxymethylcellulose (CMC) than on Avicel and showed lower processivity than the others. Structural modeling predicts an open catalytic cleft, suggesting that PaCel6B is an endoglucanase. PMID:23645193

  12. Processivity and enzymatic mode of a glycoside hydrolase family 5 endoglucanase from Volvariella volvacea.

    PubMed

    Zheng, Fei; Ding, Shaojun

    2013-02-01

    EG1 is a modular glycoside hydrolase family 5 endoglucanase from Volvariella volvacea consisting of an N-terminal carbohydrate-binding module (CBM1) and a catalytic domain (CD). The ratios of soluble to insoluble reducing sugar produced from filter paper after 8 and 24 h of exposure to EG1 were 6.66 and 8.56, respectively, suggesting that it is a processive endoglucanase. Three derivatives of EG1 containing a core domain only or additional CBMs were constructed in order to evaluate the contribution of the CBM to the processivity and enzymatic mode of EG1 under stationary and agitated conditions. All four enzymatic forms exhibited the same mode of action on both soluble and insoluble cellulosic substrates with cellobiose as a main end product. An additional CBM fused at either the N or C terminus reduced specific activity toward soluble and insoluble celluloses under stationary reaction conditions. Deletion of the CBM significantly decreased enzyme processivity. Insertion of an additional CBM also resulted in a dramatic decrease in processivity in enzyme-substrate reaction mixtures incubated for 0.5 h, but this effect was reversed when reactions were allowed to proceed for longer periods (24 h). Further significant differences were observed in the substrate adsorption/desorption patterns of EG1 and enzyme derivatives equipped with an additional CBM under agitated reaction conditions. An additional family 1 CBM improved EG1 processivity on insoluble cellulose under highly agitated conditions. Our data indicate a strong link between high adsorption levels and low desorption levels in the processivity of EG1 and possibly other processive endoglucanses. PMID:23204424

  13. Oligomeric structure of proclavaminic acid amidino hydrolase: evolution of a hydrolytic enzyme in clavulanic acid biosynthesis.

    PubMed Central

    Elkins, Jonathan M; Clifton, Ian J; Hernández, Helena; Doan, Linh X; Robinson, Carol V; Schofield, Christopher J; Hewitson, Kirsty S

    2002-01-01

    During biosynthesis of the clinically used beta-lactamase inhibitor clavulanic acid, one of the three steps catalysed by clavaminic acid synthase is separated from the other two by a step catalysed by proclavaminic acid amidino hydrolase (PAH), in which the guanidino group of an intermediate is hydrolysed to give proclavaminic acid and urea. PAH shows considerable sequence homology with the primary metabolic arginases, which hydrolyse arginine to ornithine and urea, but does not accept arginine as a substrate. Like other members of the bacterial sub-family of arginases, PAH is hexameric in solution and requires Mn2+ ions for activity. Other metal ions, including Co2+, can substitute for Mn2+. Two new substrates for PAH were identified, N-acetyl-(L)-arginine and (3R)-hydroxy-N-acetyl-(L)-arginine. Crystal structures of PAH from Streptomyces clavuligerus (at 1.75 A and 2.45 A resolution, where 1 A=0.1 nm) imply how it binds beta-lactams rather than the amino acid substrate of the arginases from which it evolved. The structures also suggest how PAH selects for a particular alcohol intermediate in the clavam biosynthesis pathway. As observed for the arginases, each PAH monomer consists of a core of beta-strands surrounded by alpha-helices, and its active site contains a di-Mn2+ centre with a bridging water molecule responsible for hydrolytic attack on to the guanidino group of the substrate. Comparison of structures obtained under different conditions reveals different conformations of a flexible loop, which must move to allow substrate binding. PMID:12020346

  14. Opposite Effects of Gene Deficiency and Pharmacological Inhibition of Soluble Epoxide Hydrolase on Cardiac Fibrosis

    PubMed Central

    Zhang, Xu; Hammock, Bruce D.; Ai, Ding; Zhu, Yi

    2014-01-01

    Arachidonic acid-derived epoxyeicosatrienoic acids (EETs) are important regulators of cardiac remodeling; manipulation of their levels is a potentially useful pharmacological strategy. EETs are hydrolyzed by soluble epoxide hydrolase (sEH) to form the corresponding diols, thus altering and reducing the activity of these oxylipins. To better understand the phenotypic impact of sEH disruption, we compared the effect of EPHX2 gene knockout (EPHX2−/−) and sEH inhibition in mouse models. Measurement of plasma oxylipin profiles confirmed that the ratio of EETs/DHETs was increased in EPHX2−/− and sEH-inhibited mice. However, plasma concentrations of 9, 11, 15, 19-HETE were elevated in EPHX2−/− but not sEH-inhibited mice. Next, we investigated the role of this difference in cardiac dysfunction induced by Angiotensin II (AngII). Both EPHX2 gene deletion and inhibition protected against AngII-induced cardiac hypertrophy. Interestingly, cardiac dysfunction was attenuated by sEH inhibition rather than gene deletion. Histochemical staining revealed that compared with pharmacological inhibition, EPHX2 deletion aggravated AngII-induced myocardial fibrosis; the mRNA levels of fibrotic-related genes were increased. Furthermore, cardiac inflammatory response was greater in EPHX2−/− than sEH-inhibited mice with AngII treatment, as evidenced by increased macrophage infiltration and expression of MCP-1 and IL-6. In vitro, AngII-upregulated MCP-1 and IL-6 expression was significantly attenuated by sEH inhibition but promoted by EPHX2 deletion in cardiofibroblasts. Thus, compared with pharmacological inhibition of sEH, EPHX2 deletion caused the shift in arachidonic acid metabolism, which may led to pathological cardiac remodeling, especially cardiac fibrosis. PMID:24718617

  15. Highly active β-xylosidases of glycoside hydrolase family 43 operating on natural and artificial substrates.

    PubMed

    Jordan, Douglas B; Wagschal, Kurt; Grigorescu, Arabela A; Braker, Jay D

    2013-05-01

    The hemicellulose xylan constitutes a major portion of plant biomass, a renewable feedstock available for conversion to biofuels and other bioproducts. β-xylosidase operates in the deconstruction of the polysaccharide to fermentable sugars. Glycoside hydrolase family 43 is recognized as a source of highly active β-xylosidases, some of which could have practical applications. The biochemical details of four GH43 β-xylosidases (those from Alkaliphilus metalliredigens QYMF, Bacillus pumilus, Bacillus subtilis subsp. subtilis str. 168, and Lactobacillus brevis ATCC 367) are examined here. Sedimentation equilibrium experiments indicate that the quaternary states of three of the enzymes are mixtures of monomers and homodimers (B. pumilus) or mixtures of homodimers and homotetramers (B. subtilis and L. brevis). k cat and k cat/K m values of the four enzymes are higher for xylobiose than for xylotriose, suggesting that the enzyme active sites comprise two subsites, as has been demonstrated by the X-ray structures of other GH43 β-xylosidases. The K i values for D-glucose (83.3-357 mM) and D-xylose (15.6-70.0 mM) of the four enzymes are moderately high. The four enzymes display good temperature (K t (0.5) ∼ 45 °C) and pH stabilities (>4.6 to <10.3). At pH 6.0 and 25 °C, the enzyme from L. brevis ATCC 367 displays the highest reported k cat and k cat/K m on natural substrates xylobiose (407 s(-1), 138 s(-1) mM(-1)), xylotriose (235 s(-1), 80.8 s(-1) mM(-1)), and xylotetraose (146 s(-1), 32.6 s(-1) mM(-1)). PMID:23053115

  16. Renal Ischemia/Reperfusion Injury in Soluble Epoxide Hydrolase-Deficient Mice

    PubMed Central

    Zhu, Ye; Blum, Maximilian; Hoff, Uwe; Wesser, Tim; Fechner, Mandy; Westphal, Christina; Gürgen, Dennis; Catar, Rusan Ali; Philippe, Aurelie; Wu, Kaiyin; Bubalo, Gordana; Rothe, Michael; Weldon, Steven M.; Dragun, Duska; Schunck, Wolf-Hagen

    2016-01-01

    Aim 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrienoic acids (EETs) are cytochrome P450 (CYP)-dependent eicosanoids that play opposite roles in the regulation of vascular tone, inflammation, and apoptosis. 20-HETE aggravates, whereas EETs ameliorate ischemia/reperfusion (I/R)-induced organ damage. EETs are rapidly metabolized to dihydroxyeicosatrienoic acids (DHETs) by the soluble epoxide hydrolase (sEH). We hypothesized that sEH gene (EPHX2) deletion would increase endogenous EET levels and thereby protect against I/R-induced acute kidney injury (AKI). Methods Kidney damage was evaluated in male wildtype (WT) and sEH-knockout (KO)-mice that underwent 22-min renal ischemia followed by two days of reperfusion. CYP-eicosanoids were analyzed by liquid chromatography tandem mass spectrometry. Results Contrary to our initial hypothesis, renal function declined more severely in sEH-KO mice as indicated by higher serum creatinine and urea levels. The sEH-KO-mice also featured stronger tubular lesion scores, tubular apoptosis, and inflammatory cell infiltration. Plasma and renal EET/DHET-ratios were higher in sEH-KO than WT mice, thus confirming the expected metabolic consequences of sEH deficiency. However, CYP-eicosanoid profiling also revealed that renal, but not plasma and hepatic, 20-HETE levels were significantly increased in sEH-KO compared to WT mice. In line with this finding, renal expression of Cyp4a12a, the murine 20-HETE-generating CYP-enzyme, was up-regulated both at the mRNA and protein level, and Cyp4a12a immunostaining was more intense in the renal arterioles of sEH-KO compared with WT mice. Conclusion These results indicate that the potential beneficial effects of reducing EET degradation were obliterated by a thus far unknown mechanism leading to kidney-specific up-regulation of 20-HETE formation in sEH-KO-mice. PMID:26727266

  17. Lactase phlorhizin hydrolase turnover in vivo in water-fed and colostrum-fed newborn pigs.

    PubMed Central

    Dudley, M A; Burrin, D G; Quaroni, A; Rosenberger, J; Cook, G; Nichols, B L; Reeds, P J

    1996-01-01

    We have estimated the synthesis rates in vivo of precursor and brush-border (BB) polypeptides of lactase phlorhizin hydrolase (LPH) in newborn pigs fed with water or colostrum for 24h post partum. At the end of the feeding period, piglets were anaesthetized and infused intravenously for 3h with L-[4-3H]- phenylalanine. Blood and jejunal samples were collected at timed intervals. The precursor and BB forms of LPH were isolated from jejunal mucosa by immunoprecipitation followed by SDS/PAGE, and their specific radioactivity in Phe determined. The kinetics of precursor and BB LPH labelling were analysed by using a linear compartmental model. Immunoisolated LPH protein consisted of five polypeptides [high-mannose LPH precursor (proLPHh), complex glycosylated LPH precursor (proLPHe), intermediate complex glycosylated LPH precursor (proLPH1i) and two forms of BB LPH]. The fractional synthesis rate (Ks) of proLPHh and proLPHc (approx. 5%/min) were the same in the two groups but the absolute synthesis rate (in arbitrary units, min-1) of proLPHh in the colostrum-fed animals was twice that of the water-fed animals. The Ks values of proLPHi polypeptides were significantly different (water-fed, 3.89%/min; colostrum-fed, 1.6%/min), but the absolute synthesis rates did not differ. The Ks of BB LPH was not different between experimental treatment groups (on average 0.037%/min). However, the proportion of newly synthesized proLPHh processed to BB LPH was 48% lower in colostrum-fed than in water-fed animals. We conclude that in neonatal pigs, the ingestion of colostrum stimulates the synthesis of proLPHh but, at least temporarily, disrupts the processing of proLPH polypeptides to the BB enzyme. PMID:9003357

  18. Characterization of two juvenile hormone epoxide hydrolases by RNA interference in the Colorado potato beetle.

    PubMed

    Lü, Feng-Gong; Fu, Kai-Yun; Guo, Wen-Chao; Li, Guo-Qing

    2015-10-10

    In insect, juvenile hormone (JH) titers are tightly regulated in different development stages through synthesis and degradation pathways. During JH degradation, JH epoxide hydrolase (JHEH) converts JH to JH diol, and hydrolyses JH acid to JH acid diol. In this study, two full length LdJHEH cDNAs were cloned from Leptinotarsa decemlineata, and were provisionally designated LdJHEH1 and LdJHEH2. Both mRNAs were detectable in the thoracic muscles, brain-corpora cardiaca-corpora allata complex, foregut, midgut, hindgut, ventral ganglia, Malpighian tubules, fat bodies, epidermis, and hemocytes of the day 2 fourth-instar larvae, and in female ovaries as well as male reproductive organs of the adults. Moreover, both LdJHEH1 and LdJHEH2 were expressed throughout all larval life, with the highest peaks occurring 32h after ecdysis of the final (fourth) instar larvae. Four double-stranded RNAs (dsRNAs) (dsJHEH1-1, dsJHEH1-2, dsJHEH2-1, dsJHEH2-2) respectively targeting LdJHEH1 and LdJHEH2 were constructed and bacterially expressed. Ingestion of dsJHEH1-1, dsJHEH1-2, dsJHEH2-1, dsJHEH2-2, and a mixture of dsJHEH1-1+dsJHEH2-1 successfully knocked down corresponding target gene function, and significantly increased JH titer and upregulated Krüppel homolog 1 (LdKr-h1) mRNA level. Knockdown of either LdJHEH1 or LdJHEH2, or both genes slightly reduced larval weight and delayed larval development, and significantly impaired adult emergence. Therefore, it is suggested that knockdown LdJHEH1 and LdJHEH2 affected JH degradation in the Colorado potato beetle. PMID:26079572

  19. Processivity and Enzymatic Mode of a Glycoside Hydrolase Family 5 Endoglucanase from Volvariella volvacea

    PubMed Central

    Zheng, Fei

    2013-01-01

    EG1 is a modular glycoside hydrolase family 5 endoglucanase from Volvariella volvacea consisting of an N-terminal carbohydrate-binding module (CBM1) and a catalytic domain (CD). The ratios of soluble to insoluble reducing sugar produced from filter paper after 8 and 24 h of exposure to EG1 were 6.66 and 8.56, respectively, suggesting that it is a processive endoglucanase. Three derivatives of EG1 containing a core domain only or additional CBMs were constructed in order to evaluate the contribution of the CBM to the processivity and enzymatic mode of EG1 under stationary and agitated conditions. All four enzymatic forms exhibited the same mode of action on both soluble and insoluble cellulosic substrates with cellobiose as a main end product. An additional CBM fused at either the N or C terminus reduced specific activity toward soluble and insoluble celluloses under stationary reaction conditions. Deletion of the CBM significantly decreased enzyme processivity. Insertion of an additional CBM also resulted in a dramatic decrease in processivity in enzyme-substrate reaction mixtures incubated for 0.5 h, but this effect was reversed when reactions were allowed to proceed for longer periods (24 h). Further significant differences were observed in the substrate adsorption/desorption patterns of EG1 and enzyme derivatives equipped with an additional CBM under agitated reaction conditions. An additional family 1 CBM improved EG1 processivity on insoluble cellulose under highly agitated conditions. Our data indicate a strong link between high adsorption levels and low desorption levels in the processivity of EG1 and possibly other processive endoglucanses. PMID:23204424

  20. Evolution, substrate specificity and subfamily classification of glycoside hydrolase family 5 (GH5)

    PubMed Central

    2012-01-01

    Background The large Glycoside Hydrolase family 5 (GH5) groups together a wide range of enzymes acting on β-linked oligo- and polysaccharides, and glycoconjugates from a large spectrum of organisms. The long and complex evolution of this family of enzymes and its broad sequence diversity limits functional prediction. With the objective of improving the differentiation of enzyme specificities in a knowledge-based context, and to obtain new evolutionary insights, we present here a new, robust subfamily classification of family GH5. Results About 80% of the current sequences were assigned into 51 subfamilies in a global analysis of all publicly available GH5 sequences and associated biochemical data. Examination of subfamilies with catalytically-active members revealed that one third are monospecific (containing a single enzyme activity), although new functions may be discovered with biochemical characterization in the future. Furthermore, twenty subfamilies presently have no characterization whatsoever and many others have only limited structural and biochemical data. Mapping of functional knowledge onto the GH5 phylogenetic tree revealed that the sequence space of this historical and industrially important family is far from well dispersed, highlighting targets in need of further study. The analysis also uncovered a number of GH5 proteins which have lost their catalytic machinery, indicating evolution towards novel functions. Conclusion Overall, the subfamily division of GH5 provides an actively curated resource for large-scale protein sequence annotation for glycogenomics; the subfamily assignments are openly accessible via the Carbohydrate-Active Enzyme database at http://www.cazy.org/GH5.html. PMID:22992189