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Sample records for recombinant polyester-cleaving hydrolase

  1. Physarum polymalic acid hydrolase: Recombinant expression and enzyme activation.

    PubMed

    Mueller, Wolfgang; Haindl, Markus; Holler, Eggehard

    2008-12-19

    As a platform for syntheses of nanoconjugates in antitumor drug delivery, polymalic acid together with its tailoring specific exohydrolase is purified from plasmodium cultures of the slime mold Physarum polycephalum, a member of the phylum myxomycota. Polymalic acid hydrolase is expressed in an inactive form that functions as a molecular adapter for polymalic acid trafficking within the plasmodium and is activated only during secretion. Activation follows specific protein tyrosine phosphorylation and dissociation from plasma membranes. Purified inactive Physarum polymalic acid hydrolase, recombinantly expressed in yeast Saccharomyces, is activated on a preparative basis by the addition of plasma membrane fragments from plasmodia of P. polycephalum. Activation of polymalic acid hydrolase and inhibition of polymalic acid synthesis by protein tyrosine phosphorylation are complementary events and could indicate a joint signal response to plasma membrane damage.

  2. Trigger factor assisted folding of the recombinant epoxide hydrolases identified from C. pelagibacter and S. nassauensis.

    PubMed

    Saini, Priya; Wani, Shadil Ibrahim; Kumar, Ranjai; Chhabra, Ravneet; Chimni, Swapandeep Singh; Sareen, Dipti

    2014-12-01

    Epoxide hydrolases (EHs), are enantioselective enzymes as they catalyze the kinetic resolution of racemic epoxides into the corresponding enantiopure vicinal diols, which are useful precursors in the synthesis of chiral pharmaceutical compounds. Here, we have identified and cloned two putative epoxide hydrolase genes (cpeh and sneh) from marine bacteria, Candidatus pelagibacter ubique and terrestrial bacteria, Stackebrandtia nassauensis, respectively and overexpressed them in pET28a vector in Escherichia coli BL21(DE3). The CPEH protein (42kDa) was found to be overexpressed as inactive inclusion bodies while SNEH protein (40kDa) was found to form soluble aggregates. In this study, the recombinant CPEH was successfully transformed from insoluble aggregates to the soluble and functionally active form, using pCold TF vector, though with low EH activity. To prevent the soluble aggregate formation of SNEH, it was co-expressed with GroEL/ES chaperone and was also fused with trigger factor (TF) chaperone at its N-terminus. The TF chaperone-assisted correct folding of SNEH led to a purified active EH with a specific activity of 3.85μmol/min/mg. The pure enzyme was further used to biocatalyze the hydrolysis of 10mM benzyl glycidyl ether (BGE) and α-methyl styrene oxide (MSO) with an enantiomeric excess of the product (eep) of 86% and 73% in 30 and 15min, respectively. In conclusion, this is the first report about the heterologous expression of epoxide hydrolases using TF as a molecular chaperone in pCold TF expression vector, resulting in remarkable increase in the solubility and activity of the otherwise improperly folded recombinant epoxide hydrolases.

  3. Recombinant S-adenosylhomocysteine hydrolase from Thermotoga maritima: cloning, overexpression, characterization, and thermal purification studies.

    PubMed

    Lozada-Ramírez, J D; Sánchez-Ferrer, A; García-Carmona, F

    2013-06-01

    S-Adenosylhomocysteine hydrolase (SAHase) encoded by sahase gene is a determinant when catalyzing the reversible conversion of adenosine and homocysteine to S-adenosylhomocysteine in most living organisms. The sahase gene was isolated from the genome of the highly thermostable anaerobic bacteria Thermotoga maritima, and then it was cloned, characterized, overexpressed using Escherichia coli, and partially purified by thermal precipitation. The thermal purification of the recombinant SAHase resulted in changes in the circular dichroism spectra. As a result of this analysis, it was possible to determine the structural changes in the composition of the α-helix and β-sheet content of the recombinant enzyme after purification. Moreover, a predicted secondary structure and 3D structural model was rendered by comparative molecular modeling to further understand the molecular function of this protein including its attractive biotechnological use.

  4. A colorimetric assay for determination of methyl parathion using recombinant methyl parathion hydrolase.

    PubMed

    Anh, Dau Hung; Cheunrungsikul, Kritsananporn; Wichitwechkarn, Jesdawan; Surareungchai, Werasak

    2011-05-01

    A simple, rapid and sensitive colorimetric dipstick assay for the detection of the organophosphorous insecticide methyl parathion (MPT) residue in vegetables was developed. The assay was based on the hydrolysis of MPT by a recombinant methyl parathion hydrolase (recMPH), the encoding gene of which was isolated from Burkholderia cepacia, a soil bacterium indigenous to Thailand. This reaction generates protons leading to a change in pH that correlates with the amount of MPH present. Hence, the pH indicator bromothymol blue was used to monitor the MPH hydrolysis as the associated color changes can be observed by the naked eye. The recMPH was immobilized on a PVDF membrane to establish a dipstick assay format. The assays could detect MPT residues in spiked vegetable samples at the concentration of 1 mg/L without using analytical instrumentation. The test is reusable and stable for up to 3 months in the absence of any preservatives.

  5. Expression pattern of recombinant organophosphorus hydrolase from Flavobacterium sp. ATCC 27551 in Escherichia coli.

    PubMed

    Kwak, Yunyoung; Rhee, In-Koo; Shin, Jae-Ho

    2013-09-01

    Concerned with the influence of tagging system on the expression of heterogeneous protein in Escherichia coli, we attempted to express the organophosphorus hydrolase (OPH) of Flavobacterium sp. ATCC 27551 in E. coli. Recombinant OPH was overproduced successfully in E. coli when modified without the use of a tobacco etch virus (TEV) protease cleavage sequence. In addition, though there has never been a report on the extracellular secretion of recombinant OPH harboring native Tat signal peptides in E. coli, the produced protein was observed to be secreted extracellularly. Through the use of reverse transcriptional quantitative real-time PCR and comparison of the predicted folding rate, it was determined that OPH expression may be affected by the existence of a TEV protease cleavage sequence at the C-terminus during the process of translated protein folding, leading to the suppressed OPH activity. With the potential compatibility between native Tat signal peptides of OPH and E. coli Tat pathway secretion system, we report a successful expression of recombinant OPH harboring native Tat signal peptides in E. coli, for the first time.

  6. Cloning and expression of a phloretin hydrolase gene from Eubacterium ramulus and characterization of the recombinant enzyme.

    PubMed

    Schoefer, Lilian; Braune, Annett; Blaut, Michael

    2004-10-01

    Phloretin hydrolase catalyzes the hydrolytic C-C cleavage of phloretin to phloroglucinol and 3-(4-hydroxyphenyl)propionic acid during flavonoid degradation in Eubacterium ramulus. The gene encoding the enzyme was cloned by screening a gene library for hydrolase activity. The insert of a clone conferring phloretin hydrolase activity was sequenced. Sequence analysis revealed an open reading frame of 822 bp (phy), a putative promoter region, and a terminating stem-loop structure. The deduced amino acid sequence of phy showed similarities to a putative protein of the 2,4-diacetylphloroglucinol biosynthetic operon from Pseudomonas fluorescens. The phloretin hydrolase was heterologously expressed in Escherichia coli and purified. The molecular mass of the native enzyme was approximately 55 kDa as determined by gel filtration. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of phy indicated molecular masses of 30 and 30.8 kDa, respectively, suggesting that the enzyme is a homodimer. The recombinant phloretin hydrolase catalyzed the hydrolysis of phloretin to equimolar amounts of phloroglucinol and 3-(4-hydroxyphenyl)propionic acid. The optimal temperature and pH of the catalyzed reaction mixture were 37 degrees C and 7.0, respectively. The K(m) for phloretin was 13 +/- 3 microM and the k(cat) was 10 +/- 2 s(-1). The enzyme did not transform phloretin-2'-glucoside (phloridzin), neohesperidin dihydrochalcone, 1,3-diphenyl-1,3-propandione, or trans-1,3-diphenyl-2,3-epoxy-propan-1-one. The catalytic activity of the phloretin hydrolase was reduced by N-bromosuccinimide, o-phenanthroline, N-ethylmaleimide, and CuCl(2) to 3, 20, 35, and 85%, respectively. Phloroglucinol and 3-(4-hydroxyphenyl)propionic acid reduced the activity to 54 and 70%, respectively.

  7. High-level expression of active recombinant ubiquitin carboxyl-terminal hydrolase of Drosophila melanogaster in Pichia pastoris.

    PubMed

    Jin, Feng-liang; Xu, Xiao-xia; Yu, Xiao-qiang; Ren, Shun-xiang

    2009-06-01

    Ubiquitin carboxyl-terminal hydrolases (UCHs) are implicated in the proteolytic processing of polymeric ubiquitin. The high specificity for the recognition site makes UCHs useful enzymes for in vitro cleavage of ubiquitin fusion proteins. In this work, an active C-terminal His-tagged UCH from Drosophila melanogaster (DmUCH) was produced as a secretory form in a recombinant strain of the methylotrophic yeast Pichia pastoris. The production of recombinant DmUCH by Mut(s) strain was much higher than that by Mut(+) strain, which was confirmed by Western blot analysis. When expression was induced at pH 6.0 in a BMMY/methanol medium, the concentration of recombinant DmUCH reached 210 mg l(-1). With the (His)(6)-tag, the recombinant DmUCH was easily purified by Ni-NTA chromatography and 18 mg pure active DmUCH were obtained from 100ml culture broth supernatant. Ubiquitin-magainin fusion protein was efficiently cleaved by DmUCH, yielding recombinant magainin with high antimicrobial activity. After removing the contaminants by Ni-NTA chromatography, recombinant magainin was purified to homogeneity easily by reversed-phase HPLC. Analysis of the recombinant magainin by ESI-MS showed that the molecular weight of the purified recombinant magainin was 2465 Da, which perfectly matches the mass calculated from the amino acid sequence. The result of mass spectrometry confirmed that the purified His-tagged DmUCH can recognize the ubiquitin-magainin fusion protein and cleave it at the carboxyl terminus of ubiquitin precisely. Our results showed that P. pastoris is a robust system to express the secreted form of DmUCH.

  8. 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.

  9. Glycoside hydrolases having multiple hydrolase activities

    DOEpatents

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

    2017-08-08

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

  10. Crystallization and preliminary X-ray diffraction analysis of recombinant hydrolase domain of 10-formyltetrahydrofolate dehydrogenase.

    PubMed

    Chumanevich, Alexander A; Davies, Christopher; Krupenko, Sergey A

    2002-10-01

    10-Formyltetrahydrofolate dehydrogenase (FDH) is an abundant enzyme in liver cytosol. It is important for the regulation of 10-formyltetrahydrofolate/tetrahydrofolate pools, for de novo purine biosynthesis and for the removal of formate in the form of CO(2). The enzyme is a natural fusion of two unrelated genes and consists of two functional catalytic domains. Here, the crystallization of the N-terminal domain of FDH is reported. This domain binds folate and functions as a 10-formyltetrahydrofolate hydrolase. The crystals grow as either spear-shaped needles or large plates, with the largest crystals reaching dimensions of 1.2 x 0.2 x 0.05 mm. Diffraction analysis revealed the space group to be P2(1)2(1)2, with unit-cell parameters a = 100.00, b = 64.63, c = 64.59 A. Based on the estimated solvent content, there is one 34 kDa molecule in the asymmetric unit. A native data set extending to 2.3 A resolution has been collected with good merging statistics.

  11. Enzymatic synthesis of S-adenosylhomocysteine: immobilization of recombinant S-adenosylhomocysteine hydrolase from Corynebacterium glutamicum (ATCC 13032).

    PubMed

    Lozada-Ramírez, J D; Sánchez-Ferrer, A; García-Carmona, F

    2012-03-01

    Recombinant S-adenosylhomocysteine hydrolase from Corynebacterium glutamicum (CgSAHase) was covalently bound to Eupergit® C. The maximum yield of bound protein was 91% and the catalytic efficiency was 96.9%. When the kinetic results for the immobilized enzyme were compared with those for the soluble enzyme, no decrease in the catalytic efficiency of the former was detected. Both soluble and immobilized enzymes showed similar optimum pH and temperature ranges. The reuse of immobilized CgSAHase caused a loss of synthetic activity due to NAD(+) release, although the binding to the support was sufficiently strong for up to 5 cycles with 95% conversion efficiency. The immobilized enzyme was incubated every 3 cycles with 100 μM NAD(+) to recover the loss of activity after 5 cycles. This maintained the activity for another 50 cycles. The purification of S-adenosylhomocysteine (SAH) provided an overall yield of 76% and 98% purity as determined by HPLC and NMR analyses. The results indicate the suitability of immobilized CgSAHase for synthesizing SAH and other important S-nucleosidylhomocysteine.

  12. Dramatic Differences in Organophosphorus Hydrolase Activity between Human and Chimeric Recombinant Mammalian Paraoxonase-1 Enzymes

    DTIC Science & Technology

    2009-01-01

    organophosphorus (OP) compounds. HuPON1 and bacterially expressed chimeric recombinant PON1s (G2E6 and G3C9) differ by multiple amino acids, none of which are in the...phenyl acetate than G3C9. In contrast, bacterial PON1s are better able to promote hydrolysis of paraoxon, whereas HuPON1 is considerably better at...catalyzing the hydrolysis of nerve agents VX and VR. These studies demonstrate that mutations distant from the active site of PON1 have large and

  13. Aspergillus fumigatus Produces Two Arabinofuranosidases From Glycosyl Hydrolase Family 62: Comparative Properties of the Recombinant Enzymes.

    PubMed

    Pérez, Rodrigo; Eyzaguirre, Jaime

    2016-04-01

    The genes of two α-L-arabinofuranosidases (AbfI and II) from family GH 62 have been identified in the genome of Aspergillus fumigatus wmo. Both genes have been expressed in Pichia pastoris and the enzymes have been purified and characterized. AbfI is composed of 999 bp, does not contain introns and codes for a protein (ABFI) of 332 amino acid residues. abfII has 1246 bp, including an intron of 51 bp; the protein ABFII has 396 amino acid residues; it includes a family 1 carbohydrate-binding module (CBM) in the N-terminal region, followed by a catalytic module. The sequence of ABFI and the catalytic module of ABFII show a 79 % identity. Both enzymes are active on p-nitrophenyl α-L-arabinofuranoside (pNPAra) with KM of 94.2 and 3.9 mM for ABFI and II, respectively. Optimal temperature for ABFI is 37 °C and for ABFII 42 °C, while the pH optimum is about 4.5 to 5 for both enzymes. ABFII shows a higher thermostability. When assayed using natural substrates, both show higher activity over rye arabinoxylan as compared to wheat arabinoxylan. ABFII only is active on sugar beet pulp arabinan and both are inactive towards debranched arabinan. The higher thermostability, higher affinity for pNPAra and wider activity over natural substrates shown by ABFII may be related to the presence of a CBM. The availability of the recombinant enzymes may be useful in biotechnological applications for the production of arabinose.

  14. Dramatic differences in organophosphorus hydrolase activity between human and chimeric recombinant mammalian paraoxonase-1 enzymes†

    PubMed Central

    Otto, Tamara C.; Harsch, Christina K.; Yeung, David T.; Magliery, Thomas J.; Cerasoli, Douglas M.; Lenz, David E.

    2009-01-01

    Human serum paraoxonase-1 (HuPON1) has the capacity to hydrolyze aryl esters, lactones, oxidized phospholipids, and organophosphorus (OP) compounds. HuPON1 and bacterially expressed chimeric recombinant PON1s (G2E6 and G3C9) differ by multiple amino acids, none of which are in the putative enzyme active site. To address the importance of these amino acid differences, the abilities of HuPON1, G2E6, G3C9, and several variants to hydrolyze phenyl acetate, paraoxon, and V-type OP nerve agents were examined. HuPON1 and G2E6 have a ten-fold greater catalytic efficiency toward phenyl acetate than G3C9. In contrast, bacterial PON1s are better able to promote hydrolysis of paraoxon, whereas HuPON1 is considerably better at catalyzing the hydrolysis of the nerve agents VX and VR. These studies demonstrate that mutations distant from the active site of PON1 have large and unpredictable effects on the substrate specificities and possibly the hydrolytic mechanisms of HuPON1, G2E6, and G3C9. The replacement of residue H115 in the putative active site with tryptophan (H115W) has highly disparate effects on HuPON1 and G2E6. In HuPON1, variant H115W loses the ability to hydrolyze VR but has improved activity toward paraoxon and VX. The H115W variant of G2E6 has similar paraoxonase activity to wild type G2E6, modest activity with phenyl acetate and VR, and increased VX hydrolysis. VR inhibits H115W HuPON1 competitively when paraoxon is the substrate and non-competitively when VX is the substrate. We have identified the first variant of HuPON1, H115W, that displays significantly enhanced catalytic activity against an authentic V-type nerve agent. PMID:19764813

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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    SciTech Connect

    Ficko-Blean, Elizabeth; Boraston, Alisdair B.

    2005-09-01

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

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

    SciTech Connect

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

    2014-10-15

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

  18. Silica Gel for Enhanced Activity and Hypochlorite Protection of Cyanuric Acid Hydrolase in Recombinant Escherichia coli

    PubMed Central

    Radian, Adi; Aukema, Kelly G.; Aksan, Alptekin

    2015-01-01

    ABSTRACT Chlorinated isocyanuric acids are widely used water disinfectants that generate hypochlorite, but with repeated application, they build up cyanuric acid (CYA) that must be removed to maintain disinfection. 3-Aminopropyltriethoxysilane (APTES)-treated Escherichia coli cells expressing cyanuric acid hydrolase (CAH) from Moorella thermoacetica exhibited significantly high CYA degradation rates and provided protection against enzyme inactivation by hypochlorite (chlorine). APTES coating or encapsulation of cells had two benefits: (i) overcoming diffusion limitations imposed by the cell wall and (ii) protecting against hypochlorite inactivation of CAH activity. Cells encapsulated in APTES gels degraded CYA three times faster than nonfunctionalized tetraethoxysilane (TEOS) gels, and cells coated with APTES degraded CYA at a rate of 29 µmol/min per mg of CAH protein, similar to the rate with purified enzyme. UV spectroscopy, fluorescence spectroscopy, and scanning electron microscopy showed that the higher rates were due to APTES increasing membrane permeability and enhancing cyanuric acid diffusion into the cytoplasm to reach the CAH enzyme. Purified CAH enzyme was shown to be rapidly inactivated by hypochlorite. APTES aggregates surrounding cells protected via the amine groups reacting with hypochlorite as shown by pH changes, zeta potential measurements, and infrared spectroscopy. APTES-encapsulated E. coli cells expressing CAH degraded cyanuric acid at high rates in the presence of 1 to 10 ppm hypochlorite, showing effectiveness under swimming pool conditions. In contrast, CAH activity in TEOS gels or free cells was completely inactivated by hypochlorite. These studies show that commercially available silica materials can selectively enhance, protect, and immobilize whole-cell biocatalysts for specialized applications. PMID:26530383

  19. Biosensor for Direct Determination of Fenitrothion and EPN Using Recombinant Pseudomonas putida JS444 with Surface Expressed Organophosphorus Hydrolase. 1. Modified Clark Oxygen Electrode

    PubMed Central

    Lei, Yu; Mulchandani, Priti; Chen, Wilfred; Mulchandani, Ashok

    2006-01-01

    This paper reports a first microbial biosensor for rapid and cost-effective determination of organophosphorus pesticides fenitrothion and EPN. The biosensor consisted of recombinant PNP-degrading/oxidizing bacteria Pseudomonas putida JS444 anchoring and displaying organophosphorus hydrolase (OPH) on its cell surface as biological sensing element and a dissolved oxygen electrode as the transducer. Surface-expressed OPH catalyzed the hydrolysis of fenitrothion and EPN to release 3-methyl-4-nitrophenol and p-nitrophenol, respectively, which were oxidized by the enzymatic machinery of Pseudomonas putida JS444 to carbon dioxide while consuming oxygen, which was measured and correlated to the concentration of organophosphates. Under the optimum operating conditions, the biosensor was able to measure as low as 277 ppb of fenitrothion and 1.6 ppm of EPN without interference from phenolic compounds and other commonly used pesticides such as carbamate pesticides, triazine herbicides and organophosphate pesticides without nitrophenyl substituent. The applicability of the biosensor to lake water was also demonstrated.

  20. Enzymic characterization of two recombinant xyloglucan endotransglucosylase/hydrolase (XTH) proteins of Arabidopsis and their effect on root growth and cell wall extension.

    PubMed

    Maris, An; Suslov, Dmitry; Fry, Stephen C; Verbelen, Jean-Pierre; Vissenberg, Kris

    2009-01-01

    Xyloglucan endotransglucosylase/hydrolases (XTHs) are enzymes involved in the modification of load-bearing cell wall components. They cleave xyloglucan chains and, often, re-form bonds to the non-reducing ends of available xyloglucan molecules in plant primary cell walls. The enzymic properties and effects on root growth of two Arabidopsis thaliana XTHs belonging to subgroup I/II, that are predominantly expressed in root hairs and in non-elongating zones of the root, were analysed here. AtXTH14 and AtXTH26 were recombinantly produced in Pichia and subsequently purified. Both proteins were found to exhibit xyloglucan endotransglucosylase (XET; EC 2.4.1.207) but not xyloglucan endohydrolase (XEH; EC 3.2.1.151) activity. Their endotransglucosylase activity was at least 70x greater on xyloglucan rather than on mixed-linkage beta-glucan. Differences were found in pH- and temperature-dependence as well as in acceptor-substrate preferences. Furthermore, the specific activity of XET was approximately equal for the two enzymes. Removal of N-linked sugar residues by Endo H treatment reduced XET activity to 60%. Constant-load extensiometry experiments revealed that the enzymes reduce the extension in a model system of heat-inactivated isolated cell walls. When given to growing roots, either of these XTH proteins reduced cell elongation in a concentration-dependent manner and caused abnormal root hair morphology. This is the first time that recombinant and purified XTHs added to growing roots have exhibited a clear effect on cell elongation. It is proposed that these specific XTH isoenzymes play a role in strengthening the side-walls of root-hairs and cell walls in the root differentiation zone after the completion of cell expansion.

  1. Expression, purification, immunogenicity and protective efficacy of a recombinant nucleoside hydrolase from Leishmania donovani, a vaccine candidate for preventing cutaneous leishmaniasis.

    PubMed

    McAtee, C Patrick; Seid, Christopher A; Hammond, Molly; Hudspeth, Elissa; Keegan, Brian P; Liu, Zhuyun; Wei, Junfei; Zhan, Bin; Arjona-Sabido, Raul; Cruz-Chan, Vladimir; Dumonteil, Eric; Hotez, Peter J; Bottazzi, Maria Elena

    2017-02-01

    The nucleoside hydrolase gene from Leishmania donovani was cloned and expressed in Escherichia coli as a full length 36-kDa protein (LdNH36). Following lysis and extraction, the protein was purified by anion exchange and gel filtration chromatography. The purified protein had a molecular mass of approximately 36-kDa and was confirmed to be >99% pure. Using a nucleoside hydrolase assay, the protein was found to exhibit a Km of 741 ± 246 μM. Protein integrity was confirmed by lithium dodecyl sulfate polyacrylamide gel electrophoresis (LDS-PAGE), mass spectrometry (MS), and enzymatic assay. Analysis of antibody levels from immunized mice indicated that LdNH36 alone or in a stable emulsion with the Toll-like receptor-4 ligand glucopyranosyl lipid adjuvant (GLA-SE) as immunostimulant induced high levels of antigen-specific IgG antibodies. The cellular immune response indicated a Th1 response in mice immunized with LdNH36, but only when formulated with GLA-SE. Mice immunized with the LdNH36 antigen in combination with the GLA-SE adjuvant and challenged with Leishmania mexicana showed significant reductions (>20 fold) in parasite burden, confirming the protective efficacy of this vaccine candidate. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. NatB Domain-Containing CRA-1 Antagonizes Hydrolase ACER-1 Linking Acetyl-CoA Metabolism to the Initiation of Recombination during C. elegans Meiosis

    PubMed Central

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

    2015-01-01

    The formation of DNA double-strand breaks (DSBs) must take place during meiosis to ensure the formation of crossovers, which are required for accurate chromosome segregation, therefore avoiding aneuploidy. However, DSB formation must be tightly regulated to maintain genomic integrity. How this regulation operates in the context of different chromatin architectures and accessibility, and how it is linked to metabolic pathways, is not understood. We show here that global histone acetylation levels undergo changes throughout meiotic progression. Moreover, perturbations to global histone acetylation levels are accompanied by changes in the frequency of DSB formation in C. elegans. We provide evidence that the regulation of histone acetylation requires CRA-1, a NatB domain-containing protein homologous to human NAA25, which controls the levels of acetyl-Coenzyme A (acetyl-CoA) by antagonizing ACER-1, a previously unknown and conserved acetyl-CoA hydrolase. CRA-1 is in turn negatively regulated by XND-1, an AT-hook containing protein. We propose that this newly defined protein network links acetyl-CoA metabolism to meiotic DSB formation via modulation of global histone acetylation. PMID:25768301

  3. Biosensor for direct determination of fenitrothion and EPN using recombinant Pseudomonas putida JS444 with surface-expressed organophosphorous hydrolase. 2. Modified carbon paste electrode.

    PubMed

    Lei, Yu; Mulchandani, Priti; Chen, Wilfred; Mulchandani, Ashok

    2007-03-01

    A whole cell-based amperometric biosensor for highly selective, sensitive, rapid, and cost-effective determination of the organophosphate pesticides fenitrothion and ethyl p-nitrophenol thio-benzene phosphonate (EPN) is discussed. The biosensor comprised genetically engineered p-nitrophenol (PNP)-degrading bacteria Pseudomonas putida JS444 anchoring and displaying organophosphorous hydrolase (OPH) on its cell surface as biological sensing element and carbon paste electrode as the amperometric transducer. Surface-expressed OPH catalyzed the hydrolysis of organophosphorous pesticides such as fenitrothion and EPN to release PNP and 3-methyl-4- nitrophenol, respectively, which were subsequently degraded by the enzymatic machinery of P. putida JS444 through electrochemically active intermediates to the TCA cycle. The electro-oxidization current of the intermediates was measured and correlated to the concentration of organophosphates. Operating at optimum conditions, 0.086 mg dry wt of cell operating at 600 mV of applied potential (vs Ag/AgCl reference) in 50 mM citrate phosphate buffer, pH 7.5, with 50 muM CoCl2 at room temperature, the biosensor measured as low as 1.4 ppb of fenitrothion and 1.6 ppb of EPN. There was no interference from phenolic compounds, carbamate pesticides, triazine herbicides, or organophosphate pesticides without nitrophenyl substituent. The service life of the biosensor and the applicability to lake water were also demonstrated.

  4. Alterations of structure and hydrolase activity of parkinsonism-associated human ubiquitin carboxyl-terminal hydrolase L1 variants.

    PubMed

    Nishikawa, Kaori; Li, Hang; Kawamura, Ryoichi; Osaka, Hitoshi; Wang, Yu-Lai; Hara, Yoko; Hirokawa, Takatsugu; Manago, Yoshimasa; Amano, Taiju; Noda, Mami; Aoki, Shunsuke; Wada, Keiji

    2003-04-25

    Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is a neuron-specific ubiquitin recycling enzyme. A mutation at residue 93 and polymorphism at residue 18 within human UCH-L1 are linked to familial Parkinson's disease and a decreased Parkinson's disease risk, respectively. Thus, we constructed recombinant human UCH-L1 variants and examined their structure (using circular dichroism) and hydrolase activities. We confirmed that an I93M substitution results in a decrease in kcat (45.6%) coincident with an alteration in alpha-helical content. These changes may contribute to the pathogenesis of Parkinson's disease. In contrast, an S18Y substitution results in an increase in kcat (112.6%) without altering the circular dichroistic spectrum. These data suggest that UCH-L1 hydrolase activity may be inversely correlated with Parkinson's disease risk and that the hydrolase activity is protective against the disease. Furthermore, we found that oxidation of UCH-L1 by 4-hydroxynonenal, a candidate for endogenous mediator of oxidative stress-induced neuronal cell death, results in a loss of hydrolase activity. Taken together, these results suggest that further studies of altered UCH-L1 hydrolase function may provide new insights into a possible common pathogenic mechanism between familial and sporadic Parkinson's disease.

  5. Variants of glycoside hydrolases

    DOEpatents

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

    2017-07-11

    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. Variants of glycoside hydrolases

    DOEpatents

    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.

  7. Variants of glycoside hydrolases

    DOEpatents

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

    2011-04-26

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

  8. 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. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  9. 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

  10. High-throughput cloning, expression and purification of glycoside hydrolases using Ligation-Independent Cloning (LIC).

    PubMed

    Camilo, Cesar M; Polikarpov, Igor

    2014-07-01

    Recent advances in DNA sequencing techniques have led to an explosion in the amount of available genome sequencing data and this provided an inexhaustible source of uncharacterized glycoside hydrolases (GH) to be studied both structurally and enzymatically. Ligation-Independent Cloning (LIC), an interesting alternative to traditional, restriction enzyme-based cloning, and commercial recombinatorial cloning, was adopted and optimized successfully for a high throughput cloning, expression and purification pipeline. Using this platform, 130 genes encoding mainly uncharacterized glycoside hydrolases from 13 different organisms were cloned and submitted to a semi-automated protein expression and solubility screening in Escherichia coli, resulting in 73 soluble targets. The high throughput approach proved to be a powerful tool for production of recombinant glycoside hydrolases for further structural and biochemical characterization and confirmed that thioredoxin fusion tag (TRX) is a better choice to increase solubility of recombinant glycoside hydrolases expressed in E. coli, when compared to His-tag alone.

  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. Polyglycine hydrolases secreted by pathogenic fungi

    USDA-ARS?s Scientific Manuscript database

    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...

  13. 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.

  14. pHluorin-based in vivo assay for hydrolase screening.

    PubMed

    Schuster, Sascha; Enzelberger, Markus; Trauthwein, Harald; Schmid, Rolf D; Urlacher, Vlada B

    2005-05-01

    pHluorin, a pH-sensitive mutant of green fluorescent protein (GFP), acts as a sensor for intracellular pH shifts, triggered by hydrolytic enzymes. This principle was used to develop a pHluorin-based in vivo assay for hydrolase screening. The presented assay was evaluated for Escherichia coli (E. coli) cells, producing heterologous pHluorin and an esterase from Geobacillus stearothermophilus which is considered as a model hydrolase. Subsequently, the utility of this detection system was also demonstrated with recombinantly expressed hydantoinase and amidase in E. coli. This in vivo assay also shows capability for readout with flow cytometric devices. Population shifts of pHluorin-expressing E. coli cells were easily recognized due to pH changes caused by substrate hydrolysis.

  15. 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

  16. Similarities between catalase and cytosolic epoxide hydrolase.

    PubMed

    Guenthner, T M; Qato, M; Whalen, R; Glomb, S

    1989-01-01

    Cytosolic epoxide hydrolase, measured as trans-stilbene oxide hydrolase activity, was isolated and purified from human and guinea pig liver cytosol. Antiserum to the guinea pig liver preparation reacted strongly with bovine liver catalase. We determined that this lack of selectivity of the antiserum was due to catalase contamination of the epoxide hydrolase preparation. We also determined that several commercial catalase preparations are contaminated with cytosolic epoxide hydrolase. Our human epoxide hydrolase preparation contained no detectable catalase contamination, yet antiserum to this protein also cross-reacted slightly with catalase, indicating some intrinsic similarity between the two enzymes. We conclude that catalase and cytosolic epoxide hydrolase contain some similar immunogenic epitopes, and we surmise that similarities between the subunits of these two enzymes may lead to their partial copurification. Functional similarities between the two enzymes are also demonstrated, as several compounds that inhibit catalase are also shown to inhibit cytosolic epoxide hydrolase activity in the same concentration range and rank order.

  17. 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.

  18. 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

  19. 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...

  20. 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...

  1. Sulfonyl Fluoride Inhibitors of Fatty Acid Amide Hydrolase

    PubMed Central

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

    2013-01-01

    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. Cotranslocation of Methyl Parathion Hydrolase to the Periplasm and of Organophosphorus Hydrolase to the Cell Surface of Escherichia coli by the Tat Pathway and Ice Nucleation Protein Display System ▿

    PubMed Central

    Yang, Chao; Freudl, Roland; Qiao, Chuanling; Mulchandani, Ashok

    2010-01-01

    A genetically engineered Escherichia coli strain coexpressing organophosphorus hydrolase (OPH) and methyl parathion hydrolase (MPH) was constructed for the first time by cotransforming two compatible plasmids. Since these two enzymes have different substrate specificities, the coexpression strain showed a broader substrate range than strains expressing either one of the hydrolases. To reduce the mass transport limitation of organophosphates (OPs) across the cell membrane, MPH and OPH were simultaneously translocated to the periplasm and cell surface of E. coli, respectively, by employing the twin-arginine translocation (Tat) pathway and ice nucleation protein (INP) display system. The resulting recombinant strain showed sixfold-higher whole-cell activity than the control strain expressing cytosolic OP hydrolases. The correct localization of MPH and OPH was demonstrated by cell fractionation, immunoblotting, and enzyme activity assays. No growth inhibition was observed for the recombinant E. coli strain, and suspended cultures retained almost 100% of the activity over a period of 2 weeks. Owing to its high level of activity and superior stability, the recombinant E. coli strain could be employed as a whole-cell biocatalyst for detoxification of OPs. This strategy of utilizing dual translocation pathways should open up new avenues for cotranslocating multiple functional moieties to different extracytosolic compartments of a bacterial cell. PMID:19933341

  3. Selective inhibition and selective induction of multiple microsomal epoxide hydrolases.

    PubMed

    Guenthner, T M

    1986-03-01

    The inhibition in vitro and induction in vivo of microsomal trans-stilbene oxide hydrolase have been studied. This microsomal epoxide hydrolase activity is distinguishable from the previously well-defined microsomal arene oxide hydrolase by a number of catalytic criteria. Two substituted chalcone oxides, 4-phenylchalcone oxide and 4'-phenylchalcone oxide, are potent inhibitors of microsomal trans-stilbene oxide hydrolase, but have no apparent activity against benzo[a]pyrene 4,5-oxide hydrolase. Conversely, compounds that are potent inhibitors of benzo[a]pyrene 4,5-oxide hydrolase, including styrene oxide, cyclohexene oxide, and trichloropropene oxide, inhibit microsomal trans-stilbene oxide hydrolase only at very high (millimolar) concentrations. The chalcone oxides inhibit microsomal trans-stilbene oxide hydrolase noncompetitively, and have micromolar or nanomolar affinity constants for the enzyme. Attempts were made to induce microsomal trans-stilbene oxide hydrolase in vivo. Compounds that induced microsomal benzo[a]pyrene 4,5-oxide hydrolase levels in mice did not simultaneously induce trans-stilbene oxide hydrolase levels. Clofibrate was an exception; it induced levels of both enzymes to a small but statistically significant degree. The two microsomal hydrolase activities have, therefore, very different catalytic sites and appear to be under separate genetic control. 4-Phenylchalcone oxide and 4'-phenylchalcone oxide are selective inhibitors of microsomal trans-stilbene oxide hydrolase and may prove to be very useful in assessing the involvement of this enzyme in the metabolism of endogenous or xenobiotic epoxides.

  4. Hydrolase-like properties of a cofactor-independent dioxygenase.

    PubMed

    Thierbach, Sven; Büldt-Karentzopoulos, Klaudia; Dreiling, Alena; Hennecke, Ulrich; König, Simone; Fetzner, Susanne

    2012-05-29

    Mechanistic promiscuity: The (2-alkyl)-3-hydroxy-4(1H)-quinolone-cleaving dioxygenase Hod has an α/β-hydrolase fold and a Ser/His/Asp triad in its active site. Isatoic anhydride, a suicide substrate of serine hydrolases, inactivates Hod by covalent modification of the active-site serine, thus indicating that the α/β-hydrolase fold can accommodate dioxygenase chemistry without completely abandoning hydrolase-like properties.

  5. 1,2,3-Triazolylalkylribitol derivatives as nucleoside hydrolase inhibitors.

    PubMed

    Goeminne, A; McNaughton, M; Bal, G; Surpateanu, G; Van der Veken, P; De Prol, S; Versées, W; Steyaert, J; Apers, S; Haemers, A; Augustyns, K

    2007-05-01

    A range of novel 1,2,3-triazolylalkylribitol derivatives were synthesized and evaluated as nucleoside hydrolase inhibitors. The most active compound (11a) has low micromolar potency and is structurally diverse from previously reported nucleoside hydrolase inhibitors, which, along with the simplicity of the chemistry involved in its synthesis, makes it a good lead for the further development of novel nucleoside hydrolase inhibitors.

  6. A modified expression of the major hydrolase activator in Hypocrea jecorina (Trichoderma reesei) changes enzymatic catalysis of biopolymer degradation.

    PubMed

    Pucher, Marion E; Steiger, Matthias G; Mach, Robert L; Mach-Aigner, Astrid R

    2011-06-10

    Hypocrea jecorina (anamorph Trichoderma reesei) is a saprophytic fungus that produces hydrolases, which are applied in different types of industries and used for the production of biofuel. A recombinant Hypocrea strain, which constantly expresses the main transcription activator of hydrolases (Xylanase regulator 1), was found to grow faster on xylan and its monomeric backbone molecule d-xylose. This strain also showed improved ability of clearing xylan medium on plates. Furthermore, this strain has a changed transcription profile concerning genes encoding for hydrolases and enzymes associated with degradation of (hemi)celluloses. We demonstrated that enzymes of this strain from a xylan cultivation favoured break down of hemicelluloses to the monomer d-xylose compared to the parental strain, while the enzymes of the latter one formed more xylobiose. Applying supernatants from cultivation on carboxymethylcellulose in enzymatic conversion of hemicelluloses, the enzymes of the recombinant strain were clearly producing more of both, d-xylose and xylobiose, compared to the parental strain. Altogether, these results point to a changed hydrolase expression profile, an enhanced capability to form the xylan-monomer d-xylose and the assumption that there is a disordered induction pattern if the Xylanase regulator 1 is de-regulated in Hypocrea.

  7. Twisting of glycosidic bonds by hydrolases

    USDA-ARS?s Scientific Manuscript database

    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...

  8. Structure and function of polyglycine hydrolases

    USDA-ARS?s Scientific Manuscript database

    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...

  9. Production of tartaric acid using immobilized recominant cis-epoxysuccinate hydrolase.

    PubMed

    Wang, Ziqiang; Su, Munan; Li, Yanliang; Wang, Yunshan; Su, Zhiguo

    2017-09-05

    To investigate the expression and immobilization of recombinant cis-epoxysuccinate hydrolase (ESH), and its application in the biological production of L-(+)-tartaric acid. E. coli BL21 (DE3)/pET11a-ESH (His) was engineered to express recombinant ESH. The enzyme had an activity of 262 U mg(-1). The recombinant ESH was immobilized on agarose Ni-IDA matrix with metal ion affinity interaction to improve its thermostability and pH stability. The immobilization efficiency and activity yield were 94 and 95%, respectively. The specific catalytic efficiency of immobilized ESH was 104 mg U(-1) h(-1) during the continuous enzymatic production process. ESH with a histidine tag was immobilized and used for the continuous production of L-(+)-tartaric acid.

  10. Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding an alpha-amino acid ester hydrolase from Acetobacter turbidans.

    PubMed

    Polderman-Tijmes, Jolanda J; Jekel, Peter A; de Vries, Erik J; van Merode, Annet E J; Floris, René; van der Laan, Jan-Metske; Sonke, Theo; Janssen, Dick B

    2002-01-01

    The alpha-amino acid ester hydrolase from Acetobacter turbidans ATCC 9325 is capable of hydrolyzing and synthesizing beta-lactam antibiotics, such as cephalexin and ampicillin. N-terminal amino acid sequencing of the purified alpha-amino acid ester hydrolase allowed cloning and genetic characterization of the corresponding gene from an A. turbidans genomic library. The gene, designated aehA, encodes a polypeptide with a molecular weight of 72,000. Comparison of the determined N-terminal sequence and the deduced amino acid sequence indicated the presence of an N-terminal leader sequence of 40 amino acids. The aehA gene was subcloned in the pET9 expression plasmid and expressed in Escherichia coli. The recombinant protein was purified and found to be dimeric with subunits of 70 kDa. A sequence similarity search revealed 26% identity with a glutaryl 7-ACA acylase precursor from Bacillus laterosporus, but no homology was found with other known penicillin or cephalosporin acylases. There was some similarity to serine proteases, including the conservation of the active site motif, GXSYXG. Together with database searches, this suggested that the alpha-amino acid ester hydrolase is a beta-lactam antibiotic acylase that belongs to a class of hydrolases that is different from the Ntn hydrolase superfamily to which the well-characterized penicillin acylase from E. coli belongs. The alpha-amino acid ester hydrolase of A. turbidans represents a subclass of this new class of beta-lactam antibiotic acylases.

  11. Dienelactone hydrolase from Pseudomonas sp. strain B13.

    PubMed Central

    Ngai, K L; Schlömann, M; Knackmuss, H J; Ornston, L N

    1987-01-01

    Dienelactone hydrolase (EC 3.1.1.45) catalyzes the conversion of cis- or trans-4-carboxymethylenebut-2-en-4-olide (dienelactone) to maleylacetate. An approximately 24-fold purification from extracts of 3-chlorobenzoate-grown Pseudomonas sp. strain B13 yielded a homogeneous preparation of the enzyme. The purified enzyme crystallized readily and proved to be a monomer with a molecular weight of about 30,000. Each dienelactone hydrolase molecule contains two cysteinyl side chains. One of these was readily titrated by stoichiometric amounts of p-chloromercuribenzoate, resulting in inactivation of the enzyme; the inactivation could be reversed by the addition of dithiothreitol. The other cysteinyl side chain appeared to be protected in the native protein against chemical reaction with p-chloromercuribenzoate. The properties of sulfhydryl side chains in dienelactone hydrolase resembled those that have been characterized for bacterial 4-carboxymethylbut-3-en-4-olide (enol-lactone) hydrolases (EC 3.1.1.24), which also are monomers with molecular weights of about 30,000. The amino acid composition of the dienelactone hydrolase resembled the amino acid composition of enol-lactone hydrolase from Pseudomonas putida, and alignment of the NH2-terminal amino acid sequence of the dienelactone hydrolase with the corresponding sequence of an Acinetobacter calcoaceticus enol-lactone hydrolase revealed sequence identity at 8 of the 28 positions. These observations foster the hypothesis that the lactone hydrolases share a common ancestor. The lactone hydrolases differed in one significant property: the kcat of dienelactone hydrolase was 1,800 min-1, an order of magnitude below the kcat observed with enol-lactone hydrolases. The relatively low catalytic activity of dienelactone hydrolase may demand its production at the high levels observed for induced cultures of Pseudomonas sp. strain B13. PMID:3804973

  12. 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

    2015-06-05

    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.

  13. 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.

  14. Sph3 Is a Glycoside Hydrolase Required for the Biosynthesis of Galactosaminogalactan in Aspergillus fumigatus.

    PubMed

    Bamford, Natalie C; Snarr, Brendan D; Gravelat, Fabrice N; Little, Dustin J; Lee, Mark J; Zacharias, Caitlin A; Chabot, Josée C; Geller, Alexander M; Baptista, Stefanie D; Baker, Perrin; Robinson, Howard; Howell, P Lynne; Sheppard, Donald C

    2015-11-13

    Aspergillus fumigatus is the most virulent species within the Aspergillus genus and causes invasive infections with high mortality rates. The exopolysaccharide galactosaminogalactan (GAG) contributes to the virulence of A. fumigatus. A co-regulated five-gene cluster has been identified and proposed to encode the proteins required for GAG biosynthesis. One of these genes, sph3, is predicted to encode a protein belonging to the spherulin 4 family, a protein family with no known function. Construction of an sph3-deficient mutant demonstrated that the gene is necessary for GAG production. To determine the role of Sph3 in GAG biosynthesis, we determined the structure of Aspergillus clavatus Sph3 to 1.25 Å. The structure revealed a (β/α)8 fold, with similarities to glycoside hydrolase families 18, 27, and 84. Recombinant Sph3 displayed hydrolytic activity against both purified and cell wall-associated GAG. Structural and sequence alignments identified three conserved acidic residues, Asp-166, Glu-167, and Glu-222, that are located within the putative active site groove. In vitro and in vivo mutagenesis analysis demonstrated that all three residues are important for activity. Variants of Asp-166 yielded the greatest decrease in activity suggesting a role in catalysis. This work shows that Sph3 is a glycoside hydrolase essential for GAG production and defines a new glycoside hydrolase family, GH135.

  15. 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.

  16. 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

  17. Inhibition of Xenobiotic-Degrading Hydrolases by Organophosphinates

    DTIC Science & Technology

    1986-07-01

    carboxylester hydrolases and pancreatic proteinases (3). The choice of organophosphinate candidates for pretreatment agents should be made with these factors...M 4 Q r 000 44 Table 11. Purification of arylester hydrolase Specific Total Total Activity Volume Activity Proteina (Umoles/ Purifi- Fraction (mL...reaction products after the racemic mixture was exposed to arylester hydrolase (EC 3.1.1.2) or proteinases ; chiral-phase capillary gas-liquid

  18. 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

  19. 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.

  20. 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.

  1. Limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis DCL14 belongs to a novel class of epoxide hydrolases.

    PubMed

    van der Werf, M J; Overkamp, K M; de Bont, J A

    1998-10-01

    An epoxide hydrolase from Rhodococcus erythropolis DCL14 catalyzes the hydrolysis of limonene-1,2-epoxide to limonene-1,2-diol. The enzyme is induced when R. erythropolis is grown on monoterpenes, reflecting its role in the limonene degradation pathway of this microorganism. Limonene-1,2-epoxide hydrolase was purified to homogeneity. It is a monomeric cytoplasmic enzyme of 17 kDa, and its N-terminal amino acid sequence was determined. No cofactor was required for activity of this colorless enzyme. Maximal enzyme activity was measured at pH 7 and 50 degrees C. None of the tested inhibitors or metal ions inhibited limonene-1,2-epoxide hydrolase activity. Limonene-1,2-epoxide hydrolase has a narrow substrate range. Of the compounds tested, only limonene-1,2-epoxide, 1-methylcyclohexene oxide, cyclohexene oxide, and indene oxide were substrates. This report shows that limonene-1,2-epoxide hydrolase belongs to a new class of epoxide hydrolases based on (i) its low molecular mass, (ii) the absence of any significant homology between the partial amino acid sequence of limonene-1,2-epoxide hydrolase and amino acid sequences of known epoxide hydrolases, (iii) its pH profile, and (iv) the inability of 2-bromo-4'-nitroacetophenone, diethylpyrocarbonate, 4-fluorochalcone oxide, and 1, 10-phenanthroline to inhibit limonene-1,2-epoxide hydrolase activity.

  2. Norovirus recombination.

    PubMed

    Bull, Rowena A; Tanaka, Mark M; White, Peter A

    2007-12-01

    RNA recombination is a significant driving force in viral evolution. Increased awareness of recombination within the genus Norovirus of the family Calicivirus has led to a rise in the identification of norovirus (NoV) recombinants and they are now reported at high frequency. Currently, there is no classification system for recombinant NoVs and a widely accepted recombinant genotyping system is still needed. Consequently, there is duplication in reporting of novel recombinants. This has led to difficulties in defining the number and types of recombinants in circulation. In this study, 120 NoV nucleotide sequences were compiled from the current GenBank database and published literature. NoV recombinants and their recombination breakpoints were identified using three methods: phylogenetic analysis, SimPlot analysis and the maximum chi2 method. A total of 20 NoV recombinant types were identified in circulation worldwide. The recombination point is the ORF1/2 overlap in all isolates except one, which demonstrated a double recombination event within the polymerase region.

  3. Nostoc commune UTEX 584 gene expressing indole phosphate hydrolase activity in Escherichia coli.

    PubMed Central

    Xie, W Q; Whitton, B A; Simon, J W; Jäger, K; Reed, D; Potts, M

    1989-01-01

    A gene encoding an enzyme capable of hydrolyzing indole phosphate was isolated from a recombinant gene library of Nostoc commune UTEX 584 DNA in lambda gt10. The gene (designated iph) is located on a 2.9-kilobase EcoRI restriction fragment and is present in a single copy in the genome of N. commune UTEX 584. The iph gene was expressed when the purified 2.9-kilobase DNA fragment, free of any vector sequences, was added to a cell-free coupled transcription-translation system. A polypeptide with an Mr of 74,000 was synthesized when the iph gene or different iph-vector DNA templates were expressed in vitro. When carried by different multicopy plasmids and phagemids (pMP005, pBH6, pB8) the cyanobacterial iph gene conferred an Iph+ phenotype upon various strains of Escherichia coli, including a phoA mutant. Hydrolysis of 5-bromo-4-chloro-3-indolyl phosphate was detected in recombinant E. coli strains grown in phosphate-rich medium, and the activity persisted in assay buffers that contained phosphate. In contrast, indole phosphate hydrolase activity only developed in cells of N. commune UTEX 584, when they were partially depleted of phosphorus, and the activity associated with these cells was suppressed partially by the addition of phosphate to assay buffers. Indole phosphate hydrolase activity was detected in periplasmic extracts from E. coli (Iph+) transformants. Images PMID:2536677

  4. Biodegradation of an endocrine-disrupting chemical di-n-butyl phthalate by newly isolated Camelimonas sp. and enzymatic properties of its hydrolase.

    PubMed

    Chen, Xu; Zhang, Xiaolong; Yang, Yu; Yue, Dongmei; Xiao, Lin; Yang, Liuyan

    2015-04-01

    An aerobic bacterial strain M11 capable of degrading dibutyl phthalate (DBP) was isolated and identified as Camelimonas sp. This strain could not grow on dialkyl phthalates, including dimethyl, diethyl, dipropyl, dibutyl and dipentyl phthalate, but suspensions of cells could transform these compounds to phthalate via corresponding monoalkyl phthalates. The degradation kinetics of DBP was best fitted by first-order kinetic equation. During growth in Brucella Selective Medium, M11 produced the high amounts of non-DBP-induced intracellular hydrolase in the stationary phase. The DBP hydrolase gene of M11 was cloned, and the recombinant DBP hydrolase had a high optimum degradation temperature (50 °C), and a wide range of pH and temperature stability.

  5. Expression and fermentation optimization of oxidized polyvinyl alcohol hydrolase in E. coli.

    PubMed

    Yang, Yu; Zhang, Dongxu; Liu, Song; Jia, Dongxu; Du, Guocheng; Chen, Jian

    2012-01-01

    Oxidized polyvinyl alcohol (PVA) hydrolase (OPH) is a key enzyme in the degradation of PVA, suggesting that OPH has a great potential for application in textile desizing processes. In this study, the OPH gene from Sphingopyxis sp. 113P3 was modified, by artificial synthesis, for overexpression in Escherichia coli. The OPH gene, lacking the sequence encoding the original signal peptide, was inserted into pET-20b (+) expression vector, which was then used to transform E. coli BL21 (DE3). OPH expression was detected in culture medium in which the transformed E. coli BL21 (DE3) was grown. Nutritional and environmental conditions were investigated for improved production of OPH protein by the recombinant strain. The highest OPH activity measured was 47.54 U/mL and was reached after 84 h under optimal fermentation conditions; this level is 2.64-fold higher that obtained under sub-optimal conditions. The productivity of recombinant OPH reached 565.95 U/L/h. The effect of glycine on the secretion of recombinant OPH was examined by adding glycine to the culture medium to a final concentration of 200 mM. This concentration of glycine reduced the fermentation time by 24 h and increased the productivity of recombinant OPH to 733.17 U/L/h. Our results suggest that the recombinant strain reported here has great potential for use in industrial applications.

  6. Selective N-hydroxyhydantoin carbamate inhibitors of mammalian serine hydrolases

    PubMed Central

    Cognetta, Armand B.; Niphakis, Micah J.; Lee, Hyeon-Cheol; Martini, Michael L.; Hulce, Jonathan J.; Cravatt, Benjamin F.

    2015-01-01

    Serine hydrolase inhibitors, which facilitate enzyme function assignment and are used to treat a range of human disorders, often act by an irreversible mechanism that involves covalent modification of the serine hydrolase catalytic nucleophile. The portion of mammalian serine hydrolases for which selective inhibitors have been developed, however, remains small. Here, we show that N-hydroxyhydantoin (NHH) carbamates are a versatile class of irreversible serine hydrolase inhibitors that can be modified on both the staying (carbamylating) and leaving (NHH) groups to optimize potency and selectivity. Synthesis and screening of a small library of NHH carbamates by competitive activity-based protein profiling furnished selective, in vivo-active inhibitors and tailored activity-based probes for multiple mammalian serine hydrolases, including palmitoyl protein thioesterease-1 (PPT1), mutations of which cause the human disease infantile neuronal ceroid lipofuscinosis. PMID:26120000

  7. Bacterial growth does require peptidoglycan hydrolases.

    PubMed

    Vollmer, Waldemar

    2012-12-01

    Most bacteria surround their cytoplasmic membrane with a net-like, elastic heteropolymer, the peptidoglycan sacculus, to protect themselves from bursting due to the turgor and to maintain cell shape. It has been assumed that growing bacteria require peptidoglycan hydrolases to open meshes in the peptidoglycan net allowing the insertion of the newly synthesized material for surface expansion. However, peptidoglycan hydrolases essential for bacterial growth have long remained elusive. In this issue of Molecular Microbiology Singh et al. (2012) report the identification in Escherichia coli of three new DD-endopeptidases (Spr, YdhO and YebA) which are collectively required for peptidoglycan growth. Cells depleted of the three enzymes fail to incorporate new peptidoglycan, indicating that the cleavage of cross-links by the new endopeptidases is needed for surface growth of the sacculus. These results are corroborated by recent data showing that Bacillus subtilis cells require the DL-endopeptidase activity of CwlO or LytE for growth.

  8. Bacterial CS2 Hydrolases from Acidithiobacillus thiooxidans Strains Are Homologous to the Archaeal Catenane CS2 Hydrolase

    PubMed Central

    Smeulders, Marjan J.; Pol, Arjan; Venselaar, Hanka; Barends, Thomas R. M.; Hermans, John; Jetten, Mike S. M.

    2013-01-01

    Carbon disulfide (CS2) and carbonyl sulfide (COS) are important in the global sulfur cycle, and CS2 is used as a solvent in the viscose industry. These compounds can be converted by sulfur-oxidizing bacteria, such as Acidithiobacillus thiooxidans species, to carbon dioxide (CO2) and hydrogen sulfide (H2S), a property used in industrial biofiltration of CS2-polluted airstreams. We report on the mechanism of bacterial CS2 conversion in the extremely acidophilic A. thiooxidans strains S1p and G8. The bacterial CS2 hydrolases were highly abundant. They were purified and found to be homologous to the only other described (archaeal) CS2 hydrolase from Acidianus strain A1-3, which forms a catenane of two interlocked rings. The enzymes cluster in a group of β-carbonic anhydrase (β-CA) homologues that may comprise a subclass of CS2 hydrolases within the β-CA family. Unlike CAs, the CS2 hydrolases did not hydrate CO2 but converted CS2 and COS with H2O to H2S and CO2. The CS2 hydrolases of A. thiooxidans strains G8, 2Bp, Sts 4-3, and BBW1, like the CS2 hydrolase of Acidianus strain A1-3, exist as both octamers and hexadecamers in solution. The CS2 hydrolase of A. thiooxidans strain S1p forms only octamers. Structure models of the A. thiooxidans CS2 hydrolases based on the structure of Acidianus strain A1-3 CS2 hydrolase suggest that the A. thiooxidans strain G8 CS2 hydrolase may also form a catenane. In the A. thiooxidans strain S1p enzyme, two insertions (positions 26 and 27 [PD] and positions 56 to 61 [TPAGGG]) and a nine-amino-acid-longer C-terminal tail may prevent catenane formation. PMID:23836868

  9. 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

    2016-07-02

    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.

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

    PubMed Central

    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

    2016-01-01

    ABSTRACT 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

  11. 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

  12. Genetic Recombination

    ERIC Educational Resources Information Center

    Whitehouse, H. L. K.

    1973-01-01

    Discusses the mechanisms of genetic recombination with particular emphasis on the study of the fungus Sordaria brevicollis. The study of recombination is facilitated by the use of mutants of this fungus in which the color of the ascospores is affected. (JR)

  13. Genetic Recombination

    ERIC Educational Resources Information Center

    Whitehouse, H. L. K.

    1973-01-01

    Discusses the mechanisms of genetic recombination with particular emphasis on the study of the fungus Sordaria brevicollis. The study of recombination is facilitated by the use of mutants of this fungus in which the color of the ascospores is affected. (JR)

  14. Glycoside Hydrolases across Environmental Microbial Communities

    PubMed Central

    Berlemont, Renaud

    2016-01-01

    Across many environments microbial glycoside hydrolases support the enzymatic processing of carbohydrates, a critical function in many ecosystems. Little is known about how the microbial composition of a community and the potential for carbohydrate processing relate to each other. Here, using 1,934 metagenomic datasets, we linked changes in community composition to variation of potential for carbohydrate processing across environments. We were able to show that each ecosystem-type displays a specific potential for carbohydrate utilization. Most of this potential was associated with just 77 bacterial genera. The GH content in bacterial genera is best described by their taxonomic affiliation. Across metagenomes, fluctuations of the microbial community structure and GH potential for carbohydrate utilization were correlated. Our analysis reveals that both deterministic and stochastic processes contribute to the assembly of complex microbial communities. PMID:27992426

  15. Localization of Acid Hydrolases in Protoplasts

    PubMed Central

    Butcher, Henry C.; Wagner, George J.; Siegelman, Harold W.

    1977-01-01

    The development of techniques to isolate and purify relatively large quantities of intact vacuoles from mature tissues permits direct biochemical analysis of this ubiquitous mature plant cell organelle. Vacuoles and a fraction enriched in soluble cytoplasmic constituents were quantitatively prepared from Hippeastrum flower petal protoplasts. Vacuolar lysate and soluble cytoplasmic fractions were examined for acid hydrolase activities commonly associated with animal lysosomes, and pH optima were determined. Esterase, protease, carboxypeptidase, β-galactosidase, α-glycosidase and β-glycosidase, not found in the vacuole lysate fraction, were components of the soluble cytoplasmic fraction. Acid phosphatase, RNase and DNase were present in both fractions. Vacuolar enzyme activities were also examined as a function of flower development from bud through senescent stages. The data obtained are not consistent with the concept that the mature plant cell vacuole functions as a generalized lysosome. PMID:16660001

  16. 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.

  17. Starch hydrolase inhibitors from edible plants.

    PubMed

    Wang, Hongyu; Liu, Tingting; Huang, Dejian

    2013-01-01

    Type 2 diabetes is a chronic disease with gradual deterioration in glucose metabolism, which causes multiple systemic complications. Postprandial hyperglycemia is a concern in the management of type 2 diabetes. Of all the available antidiabetic therapeutic methods, inhibition of α-glucosidase and α-amylase is postulated to be a preventive treatment. Many natural products and herbal medicines have been recommended as being beneficial for mitigation of postprandial hyperglycemia. In this review, recent discoveries of α-glucosidase and α-amylase inhibitors from edible plants are described along with their chemical structures. Their inhibition mechanisms, the type of each glucosidase and amylase, and measurement methods for the inhibitory activity are also given. Finally, recent progress on low glycemic index foods incorporated with plants containing starch hydrolase inhibitors is summarized. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. Structure of Francisella tularensis peptidyl-tRNA hydrolase

    PubMed Central

    Clarke, Teresa E.; Romanov, Vladimir; Lam, Robert; Gothe, Scott A.; Peddi, Srinivasa R.; Razumova, Ekaterina B.; Lipman, Richard S. A.; Branstrom, Arthur A.; Chirgadze, Nickolay Y.

    2011-01-01

    The rational design of novel antibiotics for bacteria involves the identification of inhibitors for enzymes involved in essential biochemical pathways in cells. In this study, the cloning, expression, purification, crystallization and structure of the enzyme peptidyl-tRNA hydrolase from Francisella tularensis, the causative agent of tularemia, was performed. The structure of F. tularensis peptidyl-tRNA hydrolase is comparable to those of other bacterial peptidyl-tRNA hydrolases, with most residues in the active site conserved amongst the family. The resultant reagents, structural data and analyses provide essential information for the structure-based design of novel inhibitors for this class of proteins. PMID:21505237

  19. Activity of murein hydrolases in synchronized cultures of Escherichia coli.

    PubMed Central

    Hakenbeck, R; Messer, W

    1977-01-01

    Murein hydrolase activities were analyzed in synchronized cultures of Escherichia coli B/r. Cell wall-bound murein hydrolase activities, including the penicillin-sensitive endopeptidase, increased discontinuously during the cell cycle and showed maximum activity at a cell age of 30 to 35 min (generation time, 43 min). Maximum activity was observed at the same time that the rate of cell wall synthesis reached its maximum. These oscillations depended on the termination of replication: no increase in hydrolase activity was found if deoxyribonucleic acid synthesis was inhibited at an early time in the life cycle. In contrast, the activity of another murein hydrolase that was not tightly bound to the membrane (transglycosylase) increased exponentially with time, even when deoxyribonucleic acid synthesis was inhibited. PMID:321419

  20. Peptide hydrolase activities of the mucosa of human small intestine

    PubMed Central

    Heizer, William D.; Laster, Leonard

    1969-01-01

    Few studies have been published on peptide hydrolase activities of human small intestine mucosa. We developed methods to screen tissue extracts for such enzymes and to quantitate hydrolase activities for dipeptides containing the aromatic amino acid L-phenylalanine. The screening procedure indicated glycyl-L-proline hydrolase activity was reduced in biopsy specimens from patients with flattened intestinal mucosa. To explore this further, we established optimal assay conditions for hydrolase activities (a) glycyl-L-proline, (b) L-phenylalanyl-L-proline, (c) L-alanyl-L-phenylalanine, and (d) L-phenylalanylglycine. Biopsy specimens from patients with various intestinal disorders, but without flattened mucosa, and from three patients with flattened mucosa, showed a disproportionate reduction in activities (a) and (b), with the reduction being significantly more marked in the latter patients. We suggest that intestinal imidopeptide hydrolase activities, such as (a) and (b), are sensitive to changes in intestinal disease generally, particularly to the altered physiology associated with flattening of the mucosa, and are secondary to, rather than a cause of, the intestinal pathology. Our finding that intestinal alkaline phosphatase activity tended to parallel imidopeptide hydrolase activity, and that activity (a) was partially localized to the particulate fraction of mucosal homogenate, suggested that imidopeptide hydrolase activities may be located in the microvilli of the intestinal epithelium and that, like alkaline phosphatase activity, they may be reduced in flattened mucosae, in part at least because of the pathologic changes in the microvilli. In our studies of control subjects we did not detect peptide hydrolase activity deficiency analogous to asymptomatic disaccharidase deficiency. Images PMID:5765024

  1. 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

  2. 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. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. BpiB05, a novel metagenome-derived hydrolase acting on N-acylhomoserine lactones.

    PubMed

    Bijtenhoorn, P; Schipper, C; Hornung, C; Quitschau, M; Grond, S; Weiland, N; Streit, W R

    2011-08-20

    The N-acyl-homoserine lactones (N-AHLs) play an important role in bacterial cell-cell signaling. Up to date, however, only a few different experimentally proven classes of N-AHL ring-cleaving enzymes are known. Here we report on the isolation and biochemical characterization of a novel hydrolase derived from the soil metagenome and acting on N-AHLs. The identified protein designated BpiB05 is weakly similar to hypothetical proteins from Bacteroides fragilis, the draft genomes of two Burkholderia species as well as a marine metagenomic ORF but is otherwise not similar to any known protein. BpiB05 was overexpressed in Escherichia coli as a 10× His-tagged fusion protein. The recombinant protein revealed a molecular weight of about 70kDa and was tested for its quorum quenching (QQ) activities using a lacZ-bioassay. Additional HPLC-MS analyses confirmed the lactonolytic activity of the purified protein in the presence of Ca²⁺. Further tests suggested that BpiB05 strongly reduces motility in Pseudomonas aeruginosa, pyocyanin synthesis and biofilm formation in this microbe. Because BpiB05 is not distantly related to any of the currently known hydrolases it forms probably a novel group within the growing number of proteins acting on N-AHLs. Copyright © 2011 Elsevier B.V. All rights reserved.

  4. Identification of a novel peptidoglycan hydrolase CwlM in Mycobacterium tuberculosis.

    PubMed

    Deng, Lingyi Lynn; Humphries, Donald E; Arbeit, Robert D; Carlton, Laura E; Smole, Sandra C; Carroll, J David

    2005-02-14

    Mycobacterium tuberculosis is a major global pathogen whose threat has increased with the emergence of multidrug-resistant strains. The cell wall of M. tuberculosis is thick, rigid, and hydrophobic, which serves to protect the organism from the environment and makes it highly impermeable to conventional antimicrobial agents. There is little known about cell wall autolysins (also referred to as peptidoglycan hydrolases) of mycobacteria. We identified an open reading frame (Rv3915) in the M. tuberculosis genome designated cwlM that appeared consistent with a peptidoglycan hydrolase. The 1218-bp gene was amplified by PCR, cloned and expressed in E. coli strain HMS174(DE-3), and its gene product, a 47-kDa recombinant protein, was purified and partially characterized. Purified CwlM was able to lyse whole mycobacteria, release peptidoglycan from the cell wall of Micrococcus luteus and Mycobacterium smegmatis, and cleave N-acetylmuramoyl-L-alanyl-D-isoglutamine, releasing free N-acetylmuramic acid. These results indicate that CwlM is a novel autolysin and identify cwlM as the first, to our knowledge, autolysin gene identified and cloned from M. tuberculosis. CwlM offers a new target for a unique class of drugs that could alter the permeability of the mycobacterial cell wall and enhance the effectiveness of treatments for tuberculosis.

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

    PubMed

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

    2008-01-01

    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 A 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.

  6. Molecular and functional characterization of a unique sucrose hydrolase from Xanthomonas axonopodis pv. glycines.

    PubMed

    Kim, Hong-Suk; Park, Hyoung-Joon; Heu, Sunggi; Jung, Jin

    2004-01-01

    A novel sucrose hydrolase (SUH) from Xanthomonas axonopodis pv. glycines, a causative agent of bacterial pustule disease on soybeans, was studied at the functional and molecular levels. SUH was shown to act rather specifically on sucrose (K(m) = 2.5 mM) but not on sucrose-6-phosphate. Protein analysis of purified SUH revealed that, in this monomeric enzyme with an estimated molecular mass of 70,223 +/- 12 Da, amino acid sequences determined for several segments have corresponding nucleotide sequences in XAC3490, a protein-coding gene found in the genome of X. axonopodis pv. citri. Based on this information, the SUH gene, consisting of an open reading frame of 1,935 bp, was cloned by screening a genomic library of X. axonopodis pv. glycines 8ra. Database searches and sequence comparison revealed that SUH has significant homology to some family 13 enzymes, with all of the crucial invariant residues involved in the catalytic mechanism conserved, but it shows no similarity to known invertases belonging to family 32. suh expression in X. axonopodis pv. glycines requires sucrose induction, and insertional mutagenesis resulted in an absence of sucrose-inducible sucrose hydrolase activity in crude protein extracts and a sucrose-negative phenotype. Recombinant SUH, overproduced in Escherichia coli and purified, was shown to have the same enzymatic characteristics in terms of kinetic parameters.

  7. Purification and properties of 2-hydroxy-6-oxo-6-(2'-aminophenyl)hexa-2,4-dienoic acid hydrolase involved in microbial degradation of carbazole.

    PubMed

    Riddle, Robert R; Gibbs, Phillip R; Willson, Richard C; Benedik, Michael J

    2003-03-01

    Hydrolysis following meta-ring cleavage by a dioxygenase is a well-known step in aromatic compound metabolism. The 2-hydroxy-6-oxo-6-(2'-aminophenyl)hexa-2,4-dienoic acid hydrolase from Pseudomonas LD2 is a new member of the small group of characterized aromatic hydrolases that catalyze the cleavage of C-C bonds. In this study, the His(6)-tagged 2-hydroxy-6-oxo-6-(2'-aminophenyl)hexa-2,4-dienoic acid (HOPDA) hydrolase was purified from a recombinant Escherichia coli strain utilizing immobilized metal affinity chromatography. 2-Hydroxy-6-oxo-6-(2'-aminophenyl)hexa-2,4-dienoic acid hydrolase is a colorless homodimer with no cofactor requirement. The enzyme actively converted HOPDA into benzoic acid and 2-hydroxypenta-2,4-dienoic acid. The enzyme exhibited activity between pH 6.5 and 10.5 with a maximum activity at pH 7.0. The optimum temperature at pH 7.0 was 60 degrees C. The calculated K'(m) for HOPDA was 4.6 microM, the V(max) was 3.3 micromol min(-1), and the K(s) was 70.0 microM. This corresponds to a maximum specific turnover rate of 1300 HOPDAs(-1)dimer(-1). The deduced amino acid sequence of CarC showed 30.3, 31.3, and 31.8% identity with TodF (P. putida F1), XylF (P. putida), and DmpD (Pseudomonas sp. CF600), respectively, which are meta-cleavage compound hydrolases from other Pseudomonads. The amino acid sequence Gly-X-Ser-X-Gly, which is highly conserved in these hydrolases, is also found in CarC. Lysates from a strain expressing enzyme in which the putative active site serine is mutated to alanine showed a significant reduction in activity.

  8. Isolation and immunological characterization of a novel Cladosporium herbarum allergen structurally homologous to the alpha/beta hydrolase fold superfamily.

    PubMed

    Rid, Raphaela; Onder, Kamil; Hawranek, Thomas; Laimer, Martin; Bauer, Johann W; Holler, Claudia; Simon-Nobbe, Birgit; Breitenbach, Michael

    2010-03-01

    Because the ascomycete Cladosporium herbarum embodies one of the most important, world-wide occurring fungal species responsible for eliciting typical IgE-mediated hypersensitivity reactions ranging from rhinitis and ocular symptoms to severe involvement of the lower respiratory tract, a more comprehensive definition of its detailed allergen repertoire is unquestionably of critical medical as well as therapeutic significance. By screening a C. herbarum cDNA library with IgE antibodies pooled from 3 mold-reactive sera, we were able to identify, clone and affinity-purify a novel allergen candidate (29.9 kDa) exhibiting considerable (three-dimensional) homology to the alpha/beta hydrolase fold superfamily. The latter covers a collection of hydrolytic enzymes of widely differing phylogenetic origin as well as catalytic activity (operating in countless biological contexts) that in general exhibit only little sequence similarity yet show a remarkable conservation of structural topology. Our present study (i) characterizes recombinant non-fusion C. herbarum hydrolase as a natively folded, minor mold allergen that displays a prevalence of IgE reactivity of approximately 17% in our in vitro immunoblot experiments, (ii) proposes the existence of several putative (speculatively cross-reactive) ascomycete orthologues as determined via genome-wide in silico predictions, and (iii) finally implies that C. herbarum hydrolase could be included in forthcoming minimal testing sets when fungal allergy is suspected. Copyright 2009 Elsevier Ltd. All rights reserved.

  9. Sph3 Is a Glycoside Hydrolase Required for the Biosynthesis of Galactosaminogalactan in Aspergillus fumigatus*♦

    PubMed Central

    Bamford, Natalie C.; Snarr, Brendan D.; Gravelat, Fabrice N.; Little, Dustin J.; Lee, Mark J.; Zacharias, Caitlin A.; Chabot, Josée C.; Geller, Alexander M.; Baptista, Stefanie D.; Baker, Perrin; Robinson, Howard; Howell, P. Lynne; Sheppard, Donald C.

    2015-01-01

    Aspergillus fumigatus is the most virulent species within the Aspergillus genus and causes invasive infections with high mortality rates. The exopolysaccharide galactosaminogalactan (GAG) contributes to the virulence of A. fumigatus. A co-regulated five-gene cluster has been identified and proposed to encode the proteins required for GAG biosynthesis. One of these genes, sph3, is predicted to encode a protein belonging to the spherulin 4 family, a protein family with no known function. Construction of an sph3-deficient mutant demonstrated that the gene is necessary for GAG production. To determine the role of Sph3 in GAG biosynthesis, we determined the structure of Aspergillus clavatus Sph3 to 1.25 Å. The structure revealed a (β/α)8 fold, with similarities to glycoside hydrolase families 18, 27, and 84. Recombinant Sph3 displayed hydrolytic activity against both purified and cell wall-associated GAG. Structural and sequence alignments identified three conserved acidic residues, Asp-166, Glu-167, and Glu-222, that are located within the putative active site groove. In vitro and in vivo mutagenesis analysis demonstrated that all three residues are important for activity. Variants of Asp-166 yielded the greatest decrease in activity suggesting a role in catalysis. This work shows that Sph3 is a glycoside hydrolase essential for GAG production and defines a new glycoside hydrolase family, GH135. PMID:26342082

  10. Design and synthesis of dual modulators of soluble epoxide hydrolase and peroxisome proliferator-activated receptors.

    PubMed

    la Buscató, Estel; Blöcher, René; Lamers, Christina; Klingler, Franca-Maria; Hahn, Steffen; Steinhilber, Dieter; Schubert-Zsilavecz, Manfred; Proschak, Ewgenij

    2012-12-13

    Metabolic syndrome is a complex condition which often requires the use of multiple medications as a treatment. The resulting problems of polypharmacy are increase in side effects, drug-drug interactions, and its high economic cost. Development of multitarget compounds is a promising strategy to avoid the complications arising from administration of multiple drugs. Modulators of peroxisome proliferator-activated receptors (PPARs) are established agents in the treatment of dyslipidaemia, hyperglycaemia, and insulin resistance. Inhibitors of soluble epoxide hydrolase (sEH) are under evaluation for their use in cardiovascular diseases. In the present study, a series of dual sEH/PPAR modulators containing a pyrrole acidic headgroup and a urea pharmacophore were designed, synthesized, and evaluated in vitro using recombinant enzyme and cell-based assays. Compounds with different activity profiles were obtained which could be used in the treatment of metabolic syndrome.

  11. Catalytic scope of the thiamine-dependent multifunctional enzyme cyclohexane-1,2-dione hydrolase.

    PubMed

    Loschonsky, Sabrina; Waltzer, Simon; Fraas, Sonja; Wacker, Tobias; Andrade, Susana L A; Kroneck, Peter M H; Müller, Michael

    2014-02-10

    The thiamine diphosphate (ThDP)-dependent enzyme cyclohexane-1,2-dione hydrolase (CDH) was expressed in Escherichia coli and purified by affinity chromatography (Ni-NTA). Recombinant CDH showed the same C-C bond-cleavage and C-C bond-formation activities as the native enzyme. Furthermore, we have shown that CDH catalyzes the asymmetric cross-benzoin reaction of aromatic aldehydes and (decarboxylated) pyruvate (up to quantitative conversion, 92-99 % ee). CDH accepts also hydroxybenzaldehydes and nitrobenzaldehydes; these previously have not (or only in rare cases) been known as substrates of other ThDP-dependent enzymes. On a semipreparative scale, sterically demanding 4-(tert-butyl)benzaldehyde and 2-naphthaldehyde were transformed into the corresponding 2-hydroxy ketone products in high yields. Additionally, certain benzaldehydes with electron withdrawing substituents were identified as potential inhibitors of the ligase activity of CDH.

  12. Secretory expression of organophosphorus hydrolase OPHC2 in Yarrowia lipolytica Polg.

    PubMed

    Li, Meng; Yu, Xiaolan; Wang, Fei; Zhai, Chao; Shen, Wei; Yu, Xianhong; Wang, Xiaojuan; Ma, Lixin

    2015-01-01

    In the present study, recombinant organophosphorus hydrolase OPHC2 was successfully produced by Yarrowia lipolytica and purified. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analyses showed a major polypeptide band of 36 kDa. The purified enzyme was optimally active at 65°C and pH 8.5 and also displayed good thermal and pH stability using methyl parathion (O,O-dimethyl-O-4-p-nitrophenyl phosphorothioate) as a substrate. Moreover, as Y. lipolytica is a non-pathogenic, generally regarded as safe (GRAS) yeast, the cell culture supernatant can be used directly on vegetables and fruits that are contaminated by organophosphorus pesticides.

  13. Cell wall hydrolases and antibiotics: exploiting synergy to create efficacious new antimicrobial treatments.

    PubMed

    Wittekind, Michael; Schuch, Raymond

    2016-10-01

    Cell wall hydrolases (CWH) are enzymes that build, remodel and degrade peptidoglycan within bacterial cell walls and serve essential roles in cell-wall metabolism, bacteriophage adsorption and bacteriolysis, environmental niche expansion, as well as eukaryotic innate immune defense against bacterial infection. Some CWHs, when tested as recombinant purified proteins, have been shown to have bactericidal activities both as single agents and in combinations with other antimicrobials, displaying synergies in vitro and potent activities in animal models of infection greater than the single agents alone. We summarize in vitro, in vivo, and mechanistic studies that illustrate ACWH synergy with antibiotics, antimicrobial peptides, and other ACWHs, underscoring the overall synergistic potential of the ACWH class. Copyright © 2016. Published by Elsevier Ltd.

  14. 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.

  15. Gene Overexpression and Biochemical Characterization of the Biotechnologically Relevant Chlorogenic Acid Hydrolase from Aspergillus niger▿

    PubMed Central

    Benoit, Isabelle; Asther, Michèle; Bourne, Yves; Navarro, David; Canaan, Stéphane; Lesage-Meessen, Laurence; Herweijer, Marga; Coutinho, Pedro M.; Asther, Marcel; Record, Eric

    2007-01-01

    The full-length gene that encodes the chlorogenic acid hydrolase from Aspergillus niger CIRM BRFM 131 was cloned by PCR based on the genome of the strain A. niger CBS 513.88. The complete gene consists of 1,715 bp and codes for a deduced protein of 512 amino acids with a molecular mass of 55,264 Da and an acidic pI of 4.6. The gene was successfully cloned and overexpressed in A. niger to yield 1.25 g liter−1, i.e., 330-fold higher than the production of wild-type strain A. niger CIRM BRFM131. The histidine-tagged recombinant ChlE protein was purified to homogeneity via a single chromatography step, and its main biochemical properties were characterized. The molecular size of the protein checked by mass spectroscopy was 74,553 Da, suggesting the presence of glycosylation. ChlE is assembled in a tetrameric form with several acidic isoforms with pIs of around 4.55 and 5.2. Other characteristics, such as optimal pH and temperature, were found to be similar to those determined for the previously characterized chlorogenic acid hydrolase of A. niger CIRM BRFM 131. However, there was a significant temperature stability difference in favor of the recombinant protein. ChlE exhibits a catalytic efficiency of 12.5 × 106 M−1 s−1 toward chlorogenic acid (CGA), and its ability to release caffeic acid from CGA present in agricultural by-products such as apple marc and coffee pulp was clearly demonstrated, confirming the high potential of this enzyme. PMID:17630312

  16. Cloning and expression of 3-deoxy-d-manno-oct-2-ulosonic acid α-ketoside hydrolase from oyster hepatopancreas†

    PubMed Central

    Nakagawa, Tetsuto; Shimada, Yoshimi; Pavlova, Nadejda V; Li, Su-Chen; Li, Yu-Teh

    2015-01-01

    We have previously reported that oyster hepatopancreas contained three unusual α-ketoside hydrolases: (i) a 3-deoxy-d-manno-oct-2-ulosonic acid α-ketoside hydrolase (α-Kdo-ase), (ii) a 3-deoxy-d-glycero-d-galacto-non-2-ulosonic acid α-ketoside hydrolase and (iii) a bifunctional ketoside hydrolase capable of cleaving both the α-ketosides of Kdn and Neu5Ac (Kdn-sialidase). After completing the purification of Kdn-sialidase, we proceeded to clone the gene encoding this enzyme. Unexpectedly, we found that instead of expressing Kdn-sialidase, our cloned gene expressed α-Kdo-ase activity. The full-length gene, consisting of 1176-bp (392 amino acids, Mr 44,604), expressed an active recombinant α-Kdo-ase (R-α-Kdo-ase) in yeast and CHO-S cells, but not in various Escherichia coli strains. The deduced amino acid sequence contains two Asp boxes (S277PDDGKTW and S328TDQGKTW) commonly found in sialidases, but is devoid of the signature FRIP-motif of sialidase. The R-α-Kdo-ase effectively hydrolyzed the Kdo in the core-oligosaccharide of the structurally defined lipopolysaccharide (LPS), Re-LPS (Kdo2-Lipid A) from Salmonella minnesota R595 and E. coli D31m4. However, Rd-LPS from S. minnesota R7 that contained an extra outer core phosphorylated heptose was only slowly hydrolyzed. The complex type LPS from Neisseria meningitides A1 and M992 that contained extra 5–6 sugar units at the outer core were refractory to R-α-Kdo-ase. This R-α-Kdo-ase should become useful for studying the structure and function of Kdo-containing glycans. PMID:26362869

  17. Cloning and expression of 3-deoxy-d-manno-oct-2-ulosonic acid α-ketoside hydrolase from oyster hepatopancreas†.

    PubMed

    Nakagawa, Tetsuto; Shimada, Yoshimi; Pavlova, Nadejda V; Li, Su-Chen; Li, Yu-Teh

    2015-12-01

    We have previously reported that oyster hepatopancreas contained three unusual α-ketoside hydrolases: (i) a 3-deoxy-d-manno-oct-2-ulosonic acid α-ketoside hydrolase (α-Kdo-ase), (ii) a 3-deoxy-D-glycero-D-galacto-non-2-ulosonic acid α-ketoside hydrolase and (iii) a bifunctional ketoside hydrolase capable of cleaving both the α-ketosides of Kdn and Neu5Ac (Kdn-sialidase). After completing the purification of Kdn-sialidase, we proceeded to clone the gene encoding this enzyme. Unexpectedly, we found that instead of expressing Kdn-sialidase, our cloned gene expressed α-Kdo-ase activity. The full-length gene, consisting of 1176-bp (392 amino acids, Mr 44,604), expressed an active recombinant α-Kdo-ase (R-α-Kdo-ase) in yeast and CHO-S cells, but not in various Escherichia coli strains. The deduced amino acid sequence contains two Asp boxes (S(277)PDDGKTW and S(328)TDQGKTW) commonly found in sialidases, but is devoid of the signature FRIP-motif of sialidase. The R-α-Kdo-ase effectively hydrolyzed the Kdo in the core-oligosaccharide of the structurally defined lipopolysaccharide (LPS), Re-LPS (Kdo(2)-Lipid A) from Salmonella minnesota R595 and E. coli D31m4. However, Rd-LPS from S. minnesota R7 that contained an extra outer core phosphorylated heptose was only slowly hydrolyzed. The complex type LPS from Neisseria meningitides A1 and M992 that contained extra 5-6 sugar units at the outer core were refractory to R-α-Kdo-ase. This R-α-Kdo-ase should become useful for studying the structure and function of Kdo-containing glycans.

  18. Recombinant allergens

    PubMed Central

    Jutel, Marek; Solarewicz-Madejek, Katarzyna; Smolinska, Sylwia

    2012-01-01

    Allergen specific immunotherapy (SIT) is the only known causative treatment of allergic diseases. Recombinant allergen-based vaccination strategies arose from a strong need to both to improve safety and enhance efficacy of SIT. In addition, new vaccines can be effective in allergies including food allergy or atopic dermatitis, which poorly respond to the current treatment with allergen extracts. A number of successful clinical studies with both wild-type and hypoallergenic derivatives of recombinant allergens vaccines have been reported for the last decade. They showed high efficacy and safety profile as well as very strong modulation of T and B cell responses to specific allergens. PMID:23095874

  19. Dynamic hydrolase activities precede hypersensitive tissue collapse in tomato seedlings.

    PubMed

    Sueldo, Daniela; Ahmed, Ali; Misas-Villamil, Johana; Colby, Tom; Tameling, Wladimir; Joosten, Matthieu H A J; van der Hoorn, Renier A L

    2014-08-01

    Hydrolases such as subtilases, vacuolar processing enzymes (VPEs) and the proteasome play important roles during plant programmed cell death (PCD). We investigated hydrolase activities during PCD using activity-based protein profiling (ABPP), which displays the active proteome using probes that react covalently with the active site of proteins. We employed tomato (Solanum lycopersicum) seedlings undergoing synchronized hypersensitive cell death by co-expressing the avirulence protein Avr4 from Cladosporium fulvum and the tomato resistance protein Cf-4. Cell death is blocked in seedlings grown at high temperature and humidity, and is synchronously induced by decreasing temperature and humidity. ABPP revealed that VPEs and the proteasome are not differentially active, but that activities of papain-like cysteine proteases and serine hydrolases, including Hsr203 and P69B, increase before hypersensitive tissue collapse, whereas the activity of a carboxypeptidase-like enzyme is reduced. Similar dynamics were observed for these enzymes in the apoplast of tomato challenged with C. fulvum. Unexpectedly, these challenged plants also displayed novel isoforms of secreted putative VPEs. In the absence of tissue collapse at high humidity, the hydrolase activity profile is already altered completely, demonstrating that changes in hydrolase activities precede hypersensitive tissue collapse. © 2014 The Authors New Phytologist © 2014 New Phytologist Trust.

  20. Spectrum Recombination.

    ERIC Educational Resources Information Center

    Greenslade, Thomas B., Jr.

    1984-01-01

    Describes several methods of executing lecture demonstrations involving the recombination of the spectrum. Groups the techniques into two general classes: bringing selected portions of the spectrum together using lenses or mirrors and blurring the colors by rapid movement or foreshortening. (JM)

  1. Recombinant gonadotropins.

    PubMed

    Lathi, R B; Milki, A A

    2001-10-01

    Recombinant DNA technology makes it possible to produce large amounts of human gene products for pharmacologic applications, supplanting the need for human tissues. The genes for the alpha and beta subunits of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and human chorionic gonadotropin (hCG) have been characterized and cloned. Recombinant FSH (rFSH) has been shown to be safe and effective in the treatment of fertility disorders. In comparison with the urinary gonadotropin products, human menopausal gonadotropins (HMG), and urinary follitropins (uFSH), rFSH is more potent and better tolerated by patients. Recombinant HCG appears to be as efficacious as urinary HCG with the benefit of improved local tolerance. Recombinant LH (rLH) is likely to be recommended as a supplement to rFSH for ovulation induction in hypogonadotropic women. It may also benefit in vitro fertilization patients undergoing controlled ovarian hyperstimulation with rFSH combined with pituitary suppression, with a gonadotropin-releasing hormone agonist or antagonist.

  2. Spectrum Recombination.

    ERIC Educational Resources Information Center

    Greenslade, Thomas B., Jr.

    1984-01-01

    Describes several methods of executing lecture demonstrations involving the recombination of the spectrum. Groups the techniques into two general classes: bringing selected portions of the spectrum together using lenses or mirrors and blurring the colors by rapid movement or foreshortening. (JM)

  3. 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.

  4. Discovery libraries targeting the major enzyme classes: the serine hydrolases.

    PubMed

    Otrubova, Katerina; Srinivasan, Venkat; Boger, Dale L

    2014-08-15

    Two libraries of modestly reactive ureas containing either electron-deficient acyl anilines or acyl pyrazoles were prepared and are reported as screening libraries for candidate serine hydrolase inhibitors. Within each library is a small but powerful subset of compounds that serve as a chemotype fragment screening library capable of subsequent structural diversification. Elaboration of the pyrazole-based ureas provided remarkably potent irreversible inhibitors of fatty acid amide hydrolase (FAAH, apparent Ki=100-200 pM) complementary to those previously disclosed enlisting electron-deficient aniline-based ureas.

  5. Discovery libraries targeting the major enzyme classes: the serine hydrolases

    PubMed Central

    Otrubova, Katerina; Srinivasan, Venkat; Boger, Dale L.

    2014-01-01

    Two libraries of modestly reactive ureas containing either electron-deficient acyl anilines or acyl pyrazoles were prepared and are reported as screening libraries for candidate serine hydrolase inhibitors. Within each library is a small but powerful subset of compounds that serve as a chemotype fragment screening library capable of subsequent diversification. Elaboration of the pyrazole-based ureas provided remarkably potent irreversible structural inhibitors of fatty acid amide hydrolase (FAAH, apparent Ki = 100-200 pM) complementary to those previously disclosed enlisting electron-deficient aniline-based ureas. PMID:25037918

  6. 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.

  7. ENGINEERING OF PEPTIDOGLYCAN HYDROLASES FOR CONTROL OF PATHOGENIC BACTERIA

    USDA-ARS?s Scientific Manuscript database

    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...

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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    SciTech Connect

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

    2016-10-25

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

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

  11. 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

  12. Novel microbial epoxide hydrolases for biohydrolysis of glycidyl derivatives.

    PubMed

    Kotik, Michael; Brichac, Jiri; Kyslík, Pavel

    2005-12-06

    Microbial isolates from biofilters and petroleum-polluted bioremediation sites were screened for the presence of enantioselective epoxide hydrolases active towards tert-butyl glycidyl ether, benzyl glycidyl ether, and allyl glycidyl ether. Out of 270 isolated strains, which comprised bacteria, yeasts, and filamentous fungi, four were selected based on the enantioselectivities of their epoxide hydrolases determined in biotransformation reactions. The enzyme of Aspergillus niger M200 preferentially hydrolyses (S)-tert-butyl glycidyl ether to (S)-3-tert-butoxy-1,2-propanediol with a relatively high enantioselectivity (the enantiomeric ratio E is about 30 at a reaction temperature of 28 degrees C). Epoxide hydrolases of Rhodotorula mucilaginosa M002 and Rhodococcus fascians M022 hydrolyse benzyl glycidyl ether with relatively low enantioselectivities, the former reacting predominantly with the (S)-enantiomer, the latter preferring the (R)-enantiomer. Enzymatic hydrolysis of allyl glycidyl ether by Cryptococcus laurentii M001 proceeds with low enantioselectivity (E=3). (R)-tert-Butyl glycidyl ether with an enantiomeric excess (ee) of over 99%, and (S)-3-tert-butoxy-1,2-propanediol with an ee-value of 86% have been prepared on a gram-scale using whole cells of A. niger M200. An enantiomeric ratio of approximately 100 has been determined under optimised biotransformation conditions with the partially purified epoxide hydrolase from A. niger M200. The regioselectivity of this enzyme was determined to be total for both (S)-tert-butyl glycidyl ether and (R)-tert-butyl glycidyl ether.

  13. Recognition of corn defense chitinases by fungal polyglycine hydrolases

    USDA-ARS?s Scientific Manuscript database

    Polyglycine hydrolases (PGH)s are secreted fungal endoproteases that cleave peptide bonds in the polyglycine interdomain linker of ChitA chitinase, an antifungal protein from domesticated corn (Zea mays ssp. mays). These target-specific endoproteases are unusual because they do not cut a defined pep...

  14. 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), ...

  15. 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), ...

  16. Human Intestinal Raf Kinase Inhibitor Protein (RKIP) Catalyzes Prasugrel as a Bioactivation Hydrolase.

    PubMed

    Kazui, Miho; Ogura, Yuji; Hagihara, Katsunobu; Kubota, Kazuishi; Kurihara, Atsushi

    2016-01-01

    Prasugrel is a thienopyridine antiplatelet prodrug that undergoes rapid hydrolysis in vivo to a thiolactone metabolite by human carboxylesterase-2 (hCE2) during gastrointestinal absorption. The thiolactone metabolite is further converted to a pharmacologically active metabolite by cytochrome P450 isoforms. The aim of the current study was to elucidate hydrolases other than hCE2 involved in the bioactivation step of prasugrel in human intestine. Using size-exclusion column chromatography of a human small intestinal S9 fraction, another peak besides the hCE2 peak was observed to have prasugrel hydrolyzing activity, and this protein was found to have a molecular weight of about 20 kDa. This prasugrel hydrolyzing protein was successfully purified from a monkey small intestinal cytosolic fraction by successive four-step column chromatography and identified as Raf-1 kinase inhibitor protein (RKIP) by liquid chromatography-tandem mass spectrometry. Second, we evaluated the enzymatic kinetic parameters for prasugrel hydrolysis using recombinant human RKIP and hCE2 and estimated the contributions of these two hydrolyzing enzymes to the prasugrel hydrolysis reaction in human intestine, which were approximately 40% for hRKIP and 60% for hCE2. Moreover, prasugrel hydrolysis was inhibited by anti-hRKIP antibody and carboxylesterase-specific chemical inhibitor (bis p-nitrophenyl phosphate) by 30% and 60%, respectively. In conclusion, another protein capable of hydrolyzing prasugrel to its thiolactone metabolite was identified as RKIP, and this protein may play a significant role with hCE2 in prasugrel bioactivation in human intestine. RKIP is known to have diverse functions in many intracellular signaling cascades, but this is the first report describing RKIP as a hydrolase involved in drug metabolism.

  17. Inhibition of soluble and microsomal epoxide hydrolase by zinc and other metals.

    PubMed

    Draper, A J; Hammock, B D

    1999-11-01

    Inhibition of xenobiotic-metabolizing enzymes by metals may represent an important mechanism in regulating enzyme activity. Fourteen cations were evaluated for inhibition of microsomal epoxide hydrolase (mEH) (mouse, rat, and human liver), soluble epoxide hydrolase (sEH) (mouse, rat, and human liver), and recombinant potato sEH. Of the metals tested, Hg2+ and Zn2+ were the strongest inhibitors of mEH, while Cd2+ and Cu2+ were also strong inhibitors of sEH (I50 for all approximately 20 microM). Nickel (divalent) and Pb2+ were moderate inhibitors, but Al2+, Ba2+, Ca2+, Co2+, Fe2+, Fe3+, Mg2+, and Mn2+ were weak inhibitors of both mEH and sEH (less than 50% inhibition by 1 mM metal). Six anions (acetate, bromide, chloride, nitrate, perchlorate, and sulfate) were tested and found to have no effect on the inhibition of sEH or mEH by cations. The kinetics and type of inhibition for zinc inhibition of sEH and mEH were examined for mouse, rat, human, and potato. Zinc inhibits mEH in a competitive manner. Inhibition of human and potato sEH was noncompetitive, but interestingly, zinc inhibition of mouse sEH was very strong and uncompetitive. Inhibition by zinc could be reversed by adding EDTA to the incubation buffer. Additionally, mouse liver microsomes and cytosol were incubated with these chelators. Following incubation at 4 degrees C, samples were dialyzed to remove chelator. Both mEH and sEH activity recovered was greater in samples treated with chelator than in control incubations. Similar treatment with the protease inhibitor Nalpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) did not affect enzyme activity recovered. During systemic inflammation, hepatic metallothionien is induced, and liver metal concentrations increase while serum metal concentrations are decreased. The inhibition of microsomal and soluble epoxide hydrolase by metals may represent a mechanism of down-regulation of enzyme activity during inflammation.

  18. Effect of alpha lipoic acid on leukotriene A4 hydrolase.

    PubMed

    Torres, María José; Fierro, Angélica; Pessoa-Mahana, C David; Romero-Parra, Javier; Cabrera, Gonzalo; Faúndez, Mario

    2017-03-15

    Leukotriene A4 hydrolase is a soluble enzyme with epoxide hydrolase and aminopeptidase activities catalysing the conversion of leukotriene A4 to leukotriene B4 and the hydrolysis of the peptide proline-glycine-proline. Imbalances in leukotriene B4 synthesis are related to several pathologic conditions. Currently there are no available drugs capable to modulate the synthesis of leukotriene B4 or to block its receptors. Here we show the inhibitory profile of alpha lipoic acid on the activity of leukotriene A4 Hydrolase. Alpha lipoic acid inhibited both activities of the enzyme at concentrations lower than 10μM. The 5-lipoxygenase inhibitor zileuton, or the 5-lipoxygenase activating protein inhibitor MK-886, were unable to inhibit the activity of the enzyme. Acute promyelocytic leukaemia HL-60 cells were differentiated to leukotriene A4 hydrolase expressing neutrophil-like cells. Alpha lipoic acid inhibited the aminopeptidase activity of the cytosolic fraction from neutrophil-like cells but had no effect on the cytosolic fraction from undifferentiated cells. Docking and molecular dynamic approximations revealed that alpha lipoic acid participates in electrostatic interactions with K-565 and R-563, which are key residues for the carboxylate group recognition of endogenous substrates by the enzyme. Alpha lipoic acid is a compound widely used in clinical practice, most of its therapeutic effects are associated with its antioxidants properties, however, antioxidant effect alone is unable to explain all clinical effects observed with alpha lipoic acid. Our results invite to evaluate the significance of the inhibitory effect of alpha lipoic acid on the catalytic activity of leukotriene A4 hydrolase using in vivo models.

  19. Consolidation of glycosyl hydrolase family 30 : a dual domain 4/7 hydrolase family consisting of two structurally distinct groups

    Treesearch

    Franz J. St John; Javier M. Gonzalez; Edwin Pozharski

    2010-01-01

    In this work glycosyl hydrolase (GH) family 30 (GH30) is analyzed and shown to consist of its currently classified member sequences as well as several homologous sequence groups currently assigned within family GH5. A large scale amino acid sequence alignment and a phylogenetic tree were generated and GH30 groups and subgroups were designated. A partial rearrangement...

  20. Polyglycine hydrolases: Fungal β-lactamase-like endoproteases that cleave polyglycine regions within plant class IV chitinases

    PubMed Central

    Naumann, Todd A; Naldrett, Michael J; Ward, Todd J; Price, Neil P J

    2015-01-01

    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 Cochliobolus carbonum (Bz-cmp). Here we report the identity of their encoding genes and the primary amino acid sequences of the proteins responsible for these activities. Peptides from a tryptic digest of Es-cmp were analyzed by LC-MS/MS and the spectra obtained were matched to a draft genome sequence of E. sorghi. From this analysis, a 642 amino acid protein containing a predicted β-lactamase catalytic region of 280 amino acids was identified. Heterologous strains of the yeast Pichia pastoris were created to express this protein and its homolog from C. carbonum from their cDNAs. Both strains produced recombinant proteins with polyglycine hydrolase activity as shown by SDS-PAGE and MALDI-MS based assays. Site directed mutagenesis was used to mutate the predicted catalytic serine of Es-cmp to glycine, resulting in loss of catalytic activity. BLAST searching of publicly available fungal genomes identified full-length homologous proteins in 11 other fungi of the class Dothideomycetes, and in three fungi of the related class Sordariomycetes while significant BLAST hits extended into the phylum Basidiomycota. Multiple sequence alignment led to the identification of a network of seven conserved tryptophans that surround the β-lactamase-like region. This is the first report of a predicted β-lactamase that is an endoprotease. PMID:25966977

  1. Identification of a plasmid-borne parathion hydrolase gene from Flavobacterium sp. by southern hybridization with opd from Pseudomonas diminuta.

    PubMed

    Mulbry, W W; Karns, J S; Kearney, P C; Nelson, J O; McDaniel, C S; Wild, J R

    1986-05-01

    Parathion hydrolases have been previously described for an American isolate of Pseudomonas diminuta and a Philippine isolate of Flavobacterium sp. (ATCC 27551). The gene which encodes the broad-spectrum organophosphate phosphotriesterase in P. diminuta has been shown by other investigators to be located on a 66-kilobase (kb) plasmid. The intact gene (opd, organophosphate-degrading gene) from this degradative plasmid was cloned into M13mp10 and found to express parathion hydrolase under control of the lac promoter in Escherichia coli. In Flavobacterium sp. strain ATCC 27551, a 43-kb plasmid was associated with the production of parathion hydrolase by curing experiments. The M13mp10-cloned fragment of the opd gene from P. diminuta was used to identify a homologous genetic region from Flavobacterium sp. strain ATCC 27551. Southern hybridization experiments demonstrated that a genetic region from the 43-kb Flavobacterium sp. plasmid possessed significant homology to the opd sequence. Similar hybridization did not occur with three other native Flavobacterium sp. plasmids (approximately 23, 27, and 51 kb) present within this strain or with genomic DNA from cured strains. Restriction mapping of various recombinant DNA molecules containing subcloned fragments of both opd plasmids revealed that the restriction maps of the two opd regions were similar, if not identical, for all restriction endonucleases tested thus far. In contrast, the restriction maps of the cloned plasmid sequences outside the opd regions were not similar. Thus, it appears that the two discrete bacterial plasmids from parathion-hydrolyzing soil bacteria possess a common but limited region of sequence homology within potentially nonhomologous plasmid structures.

  2. Identification of a plasmid-borne parathion hydrolase gene from Flavobacterium sp. by southern hybridization with opd from Pseudomonas diminuta.

    PubMed Central

    Mulbry, W W; Karns, J S; Kearney, P C; Nelson, J O; McDaniel, C S; Wild, J R

    1986-01-01

    Parathion hydrolases have been previously described for an American isolate of Pseudomonas diminuta and a Philippine isolate of Flavobacterium sp. (ATCC 27551). The gene which encodes the broad-spectrum organophosphate phosphotriesterase in P. diminuta has been shown by other investigators to be located on a 66-kilobase (kb) plasmid. The intact gene (opd, organophosphate-degrading gene) from this degradative plasmid was cloned into M13mp10 and found to express parathion hydrolase under control of the lac promoter in Escherichia coli. In Flavobacterium sp. strain ATCC 27551, a 43-kb plasmid was associated with the production of parathion hydrolase by curing experiments. The M13mp10-cloned fragment of the opd gene from P. diminuta was used to identify a homologous genetic region from Flavobacterium sp. strain ATCC 27551. Southern hybridization experiments demonstrated that a genetic region from the 43-kb Flavobacterium sp. plasmid possessed significant homology to the opd sequence. Similar hybridization did not occur with three other native Flavobacterium sp. plasmids (approximately 23, 27, and 51 kb) present within this strain or with genomic DNA from cured strains. Restriction mapping of various recombinant DNA molecules containing subcloned fragments of both opd plasmids revealed that the restriction maps of the two opd regions were similar, if not identical, for all restriction endonucleases tested thus far. In contrast, the restriction maps of the cloned plasmid sequences outside the opd regions were not similar. Thus, it appears that the two discrete bacterial plasmids from parathion-hydrolyzing soil bacteria possess a common but limited region of sequence homology within potentially nonhomologous plasmid structures. Images PMID:3015022

  3. 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.

  4. Diversity and Biocatalytic Potential of Epoxide Hydrolases Identified by Genome Analysis†

    PubMed Central

    van Loo, Bert; Kingma, Jaap; Arand, Michael; Wubbolts, Marcel G.; Janssen, Dick B.

    2006-01-01

    Epoxide hydrolases play an important role in the biodegradation of organic compounds and are potentially useful in enantioselective biocatalysis. An analysis of various genomic databases revealed that about 20% of sequenced organisms contain one or more putative epoxide hydrolase genes. They were found in all domains of life, and many fungi and actinobacteria contain several putative epoxide hydrolase-encoding genes. Multiple sequence alignments of epoxide hydrolases with other known and putative α/β-hydrolase fold enzymes that possess a nucleophilic aspartate revealed that these enzymes can be classified into eight phylogenetic groups that all contain putative epoxide hydrolases. To determine their catalytic activities, 10 putative bacterial epoxide hydrolase genes and 2 known bacterial epoxide hydrolase genes were cloned and overexpressed in Escherichia coli. The production of active enzyme was strongly improved by fusion to the maltose binding protein (MalE), which prevented inclusion body formation and facilitated protein purification. Eight of the 12 fusion proteins were active toward one or more of the 21 epoxides that were tested, and they converted both terminal and nonterminal epoxides. Four of the new epoxide hydrolases showed an uncommon enantiopreference for meso-epoxides and/or terminal aromatic epoxides, which made them suitable for the production of enantiopure (S,S)-diols and (R)-epoxides. The results show that the expression of epoxide hydrolase genes that are detected by analyses of genomic databases is a useful strategy for obtaining new biocatalysts. PMID:16597997

  5. Cytosolic and microsomal epoxide hydrolases are immunologically distinguishable from each other in the rat and mouse.

    PubMed

    Guenthner, T M; Hammock, B D; Vogel, U; Oesch, F

    1981-04-10

    Antibodies raised to homogeneous rat liver microsomal epoxide hydrolase were used to distinguish microsomal epoxide hydrolase from epoxide hydrolase of cytosolic origin in mice and rats. Using double diffusion analysis in agarose gels, we show that anti-rat liver microsomal epoxide hydrolase forms a single precipitin line with solubilized microsomes from rat and mouse liver, but no reaction is seen with the corresponding cytosolic fractions. Rat or mouse microsomal epoxide hydrolase activity (using benzo[a]pyrene 4,5-oxide as substrate) can be completely precipitated out of solubilized preparations by the antibody, which is equipotent against rat and mouse microsomal epoxide hydrolase. No precipitation of cytosolic hydrolase activity (using trans-beta-ethyl styrene oxide as substrate) is seen with any concentration of the antibody tested. Thus, in the case of microsomal epoxide hydrolase, extensive immunological cross-reactivity exists between the two species, rat and mouse. In contrast, no cross-reactivity is detectable between cytosolic and microsomal epoxide hydrolase, even when enzymes from the same species are compared. We conclude that microsomal and cytosolic epoxide hydrolase activities represent distinct and immunologically non-cross-reactive protein species.

  6. OpdA, a bacterial organophosphorus hydrolase, prevents lethality in rats after poisoning with highly toxic organophosphorus pesticides

    PubMed Central

    Bird, Steven B.; Sutherland, Tara D.; Gresham, Chip; Oakeshott, John; Scott, Colin; Eddleston, Michael

    2008-01-01

    Organophosphorus (OP) pesticides poison more than 3,000,000 people every year in the developing world, mostly through intentional self-poisoning. Advances in medical therapy for OP poisoning have lagged, and current treatment is not highly effective with mortality of up to 40% in even the most advanced Western medical facilities. Administration of a broadly active bacterial OP hydrolase to patients in order to hydrolyze OPs in circulation might allow current therapies to be more effective. The objective of this work was to evaluate the efficacy of a new recombinant bacterial OP hydrolase (OpdA), cloned from Agrobacterium radiobacter, in rat models of two chemically distinct but highly toxic and rapidly acting OP pesticides: dichlorvos and parathion. Without OpdA treatment, median time to death in rats poisoned with 3 × LD50 of dichlorvos or parathion was 6 minutes and 25.5 minutes, respectively. Administration of a single dose of OpdA immediately after dichlorvos resulted in 100% survival at 24 hours, with no additional antidotal therapy. After parathion poisoning, OpdA alone caused only a delay to death. However, an additional two doses of OpdA resulted in 62.5% survival at 24 hours after parathion poisoning. In combination with pralidoxime therapy, a single dose of OpdA increased survival to 75% after parathion poisoning. Our results demonstrate that OpdA is able to improve survival after poisoning by two chemically distinct and highly toxic OP pesticides. PMID:18378376

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

    PubMed

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

    2011-08-01

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

  8. Cloning, expression, purification and preliminary X-ray analysis of EstN2, a novel archaeal α/β-hydrolase from Candidatus Nitrososphaera gargensis.

    PubMed

    Kaljunen, Heidi; Chow, Jennifer; Streit, Wolfgang R; Mueller-Dieckmann, Jochen

    2014-10-01

    EstN2 is a novel α/β-hydrolase originating from the ammonia-oxidizing thaumarchaeon Candidatus Nitrososphaera gargensis. The genome of the organism was sequenced and genes conferring putative lipolytic activity were amplified and cloned into Escherichia coli as a heterologous host. Through function-based screening, esterase and lipase activity was detected. A recombinant enzyme designated EstN2 was successfully expressed, purified and crystallized. The crystals belonged to space group I2, with one molecule per asymmetric unit, and diffracted X-rays to 1.5 Å resolution.

  9. Production, crystallization and structure determination of a mycobacterial glucosylglycerate hydrolase.

    PubMed

    Cereija, Tatiana Barros; Alarico, Susana; Empadinhas, Nuno; Pereira, Pedro José Barbosa

    2017-09-01

    Glucosylglycerate hydrolase is highly conserved among rapidly growing mycobacteria and has been found to be involved in recovery from nitrogen starvation by promoting the rapid mobilization of the glucosylglycerate that accumulates under these conditions. Here, the production, crystallization and structure determination of glucosylglycerate hydrolase from Mycobacterium hassiacum using two-wavelength anomalous diffraction of selenomethionine-substituted crystals are described. The monoclinic (space group P21) crystals diffracted to ∼2.0 Å resolution at a synchrotron-radiation source and contained four molecules in the asymmetric unit, corresponding to a Matthews coefficient of 3.07 Å(3) Da(-1) and a solvent content of 59.9%. The quality of the experimental phases allowed the automated building of 1677 of the 1792 residues in the asymmetric unit.

  10. Cloning and biochemical characterization of the fucanase FcnA: definition of a novel glycoside hydrolase family specific for sulfated fucans.

    PubMed

    Colin, Sébastien; Deniaud, Estelle; Jam, Murielle; Descamps, Valérie; Chevolot, Yann; Kervarec, Nelly; Yvin, Jean-Claude; Barbeyron, Tristan; Michel, Gurvan; Kloareg, Bernard

    2006-11-01

    Sulfated fucans are matrix polysaccharides from marine brown algae, consisting of an alpha-L-fucose backbone substituted by sulfate-ester groups, masked with ramifications, and containing other monosaccharide residues. We here report on the characterization of a novel glycoside hydrolase (FcnA) specific for the degradation of sulfated fucans. This glycoside hydrolase was purified to electrophoretic homogeneity from a Flavobacteriaceae referred to as SW5. The gene fcnA was cloned and sequenced (3021 nucleotides), and the protein (1007 amino acids) was produced in Escherichia coli. FcnA exhibited a modular architecture consisting of a 400-residue-long N-terminal domain followed by three repeated domains predicted to adopt an immunoglobulin fold and by an 80-amino acid-long C-terminal domain. A truncated recombinant protein encompassing the N-terminal domain and the immunoglobulin-like repeats was shown to retain the enzyme activity. The N-terminal catalytic domain shared approximately 25% of sequence identity with two patented fucanase genes, and these three fucanases delineate a new family of glycoside hydrolases. As shown by size-exclusion chromatography (SEC) and 1H-NMR analyses, the fucanase FcnA proceeds according to an endolytic mode of action and cleaves the alpha-(1-->4) glycosidic linkages within the blocks of repeating motifs [-->4)-alpha-L-fucopyranosyl-2,3-disulfate-(1-->3)-alpha-L-fucopyranosyl-2-sulfate-(1-->]n.

  11. Alpha/Beta-hydrolase fold enzymes: structures, functions and mechanisms.

    PubMed

    Holmquist, M

    2000-09-01

    The alpha/beta-hydrolase fold family of enzymes is rapidly becoming one of the largest group of structurally related enzymes with diverse catalytic functions. Members in this family include acetylcholinesterase, dienelactone hydrolase, lipase, thioesterase, serine carboxypeptidase, proline iminopeptidase, proline oligopeptidase, haloalkane dehalogenase, haloperoxidase, epoxide hydrolase, hydroxynitrile lyase and others. The enzymes all have a Nucleophile-His-Acid catalytic triad evolved to efficiently operate on substrates with different chemical composition or physicochemical properties and in various biological contexts. For example, acetylcholine esterase catalyzes the cleavage of the neurotransmitter acetylcholine, at a rate close to the limits of diffusion of substrate to the active site of the enzyme. Dienelactone hydrolase uses substrate-assisted catalysis to degrade aromatic compounds. Lipases act adsorbed at the water/lipid interface of their neutral water-insoluble ester substrates. Most lipases have their active site buried under secondary structure elements, a flap, which must change conformation to allow substrate to access the active site. Thioesterases are involved in a multitude of biochemical processes including bioluminiscence, fatty acid- and polyketide biosynthesis and metabolism. Serine carboxypeptidases recognize the negatively charged carboxylate terminus of their peptide substrates. Haloalkane dehalogenase is a detoxifying enzyme that converts halogenated aliphatics to the corresponding alcohols, while haloperoxidase catalyzes the halogenation of organic compounds. Hydroxynitrile lyase cleaves carbon-carbon bonds in cyanohydrins with concomitant hydrogen cyanide formation as a defense mechanism in plants. This paper gives an overview of catalytic activities reported for this family of enzymes by discussing selected examples. The current state of knowledge of the molecular basis for catalysis and substrate specificity is outlined

  12. 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.

  13. Human lung hydrolases delineate Mycobacterium tuberculosis-macrophage interactions and the capacity to control infection.

    PubMed

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

    2011-07-01

    Pulmonary surfactant contains homeostatic and antimicrobial hydrolases. When Mycobacterium tuberculosis is initially deposited in the terminal bronchioles and alveoli, as well as following release from lysed macrophages, bacilli are in intimate contact with these lung surfactant hydrolases. We identified and measured several hydrolases in human alveolar lining fluid and lung tissue that, at their physiological concentrations, dramatically modified the M. tuberculosis cell envelope. Independent of their action time (15 min to 12 h), the effects of the hydrolases on the M. tuberculosis cell envelope resulted in a significant decrease (60-80%) in M. tuberculosis association with, and intracellular growth of the bacteria within, human macrophages. The cell envelope-modifying effects of the hydrolases also led to altered M. tuberculosis intracellular trafficking and induced a protective proinflammatory response to infection. These findings add a new concept to our understanding of M. tuberculosis-macrophage interactions (i.e., the impact of lung surfactant hydrolases on M. tuberculosis infection).

  14. 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

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

    PubMed Central

    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, at the levels of the whole genome and individual glycoside hydrolase families. Results We identified 356 glycoside hydrolase genes in Brachypodium and 404 in sorghum. The corresponding proteins fell into the same 34 families that are represented in rice, Arabidopsis, and poplar, helping to define a glycoside hydrolase family profile which may be common to flowering plants. For several glycoside hydrolase familes (GH5, GH13, GH18, GH19, GH28, and GH51), we present a detailed literature review together with an examination of the family structures. This analysis of individual families revealed both similarities and distinctions between monocots and eudicots, as well as between species. Shared evolutionary histories appear to be modified by lineage-specific expansions or deletions. Within GH families, the Brachypodium and sorghum proteins generally cluster with those from other monocots. Conclusions This work provides the foundation for further comparative and functional analyses of plant glycoside hydrolases. Defining the Brachypodium glycoside hydrolases sets the stage for

  16. Contribution of hydrolase and phosphatase domains in soluble epoxide hydrolase to vascular endothelial growth factor expression and cell growth.

    PubMed

    Oguro, Ami; Sakamoto, Koichi; Suzuki, Sachiko; Imaoka, Susumu

    2009-12-01

    Soluble epoxide hydrolase (sEH) is an important pharmacological target because it metabolizes potent bioactive substrates, epoxyeicosatrienoinc acids (EETs) and other lipid epoxide. EETs have a variety of biological functions including angiogenesis and cancer metastasis. However, the regulation and physiological function of sEH is not well understood. In this study, we found that hypoxia significantly suppressed the expression of sEH in mouse liver and a human hepatoma cell line, Hep3B. Hypoxia promotes the proliferation of vascular endothelial cells or carcinoma cells. Knockdown of sEH in Hep3B cells induced vascular endothelial growth factor (VEGF) mRNA and cell growth, both of which were suppressed by overexpression of sEH. sEH has phosphatase activity as well as epoxide hydrolase (EH) activity. We prepared mutant clones which lacking EH or phosphatase activity using the amino acid change Asp335Ser or Asp9Ala, respectively. The effects of WT sEH on cell growth were lost by mutation of either the EH domain or phosphatase domain. However, mutation of the phosphatase domain but not EH domain did not influence the expression of VEGF. These results suggest that sEH plays an important role in the physiology of cells including proliferation and that the epoxide hydrolase and phosphatase domains of sEH have different biological functions.

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

    PubMed

    Jamek, Shariza B; Nyffenegger, Christian; Muschiol, Jan; Holck, Jesper; Meyer, Anne S; Mikkelsen, Jørn D

    2017-06-01

    Type A chitinases (EC 3.2.1.14), GH family 18, attack chitin ((1 → 4)-2-acetamido-2-deoxy-β-D-glucan) and chito-oligosaccharides from the reducing end to catalyze release of chitobiose (N,N'-diacetylchitobiose) via hydrolytic cleavage of N-acetyl-β-D-glucosaminide (1 → 4)-β-linkages and are thus "exo-chitobiose hydrolases." In this study, the chitinase type A from Serratia marcescens (SmaChiA) was used as a template for identifying two novel exo-chitobiose hydrolase type A enzymes, FbalChi18A and MvarChi18A, originating from the marine organisms Ferrimonas balearica and Microbulbifer variabilis, respectively. Both FbalChi18A and MvarChi18A were recombinantly expressed in Escherichia coli and were confirmed to exert exo-chitobiose hydrolase activity on chito-oligosaccharides, but differed in temperature and pH activity response profiles. Amino acid sequence comparison of the catalytic β/α barrel domain of each of the new enzymes showed individual differences, but ~69% identity of each to that of SmaChiA and highly conserved active site residues. Superposition of a model substrate on 3D structural models of the catalytic domain of the enzymes corroborated exo-chitobiose hydrolase type A activity for FbalChi18A and MvarChi18A, i.e., substrate attack from the reducing end. A main feature of both of the new enzymes was the presence of C-terminal 5/12 type carbohydrate-binding modules (SmaChiA has no C-terminal carbohydrate binding module). These new enzymes may be useful tools for utilization of chitin as an N-acetylglucosamine donor substrate via chitobiose.

  18. Immunohistochemical study of epoxide hydrolase induced by trichloroethylene in rat liver

    SciTech Connect

    Kawamoto, T.; Hobara, T.; Ogino, K.; Takemoto, T.; Nakamura, K.; Imamura, A.; Koshiro, A.; Kobayashi, H.; Iwamoto, S.; Sakai, T.

    1987-10-01

    Epoxide hydrolase catalyzes the hydrolation of potentially toxic, electrophilic epoxides that are often generated during cytochrome P-450 catalyzed monooxigenation, forming the corresponding transdihydrodiols. It is well-known that trichloroethylene is metabolized by cytochrome P-450 containing mixed-function oxidase systems to trichloroethylene oxide, which decomposes to other metabolites. As trichloroethylene is an epoxide, epoxide hydrolase is suspected to catalyze the hydrolation of trichloroethylene oxide. No reports have appeared about the relationship between trichloroethylene and epoxide hydrolase. In this report, the authors studied the effect of trichloroethylene on epoxide hydrolase immunohistochemically.

  19. 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

  20. A high throughput fluorescent assay for measuring the activity of fatty acid amide hydrolase.

    PubMed

    Kage, Karen L; Richardson, Paul L; Traphagen, Linda; Severin, Jean; Pereda-Lopez, Ana; Lubben, Thomas; Davis-Taber, Rachel; Vos, Melissa H; Bartley, Diane; Walter, Karl; Harlan, John; Solomon, Larry; Warrior, Usha; Holzman, Thomas F; Faltynek, Connie; Surowy, Carol S; Scott, Victoria E

    2007-03-30

    Fatty acid amide hydrolase (FAAH) is the enzyme responsible for the rapid degradation of fatty acid amides such as the endocannabinoid anandamide. Inhibition of FAAH activity has been suggested as a therapeutic approach for the treatment of chronic pain, depression and anxiety, through local activation of the cannabinoid receptor CB1. We have developed a high throughput screening assay for identification of FAAH inhibitors using a novel substrate, decanoyl 7-amino-4-methyl coumarin (D-AMC) that is cleaved by FAAH to release decanoic acid and the highly fluorescent molecule 7-amino-4-methyl coumarin (AMC). This assay gives an excellent signal window for measuring FAAH activity and, as a continuous assay, inherently offers improved sensitivity and accuracy over previously reported endpoint assays. The assay was validated using a panel of known FAAH inhibitors and purified recombinant human FAAH, then converted to a 384 well format and used to screen a large library of compounds (>600,000 compounds) to identify FAAH inhibitors. This screen identified numerous novel FAAH inhibitors of diverse chemotypes. These hits confirmed using a native FAAH substrate, anandamide, and had very similar rank order potency to that obtained using the D-AMC substrate. Collectively these data demonstrate that D-AMC can be successfully used to rapidly and effectively identify novel FAAH inhibitors for potential therapeutic use.

  1. Efficient kinetic resolution of phenyl glycidyl ether by a novel epoxide hydrolase from Tsukamurella paurometabola.

    PubMed

    Wu, Kai; Wang, Hualei; Sun, Huihui; Wei, Dongzhi

    2015-11-01

    Enantioselective hydrolysis of racemic epoxides mediated by epoxide hydrolases (EHs) is one of the most promising approaches to obtain enantiopure epoxides. In this study, we identified and characterized a novel EH (TpEH1) from Tsukamurella paurometabola by analyzing the conserved catalytic residues of EH. TpEH1 was overexpressed and purified, and its catalytic properties were studied using racemic phenyl glycidyl ether (PGE) and its derivatives as substrates. TpEH1 showed excellent enantioselectivity to the substrates PGE, 3-methylPGE, and 3-nitroPGE. The highest enantioselectivity (E > 100) was achieved when 3-nitroPGE was used as the substrate. The recombinant Escherichia coli TpEH1 demonstrated high substrate tolerance toward PGE and could hydrolyze PGE at concentrations of up to 400 mM (60 g/L) with high enantioselectivity (E = 65), giving (R)-PGE with enantiomeric excess of more than 99 % ee and 45 % yield within 1 h. This concentration of PGE is the highest reported concentration catalyzed by native EHs to date. Thus, the easily available and highly active E. coli TpEH1 showed great potential for the practical preparation of optically pure (R)-PGE.

  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. 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.

  4. 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

  5. Cloning and characterization of three epoxide hydrolases from a marine bacterium, Erythrobacter litoralis HTCC2594.

    PubMed

    Woo, Jung-Hee; Hwang, Young-Ok; Kang, Sung Gyun; Lee, Hyun Sook; Cho, Jang-Cheon; Kim, Sang-Jin

    2007-08-01

    Previously, we reported that ten strains belonging to Erythrobacter showed epoxide hydrolase (EHase) activities toward various epoxide substrates. Three genes encoding putative EHases were identified by analyzing open reading frames of Erythrobacter litoralis HTCC2594. Despite low similarities to reported EHases, the phylogenetic analysis of the three genes showed that eeh1 was similar to microsomal EHase, while eeh2 and eeh3 could be grouped with soluble EHases. The three EHase genes were cloned, and the recombinant proteins (rEEH1, rEEH2, and rEEH3) were purified. The functionality of purified proteins was proved by hydrolytic activities toward styrene oxide. EEH1 preferentially hydrolyzed (R)-styrene oxide, whereas EEH3 preferred to hydrolyze (S)-styrene oxide, representing enantioselective hydrolysis of styrene oxide. On the other hand, EEH2 could hydrolyze (R)- and (S)-styrene oxide at an equal rate. The optimal pH and temperature for the EHases occurred largely at neutral pHs and 40-55 degrees C. The substrate selectivity of rEEH1, rEEH2, and rEEH3 toward various epoxide substrates were also investigated. This is the first representation that a strict marine microorganism possessed three EHases with different enantioselectivity toward styrene oxide.

  6. Molecular cloning of glycoside hydrolase family 45 cellulase genes from brackish water clam Corbicula japonica.

    PubMed

    Sakamoto, Kentaro; Toyohara, Haruhiko

    2009-04-01

    We previously reported endogenous Glycoside Hydrolase Family (GHF) 9 beta-1,4-glucanase gene, CjCel9A, from common Japanese freshwater clam Corbicula japonica. Here we identified another endogenous beta-1,4-glucanase genes which belong to GHF45 (CjCel45A, CjCel45B). Both genes encode ORF of 627 bp corresponding to 208 amino acids. CjCel45A and CjCel45B are different in 5' and 3'-untranslated regions and six nucleotides in the ORF. CjCEL45 has only one GHF45 catalytic domain without any carbohydrate binding modules as is the case with other molluskan GHF45 enzymes. Phylogenetic analysis and genomic structure of CjCel45 gene implies that this gene is likely to be acquired from fungi by common ancestor of mollusks. Reverse transcription (RT)-PCR analysis and in situ hybridization revealed that CjCel45A is likely to be expressed in the secretory cells in the digestive gland, suggesting that this cellulase is produced in the same site as CjCEL9A. CjCEL45A was successfully expressed in E. coli cells and zymographic analysis of the recombinant CjCEL45A showed that CjCEL45A is a functional beta-1,4-glucanase. The finding of multiple cellulase genes in C. japonica strongly supports our hypothesis that this species function as a cellulose decomposer in estuarine environments.

  7. 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. Copyright © 2015. Published by Elsevier Ireland Ltd.

  8. Purification and characterization of methyl parathion hydrolase from Burkholderia cepacia capable of degrading organophosphate insecticides.

    PubMed

    Ekkhunnatham, Anirut; Jongsareejit, Boonsri; Yamkunthong, Wanphen; Wichitwechkarn, Jesdawan

    2012-04-01

    Methyl parathion hydrolase (MPH) from a methyl parathion-degrading Burkholderia cepacia indigenous to Thailand was purified to apparent homogeneity by three steps of column chromatography using Resource S, Sephadex G100, and Octyl Sepharose 4FF columns. Its molecular mass was determined to be 35 kDa, and the pI to be 8.5. The recombinant plasmid pGT1, containing the MPH-encoding gene, mpdB, cloned into pGEX-4T-2 was over-expressed in Escherichia coli as GST-MPH fusion protein. The recombinant MPH was purified to homogeneity by a single step, using GSTPrep FF affinity column, with the molecular mass identical to that of the native enzyme. The purified enzyme had the specific activity of about 1,600 unit mg(-1) protein and the yield of about 75%, a 39-fold increase in recovery compared to that of the native enzyme. The optimal temperature and pH were 25°C and 9.0, respectively. The MPH was stable, with its activity unchanged for 48 h at 4°C, and reduced to 50% after 5 h and to 45% after 48 h at 25°C. The enzyme activity remained 80-90% after 8-15 h at pH 6-7. Cd(2+), Co(2+), and Zn(2+) ions at the concentration of 1 mM enhanced the activity; while sodium dodecyl sulfate (SDS), dithiothreitol (DTT) and ethylenediaminetetraacetate (EDTA) reduced it. The enzyme also showed cross reactivity with other insecticides within the organophosphate group, and the kinetic parameters for individual substrates were investigated. Since MPH from B. cepacia has wide potential applications in detoxification and detection of organophosphate compounds, this study provides important basis for its future use.

  9. 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

  10. 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

  11. Structure of a soluble epoxide hydrolase identified in Trichoderma reesei.

    PubMed

    Wilson, Carolina; De Oliveira, Gabriel S; Adriani, Patrícia P; Chambergo, Felipe S; Dias, Marcio V B

    2017-08-01

    Epoxide hydrolases (EHs) are enzymes that have high biotechnological interest for the fine and transformation industry. Several of these enzymes have enantioselectivity, which allows their application in the separation of enantiomeric mixtures of epoxide substrates. Although two different families of EHs have been described, those that have the α/β-hidrolase fold are the most explored for biotechnological purpose. These enzymes are functionally very well studied, but only few members have three-dimensional structures characterised. Recently, a new EH from the filamentous fungi Trichoderma reseei (TrEH) has been discovered and functionally studied. This enzyme does not have high homology to any other EH structure and have an enatiopreference for (S)-(-) isomers. Herein we described the crystallographic structure of TrEH at 1.7Å resolution, which reveals features of its tertiary structure and active site. TrEH has a similar fold to the other soluble epoxide hydrolases and has the two characteristic hydrolase and cap domains. The enzyme is predominantly monomeric in solution and has also been crystallised as a monomer in the asymmetric unit. Although the catalytic residues are conserved, several other residues of the catalytic groove are not, and might be involved in the specificity for substrates and in the enantioselectivy of this enzyme. In addition, the determination of the crystallographic structure of TrEH might contribute to the rational site direct mutagenesis to generate an even more stable enzyme with higher efficiency to be used in biotechnological purposes. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Enzymatic degradation of monocrotophos by extracellular fungal OP hydrolases.

    PubMed

    Jain, Rachna; Garg, Veena

    2013-11-01

    The present study explores the potential of extracellular fungal organophosphate (OP) hydrolase for the degradation of monocrotophos. Extracellular OP hydrolases were isolated and purified from five different fungal isolates viz. Aspergillus niger (M1), Aspergillus flavus (M2), Penicillium aculeatum (M3), Fusarium pallidoroseum (M4), and Macrophomina sp. (M5) by AmSO4 precipitation, dialysis, and G-100 chromatography. M3 showed highest percentage yield of 68.81 followed by 55.41 % for M1. Each of the purified enzyme fraction constituted of two different subunits of 33- and 67-kDa molecular weight. Optimum enzyme fraction (150 μg ml(-1)) rapidly degraded monocrotophos within 120 h in phosphorus-free liquid culture medium (CZM) with K deg of 0.0368, 0.0138, 0.048, 0.016, 0.0138, and 0.048 day(-1) and half-life of 0.79, 2.11, 0.6, 1.8, and 2.11 days for M1, M2, M3, M4, and M5, respectively. The results were further confirmed by high performance thin layer chromatography and Fourier transform infrared which indicate the disappearance of monocrotophos by hydrolytic cleavage of vinyl phosphate bond. The overall order of enzymatic degradation was found to be P. aculeatum > A. niger > F. pallidoroseum > A. flavus = Macrophomina sp. Hence, the study concludes that extracellular OP hydrolases efficiently degraded monocrotophos and could be used as a potential candidate for the detoxification of this neurotoxin pesticide.

  13. 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

  14. Inhibiting an epoxide hydrolase virulence strategy protects CFTR**

    PubMed Central

    Bahl, Christopher D.; Hvorecny, Kelli L.; Bomberger, Jennifer M.; Stanton, Bruce A.; Hammock, Bruce D.; Morisseau, Christophe; Madden, Dean R.

    2015-01-01

    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, Cif's mechanism of action has remained unclear. It catalyzes epoxide hydrolysis, but there is no known role for natural epoxides in CFTR regulation. Here, we show that Cif's hydrolase activity is strictly required for its effects on CFTR. We also uncover a small-molecule inhibitor that protects this key component of the mucociliary defense system. Our results provide a basis for targeting Cif's distinctive virulence chemistry and suggest an unanticipated role of physiological epoxides in intracellular protein trafficking. PMID:26136396

  15. 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

  16. Proteome-wide reactivity profiling identifies diverse carbamate chemotypes tuned for serine hydrolase inhibition.

    PubMed

    Chang, Jae Won; Cognetta, Armand B; Niphakis, Micah J; Cravatt, Benjamin F

    2013-07-19

    Serine hydrolases are one of the largest and most diverse enzyme classes in Nature. Inhibitors of serine hydrolases are used to treat many diseases, including obesity, diabetes, cognitive dementia, and bacterial and viral infections. Nonetheless, the majority of the 200+ serine hydrolases in mammals still lack selective inhibitors for their functional characterization. We and others have shown that activated carbamates, through covalent reaction with the conserved serine nucleophile of serine hydrolases, can serve as useful inhibitors for members of this enzyme family. The extent to which carbamates, however, cross-react with other protein classes remains mostly unexplored. Here, we address this problem by investigating the proteome-wide reactivity of a diverse set of activated carbamates in vitro and in vivo, using a combination of competitive and click chemistry (CC)-activity-based protein profiling (ABPP). We identify multiple classes of carbamates, including O-aryl, O-hexafluoroisopropyl (HFIP), and O-N-hydroxysuccinimidyl (NHS) carbamates that react selectively with serine hydrolases across entire mouse tissue proteomes in vivo. We exploit the proteome-wide specificity of HFIP carbamates to create in situ imaging probes for the endocannabinoid hydrolases monoacylglycerol lipase (MAGL) and α-β hydrolase-6 (ABHD6). These findings, taken together, designate the carbamate as a privileged reactive group for serine hydrolases that can accommodate diverse structural modifications to produce inhibitors that display exceptional potency and selectivity across the mammalian proteome.

  17. Proteome-wide reactivity profiling identifies diverse carbamate chemotypes tuned for serine hydrolase inhibition

    PubMed Central

    Chang, Jae Won; Cognetta, Armand B.; Niphakis, Micah J.; Cravatt, Benjamin F.

    2013-01-01

    Serine hydrolases are one of the largest and most diverse enzyme classes in Nature. Inhibitors of serine hydrolases are used to treat many diseases, including obesity, diabetes, cognitive dementia, and bacterial and viral infections. Nonetheless, the majority of the 200+ serine hydrolases in mammals still lack selective inhibitors for their functional characterization. We and others have shown that activated carbamates, through covalent reaction with the conserved serine nucleophile of serine hydrolases, can serve as useful inhibitors for members of this enzyme family. The extent to which carbamates, however, cross-react with other protein classes remains mostly unexplored. Here, we address this problem by investigating the proteome-wide reactivity of a diverse set of activated carbamates in vitro and in vivo using a combination of competitive and click chemistry (CC)-activity-based protein profiling (ABPP). We identify multiple classes of carbamates, including O-aryl, O-hexafluoroisopropyl (HFIP), and O-N-hydroxysuccinimidyl (NHS) carbamates that react selectively with serine hydrolases across entire mouse tissue proteomes in vivo. We exploit the proteome-wide specificity of HFIP carbamates to create in situ imaging probes for the endocannabinoid hydrolases monoacylglycerol lipase (MAGL) and alpha-beta hydrolase-6 (ABHD6). These findings, taken together, designate the carbamate as a privileged reactive group for serine hydrolases that can accommodate diverse structural modifications to produce inhibitors that display exceptional potency and selectivity across the mammalian proteome. PMID:23701408

  18. Parathion hydrolase specified by the Flavobacterium opd gene: relationship between the gene and protein.

    PubMed Central

    Mulbry, W W; Karns, J S

    1989-01-01

    The sequence of a 1,693-base-pair plasmid DNA fragment from Flavobacterium sp. strain ATCC 27551 containing the parathion hydrolase gene (opd) was determined. Within this sequence, there is only one open reading frame large enough to encode the 35,000-dalton membrane-associated hydrolase protein purified from Flavobacterium extracts. Amino-terminal sequence analysis of the purified Flavobacterium hydrolase demonstrated that serine is the amino-terminal residue of the hydrolase protein. The amino-terminal serine corresponds to a TCG codon located 87 base pairs downstream of the presumptive ATG initiation codon in the nucleotide sequence. The amino acid composition of the purified protein agrees well with that predicted from the nucleotide sequence, using serine as the amino-terminal residue. These data suggest that the parathion hydrolase protein is processed at its amino terminus in Flavobacterium sp. Construction in Escherichia coli of a lacZ-opd gene fusion in which the first 33 amino-terminal residues of opd were replaced by the first 5 residues of lacZ resulted in the production of an active hydrolase identical in molecular mass to the hydrolase isolated from Flavobacterium sp. E. coli cells containing the lacZ-opd fusion showed higher levels of hydrolase activity than did cells containing the parent plasmid. Images PMID:2556372

  19. Parathion hydrolase specified by the Flavobacterium opd gene: relationship between the gene and protein.

    PubMed

    Mulbry, W W; Karns, J S

    1989-12-01

    The sequence of a 1,693-base-pair plasmid DNA fragment from Flavobacterium sp. strain ATCC 27551 containing the parathion hydrolase gene (opd) was determined. Within this sequence, there is only one open reading frame large enough to encode the 35,000-dalton membrane-associated hydrolase protein purified from Flavobacterium extracts. Amino-terminal sequence analysis of the purified Flavobacterium hydrolase demonstrated that serine is the amino-terminal residue of the hydrolase protein. The amino-terminal serine corresponds to a TCG codon located 87 base pairs downstream of the presumptive ATG initiation codon in the nucleotide sequence. The amino acid composition of the purified protein agrees well with that predicted from the nucleotide sequence, using serine as the amino-terminal residue. These data suggest that the parathion hydrolase protein is processed at its amino terminus in Flavobacterium sp. Construction in Escherichia coli of a lacZ-opd gene fusion in which the first 33 amino-terminal residues of opd were replaced by the first 5 residues of lacZ resulted in the production of an active hydrolase identical in molecular mass to the hydrolase isolated from Flavobacterium sp. E. coli cells containing the lacZ-opd fusion showed higher levels of hydrolase activity than did cells containing the parent plasmid.

  20. Recognition and cleavage of corn defense chitinases by fungal polyglycine hydrolases

    USDA-ARS?s Scientific Manuscript database

    Polyglycine hydrolases are secreted fungal endoproteases that cleave peptide bonds in the polyglycine interdomain linker of ChitA chitinase, an antifungal protein from domesticated corn. Polyglycine hydrolases are novel proteins in terms of activity and sequence. The objective of the study is to und...

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

    USDA-ARS?s Scientific Manuscript database

    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...

  2. Hydration of vinyl ether groups by unsaturated glycoside hydrolases and their role in bacterial pathogenesis.

    PubMed

    Hashimoto, Wataru; Itoh, Takafumi; Maruyama, Yukie; Mikami, Bunzo; Murata, Kousaku

    2007-12-01

    Many pathogenic microorganisms invade mammalian and/or plant cells by producing polysaccharide-degrading enzymes (lyases and hydrolases). Mammalian glycosaminoglycans and plant pectins that form part of the cell surface matrix are typical targets for these microbial enzymes. Unsaturated glycoside hydrolase catalyzes the hydrolytic release of an unsaturated uronic acid from oligosaccharides, which are produced through the reaction of matrix-degrading polysaccharide lyase. This enzymatic ability suggests that unsaturated glycoside hydrolases function as virulence factors in microbial infection. This review focuses on the molecular identification, bacterial distribution, and structure/function relationships of these enzymes. In contrast to general glycoside hydrolases, in which the catalytic mechanism involves the retention or inversion of an anomeric configuration, unsaturated glycoside hydrolases uniquely trigger the hydrolysis of vinyl ether groups in unsaturated saccharides but not of their glycosidic bonds.

  3. 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

  4. A constitutive expression system for glycosyl hydrolase family 7 cellobiohydrolases in Hypocrea jecorina

    DOE PAGES

    Linger, Jeffrey G.; Taylor, II, Larry E.; Baker, John O.; ...

    2015-03-18

    One of the primary industrial-scale cellulase producers is the ascomycete fungus, Hypocrea jecorina, which produces and secretes large quantities of diverse cellulolytic enzymes. Perhaps the single most important biomass degrading enzyme is cellobiohydrolase I (cbh1or Cel7A) due to its enzymatic proficiency in cellulose depolymerization. However, production of Cel7A with native-like properties from heterologous expression systems has proven difficult. In this study, we develop a protein expression system in H. jecorina (Trichoderma reesei) useful for production and secretion of heterologous cellobiohydrolases from glycosyl hydrolase family 7. Building upon previous work in heterologous protein expression in filamentous fungi, we have integrated amore » native constitutive enolase promoter with the native cbh1 signal sequence. The results are the following: The constitutive eno promoter driving the expression of Cel7A allows growth on glucose and results in repression of the native cellulase system, severely reducing background endo- and other cellulase activity and greatly simplifying purification of the recombinant protein. Coupling this system to a Δcbh1 strain of H. jecorina ensures that only the recombinant Cel7A protein is produced. Two distinct transformant colony morphologies were observed and correlated with high and null protein production. Production levels in ‘fast’ transformants are roughly equivalent to those in the native QM6a strain of H. jecorina, typically in the range of 10 to 30 mg/L when grown in continuous stirred-tank fermenters. ‘Slow’ transformants showed no evidence of Cel7A production. Specific activity of the purified recombinant Cel7A protein is equivalent to that of native protein when assayed on pretreated corn stover, as is the thermal stability and glycosylation level. Purified Cel7A produced from growth on glucose demonstrated remarkably consistent specific activity. Purified Cel7A from the same strain grown on lactose

  5. 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

  6. 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

  7. 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.

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

    SciTech Connect

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

    2015-03-09

    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. 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. Furthermore, application of this experimental method provides a new, systematic way to annotate glycoside hydrolase phylogenetic space for functional properties.

  9. Structural model for family 32 of glycosyl-hydrolase enzymes.

    PubMed

    Pons, T; Olmea, O; Chinea, G; Beldarraín, A; Márquez, G; Acosta, N; Rodríguez, L; Valencia, A

    1998-11-15

    A structural model is presented for family 32 of the glycosyl-hydrolase enzymes based on the beta-propeller fold. The model is derived from the common prediction of two different threading methods, TOPITS and THREADER. In addition, we used a correlated mutation analysis and prediction of active-site residues to corroborate the proposed model. Physical techniques (circular dichroism and differential scanning calorimetry) confirmed two aspects of the prediction, the proposed all-beta fold and the multi-domain structure. The most reliable three-dimensional model was obtained using the structure of neuraminidase (1nscA) as template. The analysis of the position of the active site residues in this model is compatible with the catalytic mechanism proposed by Reddy and Maley (J. Biol. Chem. 271:13953-13958, 1996), which includes three conserved residues, Asp, Glu, and Cys. Based on this analysis, we propose the participation of one more conserved residue (Asp 162) in the catalytic mechanism. The model will facilitate further studies of the physical and biochemical characteristics of family 32 of the glycosyl-hydrolases.

  10. Peripheral FAAH and soluble epoxide hydrolase inhibitors are synergistically antinociceptive.

    PubMed

    Sasso, Oscar; Wagner, Karen; Morisseau, Christophe; Inceoglu, Bora; Hammock, Bruce D; Piomelli, Daniele

    2015-07-01

    We need better medicines to control acute and chronic pain. Fatty acid amide hydrolase (FAAH) and soluble epoxide hydrolase (sEH) catalyze the deactivating hydrolysis of two classes of bioactive lipid mediators--fatty acid ethanolamides (FAEs) and epoxidized fatty acids (EpFAs), respectively--which are biogenetically distinct but share the ability to attenuate pain responses and inflammation. In these experiments, we evaluated the antihyperalgesic activity of small-molecule inhibitors of FAAH and sEH, administered alone or in combination, in two pain models: carrageenan-induced hyperalgesia in mice and streptozocin-induced allodynia in rats. When administered separately, the sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidine-4-yl)urea (TPPU) and the peripherally restricted FAAH inhibitor URB937 were highly active in the two models. The combination TPPU plus URB937 was markedly synergistic, as assessed using isobolographic analyses. The results of these experiments reveal the existence of a possible functional crosstalk between FAEs and EpFAs in regulating pain responses. Additionally, the results suggest that combinations of sEH and FAAH inhibitors might be exploited therapeutically to achieve greater analgesic efficacy.

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

    PubMed

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

    2015-04-03

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

  12. Acetylcarnitine hydrolase activity in bovine caudal epididymal spermatozoa

    SciTech Connect

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

    1986-05-01

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

  13. Recovering glycoside hydrolase genes from active tundra cellulolytic bacteria.

    PubMed

    Pinnell, Lee J; Dunford, Eric; Ronan, Patrick; Hausner, Martina; Neufeld, Josh D

    2014-07-01

    Bacteria responsible for cellulose hydrolysis in situ are poorly understood, largely because of the relatively recent development of cultivation-independent methods for their detection and characterization. This study combined DNA stable-isotope probing (DNA-SIP) and metagenomics for identifying active bacterial communities that assimilated carbon from glucose and cellulose in Arctic tundra microcosms. Following DNA-SIP, bacterial fingerprint analysis of gradient fractions confirmed isotopic enrichment. Sequenced fingerprint bands and clone library analysis of 16S rRNA genes identified active bacterial taxa associated with cellulose-associated labelled DNA, including Bacteroidetes (Sphingobacteriales), Betaproteobacteria (Burkholderiales), Alphaproteobacteria (Caulobacteraceae), and Chloroflexi (Anaerolineaceae). We also compared glycoside hydrolase metagenomic profiles from bulk soil and heavy DNA recovered from DNA-SIP incubations. Active populations consuming [(13)C]glucose and [(13)C]cellulose were distinct, based on ordinations of light and heavy DNA. Metagenomic analysis demonstrated a ∼3-fold increase in the relative abundance of glycoside hydrolases in DNA-SIP libraries over bulk-soil libraries. The data also indicate that multiple displacement amplification introduced bias into the resulting metagenomic analysis. This research identified DNA-SIP incubation conditions for glucose and cellulose that were suitable for Arctic tundra soil and confirmed that DNA-SIP enrichment can increase target gene frequencies in metagenomic libraries.

  14. Versatile signal peptide of Flavobacterium-originated organophosphorus hydrolase for efficient periplasmic translocation of heterologous proteins in Escherichia coli.

    PubMed

    Kang, Dong Gyun; Seo, Jeong Hyun; Jo, Byung Hoon; Kim, Chang Sup; Choi, Suk Soon; Cha, Hyung Joon

    2016-07-08

    Organophosphorus hydrolase (OPH) from Flavobacterium species is a membrane-associated homodimeric metalloenzyme and has its own signal peptide in its N-terminus. We found that OPH was translocated into the periplasmic space when the original signal peptide-containing OPH was expressed in recombinant Escherichia coli even though its translocation efficiency was relatively low. To investigate the usability of this OPH signal peptide for periplasmic expression of heterologous proteins in an E. coli system, we employed green fluorescent protein (GFP) as a cytoplasmic folding reporter and alkaline phosphatase (ALP) as a periplasmic folding reporter. We found that the OPH signal peptide was able to use both twin-arginine translocation (Tat) and general secretory (Sec) machineries by switching translocation pathways according to the nature of target proteins in E. coli. These results might be due to the lack of Sec-avoidance sequence in the c-region and a moderate hydrophobicity of the OPH signal peptide. Interestingly, the OPH signal peptide considerably enhanced the translocation efficiencies for both GFP and ALP compared with commonly used TorA and PelB signal peptides that have Tat and Sec pathway dependences, respectively. Therefore, this OPH signal peptide could be successfully used in recombinant E. coli system for efficient periplasmic production of target protein regardless of the subcellular localization where functional folding of the protein occurs. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:848-854, 2016. © 2016 American Institute of Chemical Engineers.

  15. Therapeutic Recombinant Monoclonal Antibodies

    ERIC Educational Resources Information Center

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  16. Therapeutic Recombinant Monoclonal Antibodies

    ERIC Educational Resources Information Center

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  17. Relationship between glycosyl hydrolase inventory and growth physiology of the hyperthermophile Pyrococcus furiosus on carbohydrate-based media.

    PubMed

    Driskill, L E; Kusy, K; Bauer, M W; Kelly, R M

    1999-03-01

    Utilization of a range of carbohydrates for growth by the hyperthermophile Pyrococcus furiosus was investigated by examining the spectrum of glycosyl hydrolases produced by this microorganism and the thermal labilities of various saccharides. Previously, P. furiosus had been found to grow in batch cultures on several alpha-linked carbohydrates and cellobiose but not on glucose or other beta-linked sugars. Although P. furiosus was not able to grow on any nonglucan carbohydrate or any form of cellulose in this study (growth on oat spelt arabinoxylan was attributed to glucan contamination of this substrate), significant growth at 98 degrees C occurred on beta-1,3- and beta-1,3-beta-1,4-linked glucans. Oligosaccharides generated by digestion with a recombinant laminarinase derived from P. furiosus were the compounds that were most effective in stimulating growth of the microorganism. In several cases, periodic addition of beta-glucan substrates to fed-batch cultures limited adverse thermochemical modifications of the carbohydrates (i.e., Maillard reactions and caramelization) and led to significant increases (as much as two- to threefold) in the cell yields. While glucose had only a marginally positive effect on growth in batch culture, the final cell densities nearly tripled when glucose was added by the fed-batch procedure. Nonenzymatic browning reactions were found to be significant at 98 degrees C for saccharides with degrees of polymerization (DP) ranging from 1 to 6; glucose was the most labile compound on a mass basis and the least labile compound on a molar basis. This suggests that for DP of 2 or greater protection of the nonreducing monosaccharide component may be a factor in substrate availability. For P. furiosus, carbohydrate utilization patterns were found to reflect the distribution of the glycosyl hydrolases which are known to be produced by this microorganism.

  18. A molecular model for the active site of S-adenosyl- l-homocysteine hydrolase

    NASA Astrophysics Data System (ADS)

    Yeh, Jerry C.; Borchardt, Ronald T.; Vedani, Angelo

    1991-06-01

    S-adenosyl- l-homocysteine hydrolase (AdoHcy hydrolase, EC 3.3.1.1.), a specific target for antiviral drug design, catalyzes the hydrolysis of AdoHcy to adenosine (Ado) and homocysteine (Hcy) as well as the synthesis of AdoHcy from Ado and Hcy. The enzyme isolated from different sources has been shown to contain tightly bound NAD+. Based on the 2.0 Å-resolution X-ray crystal structure of dogfish lactate dehydrogenase (LDH), which is functionally homologous to AdoHcy hydrolase, and the primary sequence of rat liver AdoHcy hydrolase, we have derived a molecular model of an extended active site for AdoHcy hydrolase. The computational mutation was performed using the software MUTAR (Yeh et al., University of Kansas, Lawrence), followed by molecular mechanics optimizations using the programs AMBER (Singh et al., University of California, San Francisco) and YETI (Vedani, University of Kansas). Solvation of the model structure was achieved by use of the program SOLVGEN (Jacober, University of Kansas); 56 water molecules were explicitly included in all refinements. Some of these may be involved in the catalytic reaction. We also studied a model of the complex of AdoHcy hydrolase with NAD+, as well as the ternary complexes of the redox reaction catalyzed by AdoHcy hydrolase and has been used to differentiate the relative binding strength of inhibitors.

  19. Identification and characterization of a new epoxide hydrolase from mouse liver microsomes.

    PubMed

    Guenthner, T M; Oesch, F

    1983-12-25

    A new microsomal epoxide hydrolase (mEH2) has been identified and characterized. This enzyme has properties which distinguish it from previously described cytosolic (cEH) or membrane-bound (mEH1) epoxide hydrolases. The enzyme is an integral microsomal protein which is not dissociated from the membrane by repeated washing, high ionic strength salt, or chaotropic agent solutions, or by sonication. It is very different from the normally described microsomal epoxide hydrolase (mEH1) as shown by its different substrate specificity and kinetic properties and by immunological criteria. In contrast to the hitherto described microsomal epoxide hydrolase, mEH1, the new enzyme effectively catalyzes the hydration of transdisubstituted oxiranes such as trans-stilbene oxide and trans-beta-ethyl styrene oxide and has no appreciable activity toward benzo(a)pyrene 4,5-oxide. It is also structurally distinct, in that it does not cross-react with antibodies raised against the normally described microsomal epoxide hydrolase mEH1. This newly described microsomal epoxide hydrolase probably represents an important factor in the control of reactive epoxides; its location in the membrane ensures access to lipophilic epoxides generated by membrane-bound monooxygenases, and its substrate specificity is such that it can hydrolyze epoxides poorly metabolized by the previously described microsomal epoxide hydrolase.

  20. Characterization of multiple epoxide hydrolase activities in mouse liver nuclear envelope.

    PubMed

    Guenthner, T M

    1986-10-01

    A nuclear envelope-associated epoxide hydrolase in mouse liver that hydrates trans-stilbene oxide has been identified and characterized. This epoxide hydrolase is distinct from the enzyme in nuclear envelopes that hydrates benzo[a]pyrene 4,5-oxide and other arene oxides. This distinction was demonstrated by the criteria of pH optima, response to specific inhibitors in vitro, and precipitation by specific antibodies. The new epoxide hydrolase had a pH optimum of 6.8, was poorly inhibited by trichloropropene oxide, was potently inhibited by 4-phenylchalcone oxide, and did not bind to antiserum against benzo[a]pyrene 4,5-oxide hydrolase. This nuclear enzyme is similar in many of its properties to cytosolic and microsomal trans-stilbene oxide hydrolases and may be nuclear envelope-bound form of these other epoxide hydrolases. It differed from these other trans-stilbene oxide hydrolases in that its affinities for both trans-stilbene oxide (measured as apparent Km) and 4-phenylchalcone oxide (measured as I50) were 4- to 20-fold lower than those of either the cytosolic or microsomal forms.

  1. Expanding the Cyanuric Acid Hydrolase Protein Family to the Fungal Kingdom

    PubMed Central

    Dodge, Anthony G.; Preiner, Chelsea S.

    2013-01-01

    The known enzymes that open the s-triazine ring, the cyanuric acid hydrolases, have been confined almost exclusively to the kingdom Bacteria and are all homologous members of the rare cyanuric acid hydrolase/barbiturase protein family. In the present study, a filamentous fungus, Sarocladium sp. strain CA, was isolated from soil by enrichment culturing using cyanuric acid as the sole source of nitrogen. A reverse-genetic approach identified a fungal cyanuric acid hydrolase gene composed of two exons and one intron. The translated spliced sequence was 39 to 53% identical to previously characterized bacterial cyanuric acid hydrolases. The sequence was used to generate a gene optimized for expression in Escherichia coli and encoding an N-terminally histidine-tagged protein. The protein was purified by nickel affinity and anion-exchange chromatography. The purified protein was shown by 13C nuclear magnetic resonance (13C-NMR) to produce carboxybiuret as the product, which spontaneously decarboxylated to yield biuret and carbon dioxide. The protein was very narrow in substrate specificity, showing activity only with cyanuric acid and N-methyl cyanuric acid. Barbituric acid was an inhibitor of enzyme activity. Sequence analysis identified genes with introns in other fungi from the Ascomycota that, if spliced, are predicted to encode proteins with cyanuric acid hydrolase activity. The Ascomycota cyanuric acid hydrolase homologs are most closely related to cyanuric acid hydrolases from Actinobacteria. PMID:24039269

  2. 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

  3. Multidomain, Surface Layer-associated Glycoside Hydrolases Contribute to Plant Polysaccharide Degradation by Caldicellulosiruptor Species.

    PubMed

    Conway, Jonathan M; Pierce, William S; Le, Jaycee H; Harper, George W; Wright, John H; Tucker, Allyson L; Zurawski, Jeffrey V; Lee, Laura L; Blumer-Schuette, Sara E; Kelly, Robert M

    2016-03-25

    The genome of the extremely thermophilic bacterium Caldicellulosiruptor kronotskyensisencodes 19 surface layer (S-layer) homology (SLH) domain-containing proteins, the most in any Caldicellulosiruptorspecies genome sequenced to date. These SLH proteins include five glycoside hydrolases (GHs) and one polysaccharide lyase, the genes for which were transcribed at high levels during growth on plant biomass. The largest GH identified so far in this genus, Calkro_0111 (2,435 amino acids), is completely unique toC. kronotskyensisand contains SLH domains. Calkro_0111 was produced recombinantly inEscherichia colias two pieces, containing the GH16 and GH55 domains, respectively, as well as putative binding and spacer domains. These displayed endo- and exoglucanase activity on the β-1,3-1,6-glucan laminarin. A series of additional truncation mutants of Calkro_0111 revealed the essential architectural features required for catalytic function. Calkro_0402, another of the SLH domain GHs inC. kronotskyensis, when produced inE. coli, was active on a variety of xylans and β-glucans. Unlike Calkro_0111, Calkro_0402 is highly conserved in the genus Caldicellulosiruptorand among other biomass-degrading Firmicutes but missing from Caldicellulosiruptor bescii As such, the gene encoding Calkro_0402 was inserted into the C. besciigenome, creating a mutant strain with its S-layer extensively decorated with Calkro_0402. This strain consequently degraded xylans more extensively than wild-typeC. bescii The results here provide new insights into the architecture and role of SLH domain GHs and demonstrate that hemicellulose degradation can be enhanced through non-native SLH domain GHs engineered into the genomes of Caldicellulosiruptorspecies. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. β-Glucuronidase from Lactobacillus brevis useful for baicalin hydrolysis belongs to glycoside hydrolase family 30.

    PubMed

    Sakurama, Haruko; Kishino, Shigenobu; Uchibori, Yoshie; Yonejima, Yasunori; Ashida, Hisashi; Kita, Keiko; Takahashi, Satomi; Ogawa, Jun

    2014-05-01

    Baicalin (baicalein 7-O-β-D-glucuronide) is one of the major flavonoid glucuronides found in traditional herbal medicines. Because its aglycone, baicalein, is absorbed more quickly and shows more effective properties than baicalin, the conversion of baicalin into baicalein by β-glucuronidase (GUS) has drawn the attention of researchers. Recently, we have found that Lactobacillus brevis subsp. coagulans can convert baicalin to baicalein. Therefore, we aimed to identify and characterize the converting enzyme from L. brevis subsp. coagulans. First, we purified this enzyme from the cell-free extracts of L. brevis subsp. coagulans and cloned its gene. Surprisingly, this enzyme was found to be a GUS belonging to glycoside hydrolase (GH) family 30 (designated as LcGUS30), and its amino acid sequence has little similarity with any GUS belonging to GH families 1, 2, and 79 that have been reported so far. We then established a high-level expression and simple purification system of the recombinant LcGUS30 in Escherichia coli. The detailed analysis of the substrate specificity revealed that LcGUS30 has strict specificity toward glycon but not toward aglycones. Interestingly, LcGUS30 prefers baicalin rather than estrone 3-(β-D-glucuronide), one of the human endogenous steroid hormones. These results indicated that L. brevis subsp. coagulans and LcGUS30 should serve as powerful tools for the construction of a safe bioconversion system for baicalin. In addition, we propose that this novel type of GUS forms a new group in subfamily 3 of GH family 30.

  5. Clostridium beijerinckii cells expressing Neocallimastix patriciarum glycoside hydrolases show enhanced lichenan utilization and solvent production.

    PubMed

    López-Contreras, A M; Smidt, H; van der Oost, J; Claassen, P A; Mooibroek, H; de Vos, W M

    2001-11-01

    Growth and the production of acetone, butanol, and ethanol by Clostridium beijerinckii NCIMB 8052 on several polysaccharides and sugars were analyzed. On crystalline cellulose, growth and solvent production were observed only when a mixture of fungal cellulases was added to the medium. On lichenan growth and solvent production occurred, but this polymer was only partially utilized. To increase utilization of these polymers and subsequent solvent production, the genes for two new glycoside hydrolases, celA and celD from the fungus Neocallimastix patriciarum, were cloned separately into C. beijerinckii. To do this, a secretion vector based on the pMTL500E shuttle vector and containing the promoter and signal sequence coding region of the Clostridium saccharobutylicum NCP262 eglA gene was constructed and fused either to the celA gene or the celD gene. Stable C. beijerinckii transformants were obtained with the resulting plasmids, pWUR3 (celA) and pWUR4 (celD). The recombinant strains showed clear halos on agar plates containing carboxymethyl cellulose upon staining with Congo red. In addition, their culture supernatants had significant endoglucanase activities (123 U/mg of protein for transformants harboring celA and 78 U/mg of protein for transformants harboring celD). Although C. beijerinckii harboring either celA or celD was not able to grow, separately or in mixed culture, on carboxymethyl cellulose or microcrystalline cellulose, both transformants showed a significant increase in solvent production during growth on lichenan and more extensive degradation of this polymer than that exhibited by the wild-type strain.

  6. The Cytoplasmic and Periplasmic Expression Levels and Folding of Organophosphorus Hydrolase Enzyme in Escherichia coli

    PubMed Central

    Latifi, Ali Mohammad; Khajeh, Khosro; Farnoosh, Gholamreza; Hassanpour, Kazem; Khodi, Samaneh

    2015-01-01

    Background: Organophosphorus hydrolase (OPH) is a type of organophosphate-degrading enzyme which is widely used in the bioremediation process. Objectives: In this study, the periplasmic and cytoplasmic productions and the activity of recombinant OPH in Escherichia coli were investigated and compared using two pET systems (pET21a and pET26b). Materials and Methods: The sequence encoding the opd gene was synthesized and expressed in the form of inclusion body using pET21a-opd and in the periplasmic space in pET26b-opd. Results: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed a band of about 37 kDa with a maximum expression level at 30°C from pET21a-opd.However, the obtained results of the periplasmic space extraction of OPH (pET26b-opd) showed a very weak band, while the cytoplasmic expression of OPH (pET21a-opd) produced a strong protein band. Conclusions: The activities studied by the production of PNP were determined by following the increase at 410 nm. The maximum PNP was produced at 30°C with an optical density of 10.62 in the presence of cytoplasmic expression of OPH (pET21a-opd). Consequently, our results suggest cytoplasmic expression system as an appropriate candidate with a high amount of OPH in spite of inclusion body formation, which needs an additional refolding step. PMID:26870308

  7. Photoionization and Recombination

    NASA Technical Reports Server (NTRS)

    Nahar, Sultana N.

    2000-01-01

    Theoretically self-consistent calculations for photoionization and (e + ion) recombination are described. The same eigenfunction expansion for the ion is employed in coupled channel calculations for both processes, thus ensuring consistency between cross sections and rates. The theoretical treatment of (e + ion) recombination subsumes both the non-resonant recombination ("radiative recombination"), and the resonant recombination ("di-electronic recombination") processes in a unified scheme. In addition to the total, unified recombination rates, level-specific recombination rates and photoionization cross sections are obtained for a large number of atomic levels. Both relativistic Breit-Pauli, and non-relativistic LS coupling, calculations are carried out in the close coupling approximation using the R-matrix method. Although the calculations are computationally intensive, they yield nearly all photoionization and recombination parameters needed for astrophysical photoionization models with higher precision than hitherto possible, estimated at about 10-20% from comparison with experimentally available data (including experimentally derived DR rates). Results are electronically available for over 40 atoms and ions. Photoionization and recombination of He-, and Li-like C and Fe are described for X-ray modeling. The unified method yields total and complete (e+ion) recombination rate coefficients, that can not otherwise be obtained theoretically or experimentally.

  8. Retinyl ester hydrolases and their roles in vitamin A homeostasis.

    PubMed

    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.

  9. Ubiquitin carboxyl hydrolase L1 significance for human diseases.

    PubMed

    Suong, Dang Ngoc Anh; Thao, Dang Thi Phuong; Masamitsu, Yamaguchi; Thuoc, Tran Linh

    2014-07-01

    Ubiquitin carboxyl hydrolase L1 (UCH-L1) is an abundant multifunctional neuron protein. It plays an important role in maintaining the ubiquitin proteasome system (UPS), vital for recognizing and degrading dysfunctional proteins in organisms. In recent decades, UCH-L1 has been implicated in the pathogenesis of many diseases, including neurodegenerative disorders, cancer and diabetes. However, the mechanisms of UCH-L1 involvement have yet to be revealed in detail. Since UCH-L1 contributes many different functions to cell metabolism, its role and regulation might be more complex than previously thought and it has become a research target in many laboratories. In this review, we summarize recent findings related to the actions of UCH-L1 in several human diseases.

  10. 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

  11. Thermodynamics of Enzyme-Catalyzed Reactions. Part 3. Hydrolases

    NASA Astrophysics Data System (ADS)

    Goldberg, Robert N.; Tewari, Yadu B.

    1994-11-01

    Equilibrium constants and enthalpy changes for reactions catalyzed by the hydrolase class of enzymes have been compiled. For each reaction the following information is given: The reference for the data; the reaction studied; the name of the enzyme used and its Enzyme Commission number; the method of measurement; the conditions of measurement [temperature, pH, ionic strength, and the buffer(s) and cofactor(s) used]; the data and an evaluation of it; and, sometimes, commentary on the data and on any corrections which have been applied to it or any calculations for which the data have been used. The data from 145 references have been examined and evaluated. Chemical Abstract Service registry numbers are given for the substances involved in these various reactions. There is a cross reference between the substances and the Enzyme Commission numbers of the enzymes used to catalyze the reactions in which the substances participate.

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

    SciTech Connect

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

    1986-03-01

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

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

    DOE PAGES

    Bianchetti, Christopher M.; Takasuka, Taichi E.; Deutsch, Sam; ...

    2015-03-09

    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. 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 themore » 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. Furthermore, application of this experimental method provides a new, systematic way to annotate glycoside hydrolase phylogenetic space for functional properties.« less

  14. Analysis of Xyloglucan Endotransglycosylase/Hydrolase (XTH) Genes and Diverse Roles of Isoenzymes during Persimmon Fruit Development and Postharvest Softening

    PubMed Central

    Han, Ye; Zhu, Qinggang; Zhang, Zhengke; Meng, Kun; Hou, Yali; Ban, Qiuyan; Suo, Jiangtao; Rao, Jingping

    2015-01-01

    Xyloglucan endotransglycosylase/hydrolase (XTH) enzymes have played a role in the remodeling of cell wall hemicelluloses. To investigate the function of XTHs in persimmon (Diospyros kaki L.) fruit development and postharvest softening, five cDNAs (DkXTH1 to DkXTH5), whose putative proteins contained the conserved DEIDFEFLG motif of XTH, were cloned. Real time quantitative PCR analysis revealed that DkXTH1, DkXTH4, and DkXTH5 peaked in immature expanding fruit, and their higher expression was observed along with higher fruit firmness in cold-treated fruit or firmer cultivar fruit during storage. The opposite gene expression patterns were observed in DkXTH2 and DkXTH3, which reached maxima concomitance with pronounced fruit softening. Meanwhile, the xyloglucan endotransglycosylase (XET) enzymes play important roles in both the rapid growth and ripening of persimmon fruit. Furthermore, the recombined DkXTH1 and DkXTH2 proteins showed significant XET activity without any detected XEH activity. However, the XET activity of recombined DkXTH2 protein had a higher affinity for small acceptor molecules than that of recombined DkXTH1 protein. The former might prefer to participate in cell wall restructuring, and the latter is more inclined to participate in cell wall assembly. Besides, DKXTH proteins could function by targeting to the cell wall under regulation of a signal peptide. The data suggested that individual DKXTHs could exhibit different patterns of expression, and the encoded products possessed specific enzymatic properties conferring on their respective functions in growth and postharvest softening of persimmon fruit. PMID:25849978

  15. Analysis of xyloglucan endotransglycosylase/hydrolase (XTH) genes and diverse roles of isoenzymes during persimmon fruit development and postharvest softening.

    PubMed

    Han, Ye; Zhu, Qinggang; Zhang, Zhengke; Meng, Kun; Hou, Yali; Ban, Qiuyan; Suo, Jiangtao; Rao, Jingping

    2015-01-01

    Xyloglucan endotransglycosylase/hydrolase (XTH) enzymes have played a role in the remodeling of cell wall hemicelluloses. To investigate the function of XTHs in persimmon (Diospyros kaki L.) fruit development and postharvest softening, five cDNAs (DkXTH1 to DkXTH5), whose putative proteins contained the conserved DEIDFEFLG motif of XTH, were cloned. Real time quantitative PCR analysis revealed that DkXTH1, DkXTH4, and DkXTH5 peaked in immature expanding fruit, and their higher expression was observed along with higher fruit firmness in cold-treated fruit or firmer cultivar fruit during storage. The opposite gene expression patterns were observed in DkXTH2 and DkXTH3, which reached maxima concomitance with pronounced fruit softening. Meanwhile, the xyloglucan endotransglycosylase (XET) enzymes play important roles in both the rapid growth and ripening of persimmon fruit. Furthermore, the recombined DkXTH1 and DkXTH2 proteins showed significant XET activity without any detected XEH activity. However, the XET activity of recombined DkXTH2 protein had a higher affinity for small acceptor molecules than that of recombined DkXTH1 protein. The former might prefer to participate in cell wall restructuring, and the latter is more inclined to participate in cell wall assembly. Besides, DKXTH proteins could function by targeting to the cell wall under regulation of a signal peptide. The data suggested that individual DKXTHs could exhibit different patterns of expression, and the encoded products possessed specific enzymatic properties conferring on their respective functions in growth and postharvest softening of persimmon fruit.

  16. The Activation of Phytophthora Effector Avr3b by Plant Cyclophilin is Required for the Nudix Hydrolase Activity of Avr3b.

    PubMed

    Kong, Guanghui; Zhao, Yao; Jing, Maofeng; Huang, Jie; Yang, Jin; Xia, Yeqiang; Kong, Liang; Ye, Wenwu; Xiong, Qin; Qiao, Yongli; Dong, Suomeng; Ma, Wenbo; Wang, Yuanchao

    2015-08-01

    Plant pathogens secrete an arsenal of effector proteins to impair host immunity. Some effectors possess enzymatic activities that can modify their host targets. Previously, we demonstrated that a Phytophthora sojae RXLR effector Avr3b acts as a Nudix hydrolase when expressed in planta; and this enzymatic activity is required for full virulence of P. sojae strain P6497 in soybean (Glycine max). Interestingly, recombinant Avr3b produced by E. coli does not have the hydrolase activity unless it was incubated with plant protein extracts. Here, we report the activation of Avr3b by a prolyl-peptidyl isomerase (PPIase), cyclophilin, in plant cells. Avr3b directly interacts with soybean cyclophilin GmCYP1, which activates the hydrolase activity of Avr3b in a PPIase activity-dependent manner. Avr3b contains a putative Glycine-Proline (GP) motif; which is known to confer cyclophilin-binding in other protein substrates. Substitution of the Proline (P132) in the putative GP motif impaired the interaction of Avr3b with GmCYP1; as a result, the mutant Avr3bP132A can no longer be activated by GmCYP1, and is also unable to promote Phytophthora infection. Avr3b elicits hypersensitive response (HR) in soybean cultivars producing the resistance protein Rps3b, but Avr3bP132A lost its ability to trigger HR. Furthermore, silencing of GmCYP1 rendered reduced cell death triggered by Avr3b, suggesting that GmCYP1-mediated Avr3b maturation is also required for Rps3b recognition. Finally, cyclophilins of Nicotiana benthamiana can also interact with Avr3b and activate its enzymatic activity. Overall, our results demonstrate that cyclophilin is a "helper" that activates the enzymatic activity of Avr3b after it is delivered into plant cells; as such, cyclophilin is required for the avirulence and virulence functions of Avr3b.

  17. 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...

  18. A novel member of glycoside hydrolase family 30 subfamily 8 with altered substrate specificity

    Treesearch

    Franz J. St John; Diane Dietrich; Casey Crooks; Edwin Pozharski; Javier M. González; Elizabeth Bales; Kennon Smith; Jason C. Hurlbert

    2014-01-01

    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

  19. 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...

  20. An integrated molecular docking and rescoring method for predicting the sensitivity spectrum of various serine hydrolases to organophosphorus pesticides.

    PubMed

    Yang, Ling-Ling; Yang, Xiao; Li, Guo-Bo; Fan, Kai-Ge; Yin, Peng-Fei; Chen, Xiang-Gui

    2016-04-01

    The enzymatic chemistry method is currently the most widely used method for the rapid detection of organophosphorus (OP) pesticides, but the enzymes used, such as cholinesterases, lack sufficient sensitivity to detect low concentrations of OP pesticides present in given samples. Serine hydrolase is considered an ideal enzyme source in seeking high-sensitivity enzymes used for OP pesticide detection. However, it is difficult to systematically evaluate sensitivities of various serine hydrolases to OP pesticides by in vitro experiments. This study aimed to establish an in silico method to predict the sensitivity spectrum of various serine hydrolases to OP pesticides. A serine hydrolase database containing 219 representative serine hydrolases was constructed. Based on this database, an integrated molecular docking and rescoring method was established, in which the AutoDock Vina program was used to produce the binding poses of OP pesticides to various serine hydrolases and the ID-Score method developed recently by us was adopted as a rescoring method to predict their binding affinities. In retrospective case studies, this method showed good performance in predicting the sensitivities of known serine hydrolases to two OP pesticides: paraoxon and diisopropyl fluorophosphate. The sensitivity spectrum of the 219 collected serine hydrolases to 37 commonly used OP pesticides was finally obtained using this method. Overall, this study presented a promising in silico tool to predict the sensitivity spectrum of various serine hydrolases to OP pesticides, which will help in finding high-sensitivity serine hydrolases for OP pesticide detection. © 2015 Society of Chemical Industry.

  1. Repurposing Suzuki Coupling Reagents as a Directed Fragment Library Targeting Serine Hydrolases and Related Enzymes

    PubMed Central

    2017-01-01

    Serine hydrolases are susceptible to potent reversible inhibition by boronic acids. Large collections of chemically diverse boronic acid fragments are commercially available because of their utility in coupling chemistry. We repurposed the approximately 650 boronic acid reagents in our collection as a directed fragment library targeting serine hydrolases and related enzymes. Highly efficient hits (LE > 0.6) often result. The utility of the approach is illustrated with the results against autotaxin, a phospholipase implicated in cardiovascular disease. PMID:28564542

  2. 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

  3. 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.

  4. Binding of Pro-Gly-Pro at the active site of leukotriene A4 hydrolase/aminopeptidase and development of an epoxide hydrolase selective inhibitor

    PubMed Central

    Stsiapanava, Alena; Olsson, Ulrika; Wan, Min; Kleinschmidt, Thea; Rutishauser, Dorothea; Zubarev, Roman A.; Samuelsson, Bengt; Rinaldo-Matthis, Agnes; Haeggström, Jesper Z.

    2014-01-01

    Leukotriene (LT) A4 hydrolase/aminopeptidase (LTA4H) is a bifunctional zinc metalloenzyme that catalyzes the committed step in the formation of the proinflammatory mediator LTB4. Recently, the chemotactic tripeptide Pro-Gly-Pro was identified as an endogenous aminopeptidase substrate for LTA4 hydrolase. Here, we determined the crystal structure of LTA4 hydrolase in complex with a Pro-Gly-Pro analog at 1.72 Å. From the structure, which includes the catalytic water, and mass spectrometric analysis of enzymatic hydrolysis products of Pro-Gly-Pro, it could be inferred that LTA4 hydrolase cleaves at the N terminus of the palindromic tripeptide. Furthermore, we designed a small molecule, 4-(4-benzylphenyl)thiazol-2-amine, denoted ARM1, that inhibits LTB4 synthesis in human neutrophils (IC50 of ∼0.5 μM) and conversion of LTA4 into LTB4 by purified LTA4H with a Ki of 2.3 μM. In contrast, 50- to 100-fold higher concentrations of ARM1 did not significantly affect hydrolysis of Pro-Gly-Pro. A 1.62-Å crystal structure of LTA4 hydrolase in a dual complex with ARM1 and the Pro-Gly-Pro analog revealed that ARM1 binds in the hydrophobic pocket that accommodates the ω-end of LTA4, distant from the aminopeptidase active site, thus providing a molecular basis for its inhibitory profile. Hence, ARM1 selectively blocks conversion of LTA4 into LTB4, although sparing the enzyme’s anti-inflammatory aminopeptidase activity (i.e., degradation and inactivation of Pro-Gly-Pro). ARM1 represents a new class of LTA4 hydrolase inhibitor that holds promise for improved anti-inflammatory properties. PMID:24591641

  5. Cloning, expression, and characterization of a glycoside hydrolase family 86 beta-agarase from a deep-sea Microbulbifer-like isolate.

    PubMed

    Ohta, Yukari; Hatada, Yuji; Nogi, Yuichi; Li, Zhijun; Ito, Susumu; Horikoshi, Koki

    2004-12-01

    The gene for a novel beta-agarase from a deep-sea Microbulbifer-like isolate was cloned and sequenced. It encoded a mature protein of 126,921 Da (1146 amino acids), which was a modular protein including two tandem carbohydrate-binding module (CBM)-like sequences and a catalytic module. The catalytic module resembled a glycoside hydrolase family 86 beta-agarase, AgrA, from Pseudoalteromonas atlantica T6c with 31% amino acid identity. Its recombinant agarase was hyper-produced extracellularly using Bacillus subtilis as the host and purified to homogeneity. The activity and stability were strongly enhanced by CaCl2. The maximal enzyme activity was observed at 45 degrees C and pH 7.5 in the presence of 10 mM CaCl2. The enzyme was an endo-type beta-agarase and degraded agarose and agarose oligosaccharides more polymerized than hexamers to yield neoagarohexaose as the main product. This is the first glycoside hydrolase family 86 enzyme to be homogeneously purified and characterized.

  6. Mycobacterium hassiacum recovers from nitrogen starvation with up-regulation of a novel glucosylglycerate hydrolase and depletion of the accumulated glucosylglycerate

    PubMed Central

    Alarico, Susana; Costa, Mafalda; Sousa, Marta S.; Maranha, Ana; Lourenço, Eva C.; Faria, Tiago Q.; Ventura, M. Rita; Empadinhas, Nuno

    2014-01-01

    Some microorganisms accumulate glucosylglycerate (GG) during growth under nitrogen deprivation. However, the molecular mechanisms underlying the role of GG and the regulation of its levels in the nitrogen stress response are elusive. Since GG is required for biosynthesis of mycobacterial methylglucose lipopolysaccharides (MGLP) we examined the molecular mechanisms linking replenishment of assimilable nitrogen to nitrogen-starved M. hassiacum with depletion of GG accumulated during nitrogen deficiency. To probe the involvement of a newly identified glycoside hydrolase in GG depletion, we produced the mycobacterial enzyme recombinantly and confirmed the specific hydrolysis of GG (GG hydrolase, GgH) in vitro. We have also observed a pronounced up-regulation of GgH mRNA in response to the nitrogen shock, which positively correlates with GG depletion in vivo and growth stimulation, implicating GgH in the recovery process. Since GgH orthologs seem to be absent from most slowly-growing mycobacteria including M. tuberculosis, the disclosure of the GgH function allows reconfiguration of the MGLP pathway in rapidly-growing species and accommodation of this possible regulatory step. This new link between GG metabolism, MGLP biosynthesis and recovery from nitrogen stress furthers our knowledge on the mycobacterial strategies to endure a frequent stress faced in some environments and during long-term infection. PMID:25341489

  7. Molecular cloning and expression of a new α-neoagarobiose hydrolase from Agarivorans gilvus WH0801 and enzymatic production of 3,6-anhydro-l-galactose.

    PubMed

    Liu, Nan; Yang, Meng; Mao, Xiangzhao; Mu, Bozhong; Wei, Dongzhi

    2016-01-01

    A new α-neoagarobiose hydrolase (NABH) called AgaWH117 was cloned from Agarivorans gilvus WH0801. The gene encoding this hydrolase consists of 1,086 bp and encodes a protein containing 361 amino acids. This new NABH showed 74% amino acid sequence identity with other known NABHs. The molecular mass of the recombinant AgaWH117 was estimated to be 41 kDa. Purified AgaWH117 showed endolytic activity during neoagarobiose degradation, yielding 3,6-anhydro-l-galactose (l-AHG) and d-galactose as products. It showed a maximum activity at a temperature of 30 °C and a pH of 6.0 and was stable at temperatures below 30 °C. Its Km and Vmax values were 2.094 mg/mL and 6.982 U/mg, respectively. The cloning strategy used and AgaWH117 isolated in this study will provide information on the saccharification process of marine biomass. This study provides a method to produce l-AHG from agarose by using AgaWH117 without an acid and describes its one-step purification by using Bio-Gel P2 chromatography.

  8. Recombination of cluster ions

    NASA Technical Reports Server (NTRS)

    Johnsen, Rainer

    1993-01-01

    Some of our recent work on molecular band emissions from recombination of molecular dimer ions (N4(+) and CO(+) CO) is discussed. Much of the experimental work was done by Y. S. Cao; the results on N4(+) recombination have been published. A brief progress report is given on our ongoing measurements of neutral products of recombination using the flowing-afterglow Langmuir-probe technique in conjunction with laser-induced fluorescence.

  9. A New Family of Biuret Hydrolases Involved in S-Triazine Ring Metabolism

    PubMed Central

    Cameron, Stephan M.; Durchschein, Katharina; Richman, Jack E.; Sadowsky, Michael J.; Wackett, Lawrence P.

    2011-01-01

    Biuret is an intermediate in the bacterial metabolism of s-triazine ring compounds and is occasionally used as a ruminant feed supplement. We used bioinformatics to identify a biuret hydrolase, an enzyme that has previously resisted efforts to stabilize, purify and characterize. This newly discovered enzyme is a member of the cysteine hydrolase superfamily, a family of enzymes previously not found to be involved in s-triazine metabolism. The gene from Rhizobium leguminosarum bv. viciae strain 3841 encoding biuret hydrolase was synthesized, transformed into Escherichia coli, and expressed. The enzyme was purified and found to be stable. Biuret hydrolase catalyzed the hydrolysis of biuret to allophanate and ammonia. The kcat/KM of 1.7 × 105 M−1s−1 and the relatively low KM of 23 ± 4 μM together suggested that this enzyme acts uniquely on biuret physiologically. This is supported by the fact that of the 34 substrate analogs of biuret tested, only two demonstrated reactivity, both at less than 5% of the rate determined for biuret. Biuret hydrolase does not react with carboxybiuret, the product of the enzyme immediately preceding biuret hydrolase in the metabolic pathway for cyanuric acid. This suggests an unusual metabolic strategy of an enzymatically-produced intermediate undergoing non-enzymatic decarboxylation to produce the substrate for the next enzyme in the pathway. PMID:21897878

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

    PubMed Central

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

    2014-01-01

    Pulmonary surfactant contains homeostatic and antimicrobial hydrolases. When Mycobacterium tuberculosis is initially deposited in the terminal bronchioles and alveoli, as well as following release from lysed macrophages, bacilli are in intimate contact with these lung surfactant hydrolases. We identified and measured several hydrolases in human alveolar lining fluid and lung tissue that, at their physiological concentrations, dramatically modified the M. tuberculosis cell envelope. Independent of their action time (15 min to 12 h), the effects of the hydrolases on the M. tuberculosis cell envelope resulted in a significant decrease (60–80%) in M. tuberculosis association with, and intracellular growth of the bacteria within, human macrophages. The cell envelope-modifying effects of the hydrolases also led to altered M. tuberculosis intracellular trafficking and induced a protective proin-flammatory response to infection. These findings add a new concept to our understanding of M. tuberculosis–macrophage inter-actions (i.e., the impact of lung surfactant hydrolases on M. tuberculosis infection). PMID:21602490

  11. A Phylogenetically Informed Comparison of GH1 Hydrolases between Arabidopsis and Rice Response to Stressors

    PubMed Central

    Cao, Yun-Ying; Yang, Jing-Fang; Liu, Tie-Yuan; Su, Zhen-Feng; Zhu, Fu-Yuan; Chen, Mo-Xian; Fan, Tao; Ye, Neng-Hui; Feng, Zhen; Wang, Ling-Juan; Hao, Ge-Fei; Zhang, Jianhua; Liu, Ying-Gao

    2017-01-01

    Glycoside hydrolases Family 1 (GH1) comprises enzymes that can hydrolyze β-O-glycosidic bond from a carbohydrate moiety. The plant GH1 hydrolases participate in a number of developmental processes and stress responses, including cell wall modification, plant hormone activation or deactivation and herbivore resistance. A large number of members has been observed in this family, suggesting their potential redundant functions in various biological processes. In this study, we have used 304 sequences of plant GH1 hydrolases to study the evolution of this gene family in plant lineage. Gene duplication was found to be a common phenomenon in this gene family. Although many members of GH1 hydrolases showed a high degree of similarity in Arabidopsis and rice, they showed substantial tissue specificity and differential responses to various stress treatments. This differential regulation implies each enzyme may play a distinct role in plants. Furthermore, some of salt-responsive Arabidopsis GH1 hydrolases were selected to test their genetic involvement in salt responses. The knockout mutants of AtBGLU1 and AtBGLU19 were observed to be less-sensitive during NaCl treatment in comparison to the wild type seedlings, indicating their participation in salt stress response. In summary, Arabidopsis and rice GH1 glycoside hydrolases showed distinct features in their evolutionary path, transcriptional regulation and genetic functions. PMID:28392792

  12. Purification and characterization of three parathion hydrolases from gram-negative bacterial strains.

    PubMed

    Mulbry, W W; Karns, J S

    1989-02-01

    Three unique parathion hydrolases were purified from gram-negative bacterial isolates and characterized. All three purified enzymes had roughly comparable affinities for ethyl parathion and had broad temperature optima at ca. 40 degrees C. The membrane-bound hydrolase of Flavobacterium sp. strain ATCC 27551 was composed of a single subunit of approximately 35,000 daltons (Da) and was inhibited by sulfhydryl reagents such as dithiothreitol (DTT) and by metal salts such as CuCl2. The cytosolic hydrolase of strain B-1 was composed of a single subunit of approximately 43,000 Da and was stimulated by DTT and inhibited by CuCl2. The membrane-bound hydrolase of strain SC was composed of four identical subunits of 67,000 Da and was inhibited by DTT and stimulated by CuCl2. The substrate ranges of the three enzymes also differed, as evidenced by their relative affinities for parathion and the related organophosphate insecticide O-ethyl-O-4-nitrophenyl phenylphosphonothioate (EPN). The B-1 hydrolase displayed equal affinity for both compounds, the Flavobacterium enzyme showed twofold-lower affinity for EPN than for parathion, and the SC hydrolase displayed no activity toward EPN. The range in characteristics of these three enzymes can be exploited in different waste disposal strategies.

  13. Purification and characterization of three parathion hydrolases from gram-negative bacterial strains.

    PubMed Central

    Mulbry, W W; Karns, J S

    1989-01-01

    Three unique parathion hydrolases were purified from gram-negative bacterial isolates and characterized. All three purified enzymes had roughly comparable affinities for ethyl parathion and had broad temperature optima at ca. 40 degrees C. The membrane-bound hydrolase of Flavobacterium sp. strain ATCC 27551 was composed of a single subunit of approximately 35,000 daltons (Da) and was inhibited by sulfhydryl reagents such as dithiothreitol (DTT) and by metal salts such as CuCl2. The cytosolic hydrolase of strain B-1 was composed of a single subunit of approximately 43,000 Da and was stimulated by DTT and inhibited by CuCl2. The membrane-bound hydrolase of strain SC was composed of four identical subunits of 67,000 Da and was inhibited by DTT and stimulated by CuCl2. The substrate ranges of the three enzymes also differed, as evidenced by their relative affinities for parathion and the related organophosphate insecticide O-ethyl-O-4-nitrophenyl phenylphosphonothioate (EPN). The B-1 hydrolase displayed equal affinity for both compounds, the Flavobacterium enzyme showed twofold-lower affinity for EPN than for parathion, and the SC hydrolase displayed no activity toward EPN. The range in characteristics of these three enzymes can be exploited in different waste disposal strategies. Images PMID:2541658

  14. 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

  15. Kinetic characterization of a cocaine hydrolase engineered from mouse butyrylcholinesterase.

    PubMed

    Chen, Xiabin; Huang, Xiaoqin; Geng, Liyi; Xue, Liu; Hou, Shurong; Zheng, Xirong; Brimijoin, Stephen; Zheng, Fang; Zhan, Chang-Guo

    2015-03-01

    Mouse butyrylcholinesterase (mBChE) and an mBChE-based cocaine hydrolase (mCocH, i.e. the A¹⁹⁹S/S²²⁷A/S²⁸⁷G/A³²⁸W/Y³³²G mutant) have been characterized for their catalytic activities against cocaine, i.e. naturally occurring (-)-cocaine, in comparison with the corresponding human BChE (hBChE) and an hBChE-based cocaine hydrolase (hCocH, i.e. the A¹⁹⁹S/F²²⁷A/S²⁸⁷G/A³²⁸W/Y³³²G mutant). It has been demonstrated that mCocH and hCocH have improved the catalytic efficiency of mBChE and hBChE against (-)-cocaine by ~8- and ~2000-fold respectively, although the catalytic efficiencies of mCocH and hCocH against other substrates, including acetylcholine (ACh) and butyrylthiocholine (BTC), are close to those of the corresponding wild-type enzymes mBChE and hBChE. According to the kinetic data, the catalytic efficiency (k(cat)/K(M)) of mBChE against (-)-cocaine is comparable with that of hBChE, but the catalytic efficiency of mCocH against (-)-cocaine is remarkably lower than that of hCocH by ~250-fold. The remarkable difference in the catalytic activity between mCocH and hCocH is consistent with the difference between the enzyme-(-)-cocaine binding modes obtained from molecular modelling. Further, both mBChE and hBChE demonstrated substrate activation for all of the examined substrates [(-)-cocaine, ACh and BTC] at high concentrations, whereas both mCocH and hCocH showed substrate inhibition for all three substrates at high concentrations. The amino-acid mutations have remarkably converted substrate activation of the enzymes into substrate inhibition, implying that the rate-determining step of the reaction in mCocH and hCocH might be different from that in mBChE and hBChE.

  16. A new group of exo-acting family 28 glycoside hydrolases of Aspergillus niger that are involved in pectin degradation

    PubMed Central

    Martens-Uzunova, Elena S.; Zandleven, Joris S.; Benen, Jaques A. E.; Awad, Hanem; Kools, Harrie J.; Beldman, Gerrit; Voragen, Alphons G. J.; Van Den Berg, Johan A.; Schaap, Peter J.

    2006-01-01

    The fungus Aspergillus niger is an industrial producer of pectin-degrading enzymes. The recent solving of the genomic sequence of A. niger allowed an inventory of the entire genome of the fungus for potential carbohydrate-degrading enzymes. By applying bioinformatics tools, 12 new genes, putatively encoding family 28 glycoside hydrolases, were identified. Seven of the newly discovered genes form a new gene group, which we show to encode exoacting pectinolytic glycoside hydrolases. This group includes four exo-polygalacturonan hydrolases (PGAX, PGXA, PGXB and PGXC) and three putative exo-rhamnogalacturonan hydrolases (RGXA, RGXB and RGXC). Biochemical identification using polygalacturonic acid and xylogalacturonan as substrates demonstrated that indeed PGXB and PGXC act as exo-polygalacturonases, whereas PGXA acts as an exo-xylogalacturonan hydrolase. The expression levels of all 21 genes were assessed by microarray analysis. The results from the present study demonstrate that exo-acting glycoside hydrolases play a prominent role in pectin degradation. PMID:16822232

  17. Unique substrate specificity of a thermostable glycosyl hydrolase from an uncultured Anaerolinea, derived from bacterial mat on a subsurface geothermal water stream.

    PubMed

    Konishi, Masaaki; Nishi, Shinro; Takami, Hideto; Shimane, Yasuhiro; Nagano, Yuriko; Mori, Kozue; Ohta, Yukari; Hatada, Yuji

    2012-10-01

    To investigate novel extremozymes encoded by sequenced metagenes from a microbial community in an extreme environment, we have characterized a recombinant glycosyl hydrolase (rGH) from an uncultured bacterium within the order Chloroflexi. rGH formed insoluble bodies in an Escherichia coli protein expression system. The protein was partially dissolved by a surfactant and was enzymatically characterized. The MW of the monomeric peptide was ~62 kDa, and it formed a homodimers in buffer. It was optimally active at 65 °C and from pH 4 to 8. rGH showed hydrolytic activity for α-1,1, α-1,2 and α-1,6 linkages, including isomaltose, but not α-1,4 and β-linkages.

  18. Recombinant Baculovirus Isolation.

    PubMed

    King, Linda A; Hitchman, Richard; Possee, Robert D

    2016-01-01

    Although there are several different methods available of making recombinant baculovirus expression vectors (reviewed in Chapter 3 ), all require a stage in which insect cells are transfected with either the virus genome alone (Bac-to-Bac(®) or BaculoDirect™, Invitrogen) or virus genome and transfer vector. In the latter case, this allows the natural process of homologous recombination to transfer the foreign gene, under control of the polyhedrin or other baculovirus gene promoter, from the transfer vector to the virus genome to create the recombinant virus. Previously, many methods required a plaque-assay to separate parental and recombinant virus prior to amplification and use of the recombinant virus. Fortunately, this step is no longer required for most systems currently available. This chapter provides an overview of the historical development of increasingly more efficient systems for the isolation of recombinant baculoviruses (Chapter 3 provides a full account of the different systems and transfer vectors available). The practical details cover: transfection of insect cells with either virus DNA or virus DNA and plasmid transfer vector; a reliable plaque-assay method that can be used to separate recombinant virus from parental (nonrecombinant) virus where this is necessary; methods for the small-scale amplification of recombinant virus; and subsequent titration by plaque-assay or real-time polymerase chain reaction (PCR). Methods unique to the Bac-to-Bac(®) system are also covered and include the transformation of bacterial cells and isolation of bacmid DNA ready for transfection of insect cells.

  19. ADP-Ribosylarginine Hydrolase Regulates Cell Proliferation and Tumorigenesis

    PubMed Central

    Kato, Jiro; Zhu, Jianfeng; Liu, Chengyu; Stylianou, Mario; Hoffmann, Victoria; Lizak, Martin J.; Glasgow, Connie G.; Moss, Joel

    2012-01-01

    Protein ADP-ribosylation is a reversible posttranslational modification of uncertain significance in cancer. In this study, we evaluated the consequences for cancer susceptibility in the mouse of a genetic deletion of the enzyme responsible for removing mono-ADP–ribose moieties from arginines in cellular proteins. Specifically, we analyzed cancer susceptibility in animals lacking the ADP-ribosylarginine hydrolase (ARH1) that cleaves the ADP ribose–protein bond. ARH1−/− cells or ARH1−/− cells overexpressing an inactive mutant ARH1 protein (ARH1−/− +dm) had higher proliferation rates than either wild-type ARH1+/+ cells or ARH1−/− cells engineered to express the wild-type ARH1 enzyme. More significantly, ARH1−/− and ARH1+/− mice spontaneously developed lymphomas, adenocarcinomas, and metastases more frequently than wild-type ARH1+/+ mice. In ARH1+/− mice, we documented in all arising tumors mutation of the remaining wild-type allele (or loss of heterozygosity), illustrating the strict correlation that existed between tumor formation and absence of ARH1 gene function. Our findings show that proper control of protein ADP-ribosylation levels affected by ARH1 is essential for cancer suppression. PMID:21697277

  20. ADP-ribosylarginine hydrolase regulates cell proliferation and tumorigenesis.

    PubMed

    Kato, Jiro; Zhu, Jianfeng; Liu, Chengyu; Stylianou, Mario; Hoffmann, Victoria; Lizak, Martin J; Glasgow, Connie G; Moss, Joel

    2011-08-01

    Protein ADP-ribosylation is a reversible posttranslational modification of uncertain significance in cancer. In this study, we evaluated the consequences for cancer susceptibility in the mouse of a genetic deletion of the enzyme responsible for removing mono-ADP-ribose moieties from arginines in cellular proteins. Specifically, we analyzed cancer susceptibility in animals lacking the ADP-ribosylarginine hydrolase (ARH1) that cleaves the ADP ribose-protein bond. ARH1(-/-) cells or ARH1(-/-) cells overexpressing an inactive mutant ARH1 protein (ARH1(-/-)+dm) had higher proliferation rates than either wild-type ARH1(+/+) cells or ARH1(-/-) cells engineered to express the wild-type ARH1 enzyme. More significantly, ARH1(-/-) and ARH1(+/-) mice spontaneously developed lymphomas, adenocarcinomas, and metastases more frequently than wild-type ARH1(+/+) mice. In ARH1(+/-) mice, we documented in all arising tumors mutation of the remaining wild-type allele (or loss of heterozygosity), illustrating the strict correlation that existed between tumor formation and absence of ARH1 gene function. Our findings show that proper control of protein ADP-ribosylation levels affected by ARH1 is essential for cancer suppression.

  1. Ubiquitin C-Terminal Hydrolase L1 in Tumorigenesis

    PubMed Central

    Hurst-Kennedy, Jennifer; Chin, Lih-Shen; Li, Lian

    2012-01-01

    Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1, aka PGP9.5) is an abundant, neuronal deubiquitinating enzyme that has also been suggested to possess E3 ubiquitin-protein ligase activity and/or stabilize ubiquitin monomers in vivo. Recent evidence implicates dysregulation of UCH-L1 in the pathogenesis and progression of human cancers. Although typically only expressed in neurons, high levels of UCH-L1 have been found in many nonneuronal tumors, including breast, colorectal, and pancreatic carcinomas. UCH-L1 has also been implicated in the regulation of metastasis and cell growth during the progression of nonsmall cell lung carcinoma, colorectal cancer, and lymphoma. Together these studies suggest UCH-L1 has a potent oncogenic role and drives tumor development. Conversely, others have observed promoter methylation-mediated silencing of UCH-L1 in certain tumor subtypes, suggesting a potential tumor suppressor role for UCH-L1. In this paper, we provide an overview of the evidence supporting the involvement of UCH-L1 in tumor development and discuss the potential mechanisms of action of UCH-L1 in oncogenesis. PMID:22811913

  2. Mapping human brain fatty acid amide hydrolase activity with PET

    PubMed Central

    Rusjan, Pablo M; Wilson, Alan A; Mizrahi, Romina; Boileau, Isabelle; Chavez, Sofia E; Lobaugh, Nancy J; Kish, Stephen J; Houle, Sylvain; Tong, Junchao

    2013-01-01

    Endocannabinoid tone has recently been implicated in a number of prevalent neuropsychiatric conditions. [11C]CURB is the first available positron emission tomography (PET) radiotracer for imaging fatty acid amide hydrolase (FAAH), the enzyme which metabolizes the prominent endocannabinoid anandamide. Here, we sought to determine the most suitable kinetic modeling approach for quantifying [11C]CURB that binds selectively to FAAH. Six healthy volunteers were scanned with arterial blood sampling for 90 minutes. Kinetic parameters were estimated regionally using a one-tissue compartment model (TCM), a 2-TCM with and without irreversible trapping, and an irreversible 3-TCM. The 2-TCM with irreversible trapping provided the best identifiability of PET outcome measures among the approaches studied (coefficient of variation (COV) of the net influx constant Ki and the composite parameter λk3 (λ=K1/k2) <5%, and COV(k3)<10%). Reducing scan time to 60 minutes did not compromise the identifiability of rate constants. Arterial spin labeling measures of regional cerebral blood flow were only slightly correlated with Ki, but not with k3 or λk3. Our data suggest that λk3 is sensitive to changes in FAAH activity, therefore, optimal for PET quantification of FAAH activities with [11C]CURB. Simulations showed that [11C]CURB binding in healthy subjects is far from a flow-limited uptake. PMID:23211960

  3. Regulation of S-Adenosylhomocysteine Hydrolase by Lysine Acetylation*

    PubMed Central

    Wang, Yun; Kavran, Jennifer M.; Chen, Zan; Karukurichi, Kannan R.; Leahy, Daniel J.; Cole, Philip A.

    2014-01-01

    S-Adenosylhomocysteine hydrolase (SAHH) is an NAD+-dependent tetrameric enzyme that catalyzes the breakdown of S-adenosylhomocysteine to adenosine and homocysteine and is important in cell growth and the regulation of gene expression. Loss of SAHH function can result in global inhibition of cellular methyltransferase enzymes because of high levels of S-adenosylhomocysteine. Prior proteomics studies have identified two SAHH acetylation sites at Lys401 and Lys408 but the impact of these post-translational modifications has not yet been determined. Here we use expressed protein ligation to produce semisynthetic SAHH acetylated at Lys401 and Lys408 and show that modification of either position negatively impacts the catalytic activity of SAHH. X-ray crystal structures of 408-acetylated SAHH and dually acetylated SAHH have been determined and reveal perturbations in the C-terminal hydrogen bonding patterns, a region of the protein important for NAD+ binding. These crystal structures along with mutagenesis data suggest that such hydrogen bond perturbations are responsible for SAHH catalytic inhibition by acetylation. These results suggest how increased acetylation of SAHH may globally influence cellular methylation patterns. PMID:25248746

  4. Disrupting Dimerization Translocates Soluble Epoxide Hydrolase to Peroxisomes.

    PubMed

    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.

  5. Genetic regulation of expression of leukotriene A4 hydrolase

    PubMed Central

    Castaldi, Peter; Cho, Michael H.; Blalock, J. Edwin; Gaggar, Amit

    2016-01-01

    In chronic inflammatory lung disorders such as chronic obstructive pulmonary disease (COPD), the concurrent organ-specific and systemic inflammatory responses lead to airway remodelling and vascular dysfunction. Although a major common risk factor for COPD, cigarette smoke alone cannot explain the progression of this disease; there is increasing evidence that genetic predisposition also plays a role in COPD susceptibility and progression. A key enzyme in chronic lung inflammation is leukotriene A4 hydrolase (LTA4H). With its aminopeptidase activity, LTA4H degrades the neutrophil chemoattractant tripeptide PGP. In this study, we used the luciferase reporter gene analysis system and quantitative trait locus analysis to explore the impact of single-nucleotide polymorphisms (SNPs) in the putative promoter region of LTA4H on LTA4H expression. We show that not only is the putative promoter of LTA4H larger than previously reported but also that SNPs in the expanded promoter region regulate expression of LTA4H both in cell-based systems and in peripheral blood samples from human subjects. These findings provide significant evidence for an active region upstream of the previously reported LTA4H promoter, which may have implications related to ongoing inflammatory processes in chronic lung disease. PMID:27730172

  6. Purification and characterization of paraoxon hydrolase from rat liver.

    PubMed Central

    Rodrigo, L; Gil, F; Hernandez, A F; Marina, A; Vazquez, J; Pla, A

    1997-01-01

    Paraoxonase (paraoxon hydrolase), an enzyme that hydrolyses paraoxon (O,O-diethyl O-p-nitrophenyl phosphate), is located in mammals primarily in the serum and liver. Although considerable information is available regarding serum paraoxonase, little is known about the hepatic form of this enzyme. The present work represents the first study on the purification of rat liver paraoxonase. This enzyme has been purified 415-fold to apparent homogeneity with a final specific activity of 1370 units/mg using a protocol consisting of five steps: solubilization of the microsomal fraction, hydroxyapatite adsorption, chromatography on DEAE-Sepharose CL-6B, non-specific affinity chromatography on Cibacron Blue 3GA and anion exchange on Mono Q HR 5/5. The presence of Ca2+ and Triton X-100 in the buffers throughout the purification procedure was essential for maintaining enzyme activity. SDS/PAGE of the final preparation indicated a single protein-staining band with an apparent Mr of 45 000. N-terminal and internal amino acid sequences were determined and compared with those of paraoxonases from human and rabbit serum and mouse liver, showing a high similarity. The pH profile showed optimum activity at pH 8.5. The pH stability and heat inactivation of the enzyme were also studied. The Km for liver paraoxonase was 1.69 mM. PMID:9032442

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

    PubMed

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

    2015-10-15

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

  8. 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

  9. Investigation of the Bacillus cereus phosphonoacetaldehyde hydrolase catalytic mechanism

    SciTech Connect

    Hepburn, T.W.

    1988-01-01

    The enzyme phosphonoacetaldehyde hydrolase (phosphonatase) from Bacillus cereus catalyzes the conversion of phosphonoacetaldehyde and phosphate. We have demonstrated that phosphonatase is inactivated when incubated with either acetaldehyde or phosphonoacetaldehyde for short time periods at low temperature in the presence of NaBH{sup 4}. This result suggests that the Schiff base mechanism is operative since such treatment might be expected to inactivate the enzyme by reducing an iminium cation mechanistic intermediate. The inactivation process was shown to be highly specific for a single lysine residue. Incubation of phosphonatase with ({sup 3}H)-NaBH{sub 4} and phosphonacetaldehyde ({sup 14}C)-acetaldehyde and NaBH{sub 4} or (C{sub 2}-{sup 3}H)- phosphonoacetaldehyde and NaBH{sup 4} resulted in radiolabeled inactivated enzyme. Tryptic hydrolysis and reverse phase HPLC chromatography of the resulting digests demonstrated that the (C{sub 2} - {sup 3}H)- phosphonoacetaldehyde/NaBH{sub 4} methodology afforded the most specifically tritium labeled, inactivated phosphonatase. The radiolabeled, active site peptide was purified to homogeneity and its amino acid sequence was determined.

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

    PubMed

    Rodríguez-Rubio, Lorena; Martínez, Beatriz; Donovan, David M; Rodríguez, Ana; García, Pilar

    2013-11-01

    Virion-associated peptidoglycan hydrolases (VAPGH) are phage-encoded lytic enzymes that locally degrade the peptidoglycan (PG) of the bacterial cell wall during infection. In contrast to endolysins, PGHs that mediate lysis of the host bacteria at the end of the lytic cycle to release of phage progeny, the action of VAPGHs generates a small hole through which the phage tail tube crosses the cell envelope to eject the phage genetic material at the beginning to the infection cycle. The antimicrobial activity of VAPGHs was first discovered through the observation of the phenomenon of 'lysis from without', in which the disruption of the bacterial cell wall occurs prior to phage production and is caused by a high number of phages adsorbed onto the cell surface. Based on a unique combination of properties of VAPGHs such as high specificity, remarkable thermostability, and a modular organization, these proteins are potential candidates as new antibacterial agents, e.g. against antibiotic-resistant bacteria in human therapy and veterinary as well as biopreservatives in food safety, and as biocontrol agents of harmful bacteria in agriculture. This review provides an overview of the different VAPGHs discovered to date and their potential as novel antimicrobials.

  11. Expression of organophosphate hydrolase in the filamentous fungus Gliocladium virens.

    PubMed

    Dave, K I; Lauriano, C; Xu, B; Wild, J R; Kenerley, C M

    1994-05-01

    The broad-spectrum organophosphate hydrolase (OPH; EC 3.1.8.1) encoded by the organophosphate-degrading gene (opd) from Pseudomonas diminuta MG and Flavobacterium sp. ATCC 27551 possesses capabilities of both P-O bond hydrolysis (e.g. paraoxon) and P-F bond hydrolysis [e.g. sarin and diisopropylfluorophosphate (DFP)]. In the present study a 9.4-kb plasmid, pCL1, was used to transform the saprophytic fungus Gliocladium virens. pCL1 was derived from pJS294 by placing the fungal promoter (prom1) from Cochliobolus heterostrophus upstream and the trpC terminator from Aspergillus nidulans down-stream of the opd gene. Southern analysis of restricted genomic DNA from various transformants indicated that integration occurred non-specifically at multiple sites. Western blot analysis of mycelial extracts from transformants confirmed the production of a processed form of the enzyme in the fungus. Maximal levels of OPH activity (rate of p-nitrophenol production from paraoxon) were observed after 168 h of culture and activity levels correlated with biomass production in mature vegetative growth.

  12. Molecular Basis of Arabinobio-hydrolase Activity in Phytopathogenic Fungi

    PubMed Central

    Carapito, Raphaël; Imberty, Anne; Jeltsch, Jean-Marc; Byrns, Simon C.; Tam, Pui-Hang; Lowary, Todd L.; Varrot, Annabelle; Phalip, Vincent

    2009-01-01

    The phytopathogenic fungus Fusarium graminearum secretes a very diverse pool of glycoside hydrolases (GHs) aimed at degrading plant cell walls. α-l-Arabinanases are essential GHs participating in the complete hydrolysis of hemicellulose, a natural resource for various industrial processes, such as bioethanol or pharmaceuticals production. Arb93A, the exo-1,5-α-l-arabinanase of F. graminearum encoded by the gene fg03054.1, belongs to the GH93 family, for which no structural data exists. The enzyme is highly active (1065 units/mg) and displays a strict substrate specificity for linear α-1,5-l-arabinan. Biochemical assays and NMR experiments demonstrated that the enzyme releases α-1,5-l-arabinobiose from the nonreducing end of the polysaccharide. We determined the crystal structure of the native enzyme and its complex with α-1,5-l-arabinobiose, a degradation product of α-Me-1,5-l-arabinotetraose, at 1.85 and 2.05Å resolution, respectively. Arb93A is a monomeric enzyme, which presents the six-bladed β-propeller fold characteristic of sialidases of clan GHE. The configuration of the bound arabinobiose is consistent with the retaining mechanism proposed for the GH93 family. Catalytic residues were proposed from the structural analysis, and site-directed mutagenesis was used to validate their role. They are significantly different from those observed for GHE sialidases. PMID:19269961

  13. Ancient origin of glycosyl hydrolase family 9 cellulase genes.

    PubMed

    Davison, Angus; Blaxter, Mark

    2005-05-01

    While it is widely accepted that most animals (Metazoa) do not have endogenous cellulases, relying instead on intestinal symbionts for cellulose digestion, the glycosyl hydrolase family 9 (GHF9) cellulases found in the genomes of termites, abalone, and sea squirts could be an exception. Using information from expressed sequence tags, we show that GHF9 genes (subgroup E2) are widespread in Metazoa because at least 11 classes in five phyla have expressed GHF9 cellulases. We also demonstrate that eukaryotic GHF9 gene families are ancient, forming distinct monophyletic groups in plants and animals. As several intron positions are also conserved between four metazoan phyla then, contrary to the still widespread belief that cellulases were horizontally transferred to animals relatively recently, GHF9 genes must derive from an ancient ancestor. We also found that sequences isolated from the same animal phylum tend to group together, and in some deuterostomes, GHF9 genes are characterized by substitutions in catalytically important sites. Several paralogous subfamilies of GHF9 can be identified in plants, and genes from primitive species tend to arise basally to angiosperm representatives. In contrast, GHF9 subgroup E2 genes are relatively rare in bacteria.

  14. 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

  15. 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

  16. Recognition of Corn Defense Chitinases by Fungal Polyglycine Hydrolases.

    PubMed

    Naumann, Todd A; Bakota, Erica L; Price, Neil P J

    2017-04-06

    Polyglycine hydrolases (PGH)s are secreted fungal endoproteases that cleave peptide bonds in the polyglycine interdomain linker of ChitA chitinase, an antifungal protein from domesticated corn (Zea mays ssp. mays). These target-specific endoproteases are unusual because they do not cut a specific peptide bond but select one of many Gly-Gly bonds within the polyglycine region. Some Gly-Gly bonds are cleaved frequently while others are never cleaved. Moreover, we have previously shown that PGHs from different fungal pathogens prefer to cleave different Gly-Gly peptide bonds. It is not understood how PGHs selectively cleave the ChitA linker, especially because its polyglycine structure lacks peptide sidechains. To gain insights into this process we synthesized several peptide analogs of ChitA to evaluate them as potential substrates and inhibitors of Es-cmp, a PGH from the plant pathogenic fungus Epicoccum sorghi. Our results showed that part of the PGH recognition site for substrate chitinases is adjacent to the polyglycine linker on the carboxy side. More specifically, four amino acid residues were implicated, each spaced four residues apart on an alpha helix. Moreover, analogous peptides with selective Gly->sarcosine (N-methylglycine) mutations or a specific Ser->Thr mutation retained inhibitor activity but were no longer cleaved by PGH. Additonally, our findings suggest that peptide analogs of ChitA that inhibit PGH activity could be used to strengthen plant defenses. This article is protected by copyright. All rights reserved.

  17. Bacterial 2,4-dioxygenases: new members of the alpha/beta hydrolase-fold superfamily of enzymes functionally related to serine hydrolases.

    PubMed

    Fischer, F; Künne, S; Fetzner, S

    1999-09-01

    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 alpha/beta 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 alpha/beta 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 alpha/beta 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.

  18. Characterization of active-site residues in diadenosine tetraphosphate hydrolase from Lupinus angustifolius.

    PubMed Central

    Maksel, D; Gooley, P R; Swarbrick, J D; Guranowski, A; Gange, C; Blackburn, G M; Gayler, K R

    2001-01-01

    Site-directed mutagenesis has been used to characterize the functions of key amino acid residues in the catalytic site of the 'nudix' hydrolase, (asymmetrical) diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) hydrolase (EC 3.6.1.17) from Lupinus angustifolius, the three-dimensional solution structure of which has recently been solved. Residues within the nudix motif, Gly-(Xaa)5-Glu-(Xaa)7-Arg-Glu-Uaa-Xaa-(Glu)2-Xaa-Gly (where Xaa represents unspecified amino acids and Uaa represents the bulky aliphatic amino acids Ile, Leu or Val) conserved in 'nudix enzymes', and residues important for catalysis from elsewhere in the molecule, were mutated and the expressed proteins characterized. The results reveal a high degree of functional conservation between lupin asymmetric Ap4A hydrolase and the 8-oxo-dGTP hydrolase from Escherichia coli. Charged residues in positions equivalent to those that ligate an enzyme-bound metal ion in the E. coli 8-oxo-dGTP hydrolase [Harris, Wu, Massiah and Mildvan (2000) Biochemistry 39, 1655-1674] were shown to contribute to catalysis to similar extents in the lupin enzyme. Mutations E55Q, E59Q and E125Q all reduced kcat markedly, whereas mutations R54Q, E58Q and E122Q had smaller effects. None of the mutations produced a substantial change in the Km)for Ap4A, but several extensively modified the pH-dependence and fluoride-sensitivities of the hydrolase. It was concluded that the precisely positioned glutamate residues Glu-55, Glu-59 and Glu-125 are conserved as functionally significant components of the hydrolytic mechanism in both of these members of the nudix family of hydrolases. PMID:11439089

  19. 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

  20. Improving the secretion of a methyl parathion hydrolase in Pichia pastoris by modifying its N-terminal sequence.

    PubMed

    Wang, Ping; Huang, Lu; Jiang, Hu; Tian, Jian; Chu, Xiaoyu; Wu, Ningfeng

    2014-01-01

    Pichia pastoris is commonly used to express and secrete target proteins, although not all recombinant proteins can be successfully produced. In this study, we used methyl parathion hydrolase (MPH) from Ochrobactrum sp. M231 as a model to study the importance of the N-terminus of the protein for its secretion. While MPH can be efficiently expressed intracellularly in P. pastoris, it is not secreted into the extracellular environment. Three MPH mutants (N66-MPH, D10-MPH, and N9-MPH) were constructed through modification of its N-terminus, and the secretion of each by P. pastoris was improved when compared to wild-type MPH. The level of secreted D10-MPH was increased to 0.21 U/mL, while that of N9-MPH was enhanced to 0.16 U/mL. Although N66-MPH was not enzymatically active, it was secreted efficiently, and was identified by SDS-PAGE. These results demonstrate that the secretion of heterologous proteins in P. pastoris may be improved by modifying their N-terminal structures.

  1. High Phylogenetic Diversity of Glycosyl Hydrolase Family 10 and 11 Xylanases in the Sediment of Lake Dabusu in China

    PubMed Central

    Ng, Tzi Bun; Lin, Juan; Ye, Xiu Yun

    2014-01-01

    Soda lakes are one of the most stable naturally occurring alkaline and saline environments, which harbor abundant microorganisms with diverse functions. In this study, culture-independent molecular methods were used to explore the genetic diversity of glycoside hydrolase (GH) family 10 and GH11 xylanases in Lake Dabusu, a soda lake with a pH value of 10.2 and salinity of 10.1%. A total of 671 xylanase gene fragments were obtained, representing 78 distinct GH10 and 28 GH11 gene fragments respectively, with most of them having low homology with known sequences. Phylogenetic analysis revealed that the GH10 xylanase sequences mainly belonged to Bacteroidetes, Proteobacteria, Actinobacteria, Firmicutes and Verrucomicrobia, while the GH11 sequences mainly consisted of Actinobacteria, Firmicutes and Fungi. A full-length GH10 xylanase gene (xynAS10-66) was directly cloned and expressed in Escherichia coli, and the recombinant enzymes showed high activity at alkaline pH. These results suggest that xylanase gene diversity within Lake Dabusu is high and that most of the identified genes might be novel, indicating great potential for applications in industry and agriculture. PMID:25392912

  2. Production of (S)-4-chloro-3-hydroxybutyrate by microbial resolution using hydrolase from Rhizobium sp. DS-S-51.

    PubMed

    Nakagawa, Atsushi; Suzuki, Takahiro; Kato, Ko; Shinmyo, Atsuhiko; Suzuki, Toshio

    2008-04-01

    (S)-4-Chloro-3-hydroxybutyrate (CHB) is essential for the synthesis of biologically and pharmacologically important compounds. Rhizobium sp. DS-S-51 isolated from soil samples showed hydrolytic activity toward (R)-CHB in the racemate to (R)-3-hydroxy-gamma-butyrolactone (HL) under a simple composition of the reaction. Residual (S)-CHB was obtained with high optical purity. The gene encoding the enzyme concerned, designated CHB hydrolase, was isolated from DS-S-51, and the gene was highly expressed in Escherichia coli JM109. When the resolution of racemic methyl CHB (CHBM) as a substrate was performed using this recombinant cell, JM109 (pKK-R1), the hydrolytic activity was found to be 40-fold greater than that of DS-S-51, and the maximum concentration of the substrate added increased 2-fold. Moreover, (R)-HL was also obtained without decreasing the optical purity compared with that when (R)-CHBM was used as a substrate.

  3. Genome analysis of the staphylococcal temperate phage DW2 and functional studies on the endolysin and tail hydrolase

    PubMed Central

    Keary, Ruth; McAuliffe, Olivia; Ross, R Paul; Hill, Colin; O’Mahony, Jim; Coffey, Aidan

    2014-01-01

    This study describes the genome of temperate Siphoviridae phage DW2, which is routinely propagated on Staphylococcus aureus DPC5246. The 41941 bp genome revealed an open reading frame (ORF1) which has a high level of homology with members of the resolvase subfamily of site-specific serine recombinase, involved in chromosomal integration and excision. In contrast, the majority of staphylococcal phages reported to date encode tyrosine recombinases. Two putative genes encoded by phage DW2 (ORF15 and ORF24) were highly homologous to the NWMN0273 and NWMN0280 genes encoding virulence factors carried on the genome of ϕNM4, a prophage in the genome of S. aureus Newman. Phage DW2 also encodes proteins highly homologous to two well-characterized Staphylococcus aureus pathogenicity island derepressors encoded by the staphylococcal helper phage 80α indicating that it may similarly act as a helper phage for mobility of pathogenicity islands in S. aureus. This study also focused on the enzybiotic potential of phage DW2. The structure of the putative endolysin and tail hydrolase were investigated and used as the basis for a cloning strategy to create recombinant peptidoglycan hydrolyzing proteins. After overexpression in E. coli, four of these proteins (LysDW2, THDW2, CHAPE1-153, and CHAPE1-163) were demonstrated to have hydrolytic activity against peptidoglycan of S. aureus and thus represent novel candidates for exploitation as enzybiotics. PMID:25105056

  4. Isolation and characterization of a novel glycosyl hydrolase family 74 (GH74) cellulase from the black goat rumen metagenomic library.

    PubMed

    Song, Yun-Hee; Lee, Kyung-Tai; Baek, Jin-Young; Kim, Min-Ju; Kwon, Mi-Ra; Kim, Young-Joo; Park, Mi-Rim; Ko, Haesu; Lee, Jin-Sung; Kim, Keun-Sung

    2017-05-01

    This study aimed to isolate and characterize a novel cellulolytic enzyme from black goat rumen by using a culture-independent approach. A metagenomic fosmid library was constructed from black goat rumen contents and screened for a novel cellulase. The KG37 gene encoding a protein of 858 amino acid residues (92.7 kDa) was isolated. The deduced protein contained a glycosyl hydrolase family 74 (GH74) domain and showed 77% sequence identity to two endo-1,4-β-glucanases from Fibrobacter succinogenes. The novel GH74 cellulase gene was overexpressed in Escherichia coli, and its protein product was functionally characterized. The recombinant GH74 cellulase showed a broad substrate spectrum. The enzyme exhibited its optimum activity at pH 5.0 and temperature range of 20-50 °C. The enzyme was thermally stable at pH 5.0 and at a temperature of 20-40 °C. The novel GH74 cellulase can be practically exploited to convert lignocellulosic biomass to value-added products in various industrial applications in future.

  5. Overproduction of acyloxyacyl hydrolase by macrophages and dendritic cells prevents prolonged reactions to bacterial lipopolysaccharide in vivo.

    PubMed

    Ojogun, Noredia; Kuang, Tang-Yong; Shao, Baomei; Greaves, David R; Munford, Robert S; Varley, Alan W

    2009-12-01

    Although recognition of lipopolysaccharide (LPS) by the myeloid differentiation factor 2-Toll-like receptor 4 complex is important for triggering protective inflammatory responses in animals, terminating many of these responses requires LPS inactivation by a host lipase, acyloxyacyl hydrolase (AOAH). To test whether endogenously produced recombinant AOAH can modulate responses to LPS and gram-negative bacteria, we engineered transgenic mice that overexpress AOAH in dendritic cells and macrophages, cell types that normally produce it. Transgenic mice deacylated LPS more rapidly than did wild-type controls. They also were protected from LPS-induced hepatosplenomegaly, recovered more quickly from LPS-induced weight loss, and were more likely to survive when challenged with live Escherichia coli. Constitutive overexpression of AOAH in vivo hastened recovery from LPS exposure without interfering with the normal acute inflammatory response to this important microbial signal molecule. Our results suggest that the extent to which macrophages and dendritic cells produce AOAH may influence the outcome of many gram-negative bacterial diseases.

  6. Isolation and Characterization of a Glycosyl Hydrolase Family 16 β-Agarase from a Mangrove Soil Metagenomic Library

    PubMed Central

    Mai, Zhimao; Su, Hongfei; Zhang, Si

    2016-01-01

    A mangrove soil metagenomic library was constructed and a β-agarase gene designated as AgaML was isolated by functional screening. The gene encoded for a 659-amino-acids polypeptide with an estimated molecular mass of 71.6 kDa. The deduced polypeptide sequences of AgaML showed the highest identity of 73% with the glycoside hydrolase family 16 β-agarase from Microbulbifer agarilyticus in the GenBank database. AgaML was cloned and highly expressed in Escherichia coli BL21(DE3). The purified recombinant protein, AgaML, showed optimal activity at 50 °C and pH 7.0. The kinetic parameters of Km and Vmax values toward agarose were 4.6 mg·mL−1 and 967.5 μM·min−1·mg−1, respectively. AgaML hydrolyzed the β-1,4-glycosidic linkages of agar to generate neoagarotetraose (NA4) and neoagarohexaose (NA6) as the main products. These characteristics suggest that AgaML has potential application in cosmetic, pharmaceuticals and food industries. PMID:27548158

  7. High phylogenetic diversity of glycosyl hydrolase family 10 and 11 xylanases in the sediment of Lake Dabusu in China.

    PubMed

    Wang, Guozeng; Huang, Xiaoyun; Ng, Tzi Bun; Lin, Juan; Ye, Xiu Yun

    2014-01-01

    Soda lakes are one of the most stable naturally occurring alkaline and saline environments, which harbor abundant microorganisms with diverse functions. In this study, culture-independent molecular methods were used to explore the genetic diversity of glycoside hydrolase (GH) family 10 and GH11 xylanases in Lake Dabusu, a soda lake with a pH value of 10.2 and salinity of 10.1%. A total of 671 xylanase gene fragments were obtained, representing 78 distinct GH10 and 28 GH11 gene fragments respectively, with most of them having low homology with known sequences. Phylogenetic analysis revealed that the GH10 xylanase sequences mainly belonged to Bacteroidetes, Proteobacteria, Actinobacteria, Firmicutes and Verrucomicrobia, while the GH11 sequences mainly consisted of Actinobacteria, Firmicutes and Fungi. A full-length GH10 xylanase gene (xynAS10-66) was directly cloned and expressed in Escherichia coli, and the recombinant enzymes showed high activity at alkaline pH. These results suggest that xylanase gene diversity within Lake Dabusu is high and that most of the identified genes might be novel, indicating great potential for applications in industry and agriculture.

  8. Recombination and Replication

    PubMed Central

    Syeda, Aisha H.; Hawkins, Michelle; McGlynn, Peter

    2014-01-01

    The links between recombination and replication have been appreciated for decades and it is now generally accepted that these two fundamental aspects of DNA metabolism are inseparable: Homologous recombination is essential for completion of DNA replication and vice versa. This review focuses on the roles that recombination enzymes play in underpinning genome duplication, aiding replication fork movement in the face of the many replisome barriers that challenge genome stability. These links have many conserved features across all domains of life, reflecting the conserved nature of the substrate for these reactions, DNA. PMID:25341919

  9. Dissociative recombination in aeronomy

    NASA Technical Reports Server (NTRS)

    Fox, J. L.

    1989-01-01

    The importance of dissociative recombination in planetary aeronomy is summarized, and two examples are discussed. The first is the role of dissociative recombination of N2(+) in the escape of nitrogen from Mars. A previous model is updated to reflect new experimental data on the electronic states of N produced in this process. Second, the intensity of the atomic oxygen green line on the nightside of Venus is modeled. Use is made of theoretical rate coefficients for production of O (1S) in dissociative recombination from different vibrational levels of O2(+).

  10. 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

  11. 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

  12. 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.

  13. Mechanistic studies of ubiquitin C-terminal hydrolase L1.

    PubMed

    Case, April; Stein, Ross L

    2006-02-21

    Ubiquitin C-terminal hydrolases (UCHs) cleave Ub-X bonds (Ub is ubiquitin and X an alcohol, an amine, or a protein) through a thioester intermediate that is produced by nucleophilic attack of the Cys residue of a Cys-SH/His-Im catalytic diad. We are studying the mechanism of UCH-L1, a UCH that is implicated in Parkinson's disease, and now wish to report our initial findings. (i) Pre-steady-state kinetic studies for UCH-L1-catalyzed hydrolysis of Ub-AMC (AMC, 7-amido-4-methylcoumarin) indicate that k(cat) is rate-limited by acyl-enzyme formation. Thus, K(m) = K(s), the dissociation constant for the Michaelis complex, and k(cat) = k(2), the rate constant for acyl-enzyme formation. (ii) For K(assoc) (=K(s)(-)(1)), DeltaC(p) = -0.8 kcal mol(-)(1) deg(-)(1) and is consistent with coupling between substrate association and a conformational change of the enzyme. For k(2), DeltaS(++) = 0 and suggests that in the E-S, substrate and active site residues are precisely aligned for reaction. (iii) Solvent isotope effects are (D)K(assoc) = 0.5 and (D)k(2) = 0.9, suggesting that the substrate binds to a form of free enzyme in which the active site Cys exists as the thiol. In the resultant Michaelis complex, the diad has tautomerized to ion pair Cys-S(-)/His-ImH(+). Subsequent attack of thiolate produces the acyl-enzyme species. In contrast, isotope effects for association of UCH-L1 with transition-state analogue ubiquitin aldehyde suggest that an alternative mechanistic pathway can sometimes be available to UCH-L1 involving general base-catalyzed attack of Cys-SH by His-Im.

  14. 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. Copyright © 2016. Published by Elsevier Masson SAS.

  15. Long-acting cocaine hydrolase for addiction therapy

    PubMed Central

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

    2016-01-01

    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

  16. Soluble epoxide hydrolase: a novel therapeutic target in stroke

    PubMed Central

    Zhang, Wenri; Koerner, Ines P; Noppens, Ruediger; Grafe, Marjorie; Tsai, Hsing-Ju; Morisseau, Christophe; Luria, Ayala; Hammock, Bruce D; Falck, John R; Alkayed, Nabil J

    2009-01-01

    The P450 eicosanoids epoxyeicosatrienoic acids (EETs) are produced in brain and perform important biological functions, including protection from ischemic injury. The beneficial effect of EETs, however, is limited by their metabolism via soluble epoxide hydrolase (sEH). We tested the hypothesis that sEH inhibition is protective against ischemic brain damage in vivo by a mechanism linked to enhanced cerebral blood flow (CBF). We determined expression and distribution of sEH immunoreactivity (IR) in brain, and examined the effect of sEH inhibitor 12-(3-adamantan-1-yl-ureido)-dodecanoic acid butyl ester (AUDA-BE) on CBF and infarct size after experimental stroke in mice. Mice were administered a single intraperitoneal injection of AUDA-BE (10 mg/kg) or vehicle at 30 mins before 2-h middle cerebral artery occlusion (MCAO) or at reperfusion, in the presence and absence of P450 epoxygenase inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl) hexanamide (MS-PPOH). Immunoreactivity for sEH was detected in vascular and non-vascular brain compartments, with predominant expression in neuronal cell bodies and processes. 12-(3-Adamantan-1-yl-ureido)-dodecanoic acid butyl ester was detected in plasma and brain for up to 24 h after intraperitoneal injection, which was associated with inhibition of sEH activity in brain tissue. Finally, AUDA-BE significantly reduced infarct size at 24 h after MCAO, which was prevented by MS-PPOH. However, regional CBF rates measured by iodoantipyrine (IAP) autoradiography at end ischemia revealed no differences between AUDA-BE- and vehicle-treated mice. The findings suggest that sEH inhibition is protective against ischemic injury by non-vascular mechanisms, and that sEH may serve as a therapeutic target in stroke. PMID:17440491

  17. 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

  18. 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.

  19. Inhibition of soluble epoxide hydrolase increases coronary perfusion in mice

    PubMed Central

    Qin, Jun; Sun, Dong; Jiang, Houli; Kandhi, Sharath; Froogh, Ghezal; Hwang, Sung Hee; Hammock, Bruce D; Wolin, Michael S; Thompson, Carl I; Hintze, Thomas H; Huang, An

    2015-01-01

    Roles of soluble epoxide hydrolase (sEH), the enzyme responsible for hydrolysis of epoxyeicosatrienoic acids (EETs) to their diols (DHETs), in the coronary circulation and cardiac function remain unknown. We tested the hypothesis that compromising EET hydrolysis/degradation, via sEH deficiency, lowers the coronary resistance to promote cardiac perfusion and function. Hearts were isolated from wild type (WT), sEH knockout (KO) mice and WT mice chronically treated with t-TUCB (sEH inhibitor), and perfused with constant flow at different pre-loads. Compared to WT controls, sEH-deficient hearts required significantly greater basal coronary flow to maintain the perfusion pressure at 100 mmHg and exhibited a greater reduction in vascular resistance during tension-induced heart work, implying a better coronary perfusion during cardiac performance. Cardiac contractility, characterized by developed tension in response to changes in preload, was potentially increased in sEH-KO hearts, manifested by an enlarged magnitude at each step-wise increase in end-diastolic to peak-systolic tension. 14,15-EEZE (EET antagonist) prevented the adaptation of coronary circulation in sEH null hearts whereas responses in WT hearts were sensitive to the inhibition of NO. Cardiac expression of EET synthases (CYP2J2/2C29) was comparable in both genotypic mice whereas, levels of 14,15-, 11,12- and 8,9-EETs were significantly higher in sEH-KO hearts, accompanied with lower levels of DHETs. In conclusion, the elevation of cardiac EETs, as a function of sEH deficiency, plays key roles in the adaptation of coronary flow and cardiac function. PMID:26071213

  20. Naphthalene cytotoxicity in microsomal epoxide hydrolase deficient mice

    PubMed Central

    Carratt, SA; Morin, D; Buckpitt, AR; Edwards, PC; Van Winkle, LS

    2016-01-01

    Naphthalene (NA) is a ubiquitous pollutant to which humans are widely exposed. 1,2-Dihydro-1,2-dihydroxynaphthalene (NA-dihydrodiol) is a major metabolite of NA generated by microsomal epoxide hydrolase (mEH). To investigate the role of the NA-dihydrodiol and subsequent metabolites (ie 1,2-naphthoquinone) in cytotoxicity, we exposed both male and female wild type (WT) and mEH null mice (KO) to NA by inhalation (5, 10, 20 ppm for 4 hours). NA-dihydrodiol was ablated in the KO mice. High-resolution histopathology was used to study site-specific cytotoxicity, and formation of naphthalene metabolites was measured by HPLC in microdissected airways. Swollen and vacuolated airway epithelial cells were observed in the intra- and extrapulmonary airways of all mice at and below the current OSHA standard (10 ppm). Female mice may be more susceptible to this acute cytotoxicity. In the extrapulmonary airways, WT mice were more susceptible to damage than KO mice, indicating that the metabolites associated with mEH-mediated metabolism could be partially responsible for cytotoxicity at this site. The level of cytotoxicity in the mEH KO mice at all airway levels suggests that non-mEH metabolites are contributing to NA cellular damage in the lung. Our results indicate that the apparent contribution of mEH-dependent metabolites to toxicity differs by location in the lung. These studies suggest that metabolites generated through the mEH pathway may be of minor importance in distal airway toxicity and subsequent carcinogenesis from NA exposure. PMID:26840748

  1. Naphthalene cytotoxicity in microsomal epoxide hydrolase deficient mice.

    PubMed

    Carratt, S A; Morin, D; Buckpitt, A R; Edwards, P C; Van Winkle, L S

    2016-03-30

    Naphthalene (NA) is a ubiquitous pollutant to which humans are widely exposed. 1,2-Dihydro-1,2-dihydroxynaphthalene (NA-dihydrodiol) is a major metabolite of NA generated by microsomal epoxide hydrolase (mEH). To investigate the role of the NA-dihydrodiol and subsequent metabolites (i.e. 1,2-naphthoquinone) in cytotoxicity, we exposed both male and female wild type (WT) and mEH null mice (KO) to NA by inhalation (5, 10, 20 ppm for 4h). NA-dihydrodiol was ablated in the KO mice. High-resolution histopathology was used to study site-specific cytotoxicity, and formation of naphthalene metabolites was measured by HPLC in microdissected airways. Swollen and vacuolated airway epithelial cells were observed in the intra- and extrapulmonary airways of all mice at and below the current OSHA standard (10 ppm). Female mice may be more susceptible to this acute cytotoxicity. In the extrapulmonary airways, WT mice were more susceptible to damage than KO mice, indicating that the metabolites associated with mEH-mediated metabolism could be partially responsible for cytotoxicity at this site. The level of cytotoxicity in the mEH KO mice at all airway levels suggests that non-mEH metabolites are contributing to NA cellular damage in the lung. Our results indicate that the apparent contribution of mEH-dependent metabolites to toxicity differs by location in the lung. These studies suggest that metabolites generated through the mEH pathway may be of minor importance in distal airway toxicity and subsequent carcinogenesis from NA exposure. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  2. 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.

  3. Reaction Pathway for Cocaine Hydrolase-Catalyzed Hydrolysis of (+)-Cocaine

    PubMed Central

    Yao, Yuan; Liu, Junjun; Zheng, Fang; Zhan, Chang-Guo

    2017-01-01

    A recently designed and discovered cocaine hydrolase (CocH), engineered from human butyrylcholinesterase (BChE), has been proven promising as a novel enzyme therapy for treatment of cocaine overdose and addiction because it is highly efficient in catalyzing hydrolysis of naturally occurring (−)-cocaine. It has been known that the CocH also has a high catalytic efficiency against (+)-cocaine, a synthetic enantiomer of cocaine. Reaction pathway and the corresponding free energy profile for the CocH-catalyzed hydrolysis of (+)-cocaine have been determined, in the present study, by performing first-principles pseudobond quantum mechanical/molecular mechanical (QM/MM)-free energy (FE) calculations. Acordingt to the QM/MM-FE results, the catalytic hydrolysis process is initiated by the nucleophilic attack on carbonyl carbon of (−)-cocaine benzoyl ester via hydroxyl oxygen of S198 side chain, and the second reaction step (i.e. dissociation of benzoyl ester) is rate-determining. This finding for CocH-catalyzed hydrolysis of (+)-cocaine is remarkably different from that for the (+)-cocaine hydrolysis catalyzed by bacterial cocaine esterase in which the first reaction step of the deacylation is associated with the highest free energy barrier (~17.9 kcal/mol). The overall free energy barrier (~16.0 kcal/mol) calculated for the acylation stage of CocH-catalyzed hydrolysis of (+)-cocaine is in good agreement with the experimental free energy barrier of ~14.5 kcal/mol derivated from the experimental kinetic data. PMID:28250715

  4. Surface recombination in semiconductors

    SciTech Connect

    Langer, J.M.; Walukiewicz, W.

    1995-07-01

    We propose two general criteria for a surface defect state to act as an efficient, nonradiative recombination center. The first is that the thermal ionization energy should not deviate from the mid-gap energy by more than the relaxation energy of the defect, In this case the activation energy for the recombination is given by the barrier for the capture of the first carrier, whereas the second carrier is captured athermally. The second citerion is related to the position of the average dangling bond energy relative to the band edges. If, as in the cases of InP or InAs, it is located close to a band edge, a low surface recombination velocity is expected. However a much faster recombination is predicated and experimentally observed in the materials with the average dangling bond energy located close to the mid-gap. The relevance of these criteria for the novel wide-gap optoelectronic materials is discussed.

  5. Multiphoton Assisted Recombination

    NASA Astrophysics Data System (ADS)

    Shuman, E. S.; Jones, R. R.; Gallagher, T. F.

    2008-12-01

    We have observed multiphoton assisted recombination in the presence of a 38.8 GHz microwave field. Stimulated emission of up to ten microwave photons results in energy transfer from continuum electrons, enabling recombination. The maximum electron energy loss is far greater than the 2Up predicted by the standard “simpleman’s” model. The data are well reproduced by both an approximate analytic expression and numerical simulations in which the combined Coulomb and radiation fields are taken into account.

  6. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, C.W.; Mangel, W.F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

  7. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, Carl W.; Mangel, Walter F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  8. Recombination and chromosome segregation.

    PubMed Central

    Sherratt, David J; Søballe, Britta; Barre, François-Xavier; Filipe, Sergio; Lau, Ivy; Massey, Thomas; Yates, James

    2004-01-01

    The duplication of DNA and faithful segregation of newly replicated chromosomes at cell division is frequently dependent on recombinational processes. The rebuilding of broken or stalled replication forks is universally dependent on homologous recombination proteins. In bacteria with circular chromosomes, crossing over by homologous recombination can generate dimeric chromosomes, which cannot be segregated to daughter cells unless they are converted to monomers before cell division by the conserved Xer site-specific recombination system. Dimer resolution also requires FtsK, a division septum-located protein, which coordinates chromosome segregation with cell division, and uses the energy of ATP hydrolysis to activate the dimer resolution reaction. FtsK can also translocate DNA, facilitate synapsis of sister chromosomes and minimize entanglement and catenation of newly replicated sister chromosomes. The visualization of the replication/recombination-associated proteins, RecQ and RarA, and specific genes within living Escherichia coli cells, reveals further aspects of the processes that link replication with recombination, chromosome segregation and cell division, and provides new insight into how these may be coordinated. PMID:15065657

  9. 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

  10. Evaluation of NHS carbamates as a potent and selective class of endocannabinoid hydrolase inhibitors.

    PubMed

    Niphakis, Micah J; Cognetta, Armand B; Chang, Jae Won; Buczynski, Matthew W; Parsons, Loren H; Byrne, Frederika; Burston, James J; Chapman, Victoria; Cravatt, Benjamin F

    2013-09-18

    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.

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

    SciTech Connect

    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 product 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.

  12. Phosphonoacetic acid utilization by fungal isolates: occurrence and properties of a phosphonoacetate hydrolase in some penicillia.

    PubMed

    Forlani, Giuseppe; Klimek-Ochab, Magdalena; Jaworski, Jakub; Lejczak, Barbara; Picco, Anna M

    2006-12-01

    Among a collection of 18 fungal strains representing eight genera, only two strains (Penicillium oxalicum and P. minioluteum) were capable of growth on phosphonoacetic acid as sole phosphorous source. Enrichment liquid cultures in minimal medium with the compound as the only P-source selected four isolates, that were also identified as Penicillium spp. Phosphonoacetate metabolism did not lead to extracellular release of inorganic phosphate. In all cases phosphonoacetate hydrolase activity was detected in partially purified extracts, and a protein of the expected molecular mass reacted with polyclonal antibodies raised against the enzyme from P. oxalicum. There was no relation between phosphonoacetate hydrolase specific activity and growth rate or yield. Phosphonoacetic acid was the inducer of the hydrolase, independently of the concurrent availability of inorganic phosphate. Notwithstanding this, the utilization of the phosphonate was significantly inhibited in the presence of phosphate, suggesting an interference of the latter with phosphonoacetic acid uptake.

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

    PubMed

    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

    2016-03-01

    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 product 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. The theoretical calculations provide a guide for future experimental studies designed to produce mutant cellulases with enhanced activity.

  14. Molecular cloning and amino acid sequence of leukotriene A4 hydrolase.

    PubMed Central

    Funk, C D; Rådmark, O; Fu, J Y; Matsumoto, T; Jörnvall, H; Shimizu, T; Samuelsson, B

    1987-01-01

    A cDNA clone corresponding to leukotriene A4 hydrolase was isolated from a human lung lambda gt11 expression library by immunoscreening with a polyclonal antiserum. Several additional clones from human lung and placenta cDNA lambda g11 libraries were obtained by plaque hybridization with the 32P-labeled lung cDNA clone. One of these clones has an insert of 1910 base pairs that contains the complete protein-coding region. From the deduced primary structure, leukotriene A4 hydrolase is a 610 amino and protein with a calculated molecular weight of 69,140. No apparent homologies with microsomal epoxide hydrolases were found. RNA blot analysis indicated substantial amounts of a discrete mRNA of approximately equal to 2250 nucleotides in lung tissue and leukocytes. Images PMID:2821541

  15. Murein hydrolase activity of surface layer proteins from Lactobacillus acidophilus against Escherichia coli.

    PubMed

    Meng, Jun; Gao, Shu-Ming; Zhang, Qiu-Xiang; Lu, Rong-Rong

    2015-08-01

    The aim of this study was to investigate the murein hydrolase activities of the surface layer proteins (SLPs) from two strains of Lactobacillus acidophilus using zymography. The influence of these hydrolase activities on Escherichia coli ATCC 43893 was also evaluated by analysing their growth curve, cell morphology and physiological state. After the incubation of E. coli with SLPs, growth was inhibited, the number of viable cells was significantly reduced, examination by transmission electron microscopy showed that the cell wall was damaged and flow cytometry results indicated that the majority of the cells were sublethally injured. All of these results suggested that the SLPs of both L. acidophilus strains possessed murein hydrolase activities that were sublethal to E. coli cells. Copyright © 2015. Published by Elsevier B.V.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  17. Cloning and expression of diadenosine 5',5'''-P1,P4-tetraphosphate hydrolase from Lupinus angustifolius L.

    PubMed Central

    Maksel, D; Guranowski, A; Ilgoutz, S C; Moir, A; Blackburn, M G; Gayler, K R

    1998-01-01

    The first isolation, cloning and expression of cDNA encoding an asymmetric diadenosine 5',5'''P1,P4-tetraphosphate pyrophosphohydrolase (Ap4A hydrolase) from a higher plant is described. Ap4A hydrolase protein was purified from seeds of both Lupinus luteus and Lupinus angustifolius and partially sequenced. The Ap4A hydrolase cDNA was cloned from L. angustifolius cotyledonary polyadenylated RNA using reverse transcription and PCR with primers based on the amino acid sequence. The cDNA encoded a protein of 199 amino acids, molecular mass 22982Da. When expressed in Escherichia coli fused to a maltose-binding protein, the enzyme catalysed asymmetric cleavage of Ap4A to AMP and ATP which was inhibited at concentrations of F- as low as 3 microM. These are properties characteristic of Ap4A hydrolase (asymmetrical) (EC 3.6.1. 17). Comparison of the Ap4A hydrolase sequences derived from the four known cDNAs from pig, human, lupin and fission yeast showed that, like the mammalian hydrolase, the lupin enzyme possesses a Mut T motif but no other significant similarities. No sequence similarity to the human fragile histidine triad protein, as found in the Ap4A hydrolase from Schizosaccharomyces pombe, was detected in the Ap4A hydrolase from lupin. PMID:9425114

  18. A Novel Lipid Droplet-Associated Serine Hydrolase Regulates Macrophage Cholesterol Mobilization

    PubMed Central

    Goo, Young-Hwa; Son, Se-Hee; Kreienberg, Paul B.; Paul, Antoni

    2014-01-01

    Objective Lipid-laden macrophages or foam cells are characterized by massive cytosolic lipid droplet (LD) deposition containing mostly cholesterol ester (CE) derived from the lipoproteins cleared from the arterial wall. Cholesterol efflux from foam cells is considered to be atheroprotective. Since cholesterol is effluxed as free cholesterol (FC), CE accumulation in LDs may limit FC efflux. Our objective was to identify proteins that regulate cholesterol trafficking through LDs. Approach and results In a proteomic analysis of the LD fraction of RAW 264.7 macrophages we identified an evolutionarily conserved protein with a canonical GXSXG lipase catalytic motif and a predicted α/β-hydrolase fold, the RIKEN cDNA 1110057K04 gene, which we named lipid droplet-associated hydrolase (LDAH). LDAH association to LDs was confirmed by immunoblotting and immunocytochemistry. LDAH was labeled with a probe specific for active serine hydrolases. LDAH showed relatively weak in vitro CE hydrolase activity. However, cholesterol measurements in intact cells supported a significant role of LDAH in CE homeostasis, since LDAH upregulation and downregulation decreased and increased, respectively, intracellular cholesterol and CE in HEK293 cells and RAW 264.7 macrophages. Mutation of the putative nucleophilic serine impaired active hydrolase probe binding, in vitro CE hydrolase activity, and the cholesterol lowering effect in cells, while this mutant still localized to the LD. LDAH upregulation increased CE hydrolysis and cholesterol efflux from macrophages and, interestingly, LDAH is highly expressed in macrophage-rich areas within mouse and human atherosclerotic lesions. Conclusions The data identify a candidate target to promote reverse cholesterol transport from atherosclerotic lesions. PMID:24357060

  19. Congenital hypothyroidism mutations affect common folding and trafficking in the α/β-hydrolase fold proteins.

    PubMed

    De Jaco, Antonella; Dubi, Noga; Camp, Shelley; Taylor, Palmer

    2012-12-01

    The α/β-hydrolase fold superfamily of proteins is composed of structurally related members that, despite great diversity in their catalytic, recognition, adhesion and chaperone functions, share a common fold governed by homologous residues and conserved disulfide bridges. Non-synonymous single nucleotide polymorphisms within the α/β-hydrolase fold domain in various family members have been found for congenital endocrine, metabolic and nervous system disorders. By examining the amino acid sequence from the various proteins, mutations were found to be prevalent in conserved residues within the α/β-hydrolase fold of the homologous proteins. This is the case for the thyroglobulin mutations linked to congenital hypothyroidism. To address whether correct folding of the common domain is required for protein export, we inserted the thyroglobulin mutations at homologous positions in two correlated but simpler α/β-hydrolase fold proteins known to be exported to the cell surface: neuroligin3 and acetylcholinesterase. Here we show that these mutations in the cholinesterase homologous region alter the folding properties of the α/β-hydrolase fold domain, which are reflected in defects in protein trafficking, folding and function, and ultimately result in retention of the partially processed proteins in the endoplasmic reticulum. Accordingly, mutations at conserved residues may be transferred amongst homologous proteins to produce common processing defects despite disparate functions, protein complexity and tissue-specific expression of the homologous proteins. More importantly, a similar assembly of the α/β-hydrolase fold domain tertiary structure among homologous members of the superfamily is required for correct trafficking of the proteins to their final destination.

  20. 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.

  1. 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.

  2. A constitutive expression system for glycosyl hydrolase family 7 cellobiohydrolases in Hypocrea jecorina

    SciTech Connect

    Linger, Jeffrey G.; Taylor, II, Larry E.; Baker, John O.; Vander Wall, Todd; Hobdey, Sarah E.; Podkaminer, Kara; Himmel, Michael E.; Decker, Stephen R.

    2015-03-18

    One of the primary industrial-scale cellulase producers is the ascomycete fungus, Hypocrea jecorina, which produces and secretes large quantities of diverse cellulolytic enzymes. Perhaps the single most important biomass degrading enzyme is cellobiohydrolase I (cbh1or Cel7A) due to its enzymatic proficiency in cellulose depolymerization. However, production of Cel7A with native-like properties from heterologous expression systems has proven difficult. In this study, we develop a protein expression system in H. jecorina (Trichoderma reesei) useful for production and secretion of heterologous cellobiohydrolases from glycosyl hydrolase family 7. Building upon previous work in heterologous protein expression in filamentous fungi, we have integrated a native constitutive enolase promoter with the native cbh1 signal sequence. The results are the following: The constitutive eno promoter driving the expression of Cel7A allows growth on glucose and results in repression of the native cellulase system, severely reducing background endo- and other cellulase activity and greatly simplifying purification of the recombinant protein. Coupling this system to a Δcbh1 strain of H. jecorina ensures that only the recombinant Cel7A protein is produced. Two distinct transformant colony morphologies were observed and correlated with high and null protein production. Production levels in ‘fast’ transformants are roughly equivalent to those in the native QM6a strain of H. jecorina, typically in the range of 10 to 30 mg/L when grown in continuous stirred-tank fermenters. ‘Slow’ transformants showed no evidence of Cel7A production. Specific activity of the purified recombinant Cel7A protein is equivalent to that of native protein when assayed on pretreated corn stover, as is the thermal stability and glycosylation level. Purified Cel7A produced from growth on glucose demonstrated remarkably consistent specific activity. Purified Cel7A from the same strain grown on lactose

  3. Arachidonoylserotonin and other novel inhibitors of fatty acid amide hydrolase.

    PubMed

    Bisogno, T; Melck, D; De Petrocellis, L; Bobrov MYu; Gretskaya, N M; Bezuglov, V V; Sitachitta, N; Gerwick, W H; Di Marzo, V

    1998-07-30

    Fatty acid amide hydrolase (FAAH) catalyzes the hydrolysis of bioactive fatty acid amides and esters such as the endogenous cannabinoid receptor ligands, anandamide (N-arachidonoyl-ethanolamine) and 2-arachidonoylglycerol, and the putative sleep inducing factor cis-9-octadecenoamide (oleamide). Most FAAH blockers developed to date also inhibit cytosolic phospholipase A2 (cPLA2) and/or bind to the CB1 cannabinoid receptor subtype. Here we report the finding of four novel FAAH inhibitors, two of which, malhamensilipin A and grenadadiene, were screened out of a series of thirty-two different algal natural products, and two others, arachidonoylethylene glycol (AEG) and arachidonoyl-serotonin (AA-5-HT) were selected out of five artificially functionalized polyunsaturated fatty acids. When using FAAH preparations from mouse neuroblastoma N18TG2 cells and [14C]anandamide as a substrate, the IC50s for these compounds ranged from 12.0 to 26 microM, the most active compound being AA-5-HT. This substance was also active on FAAH from rat basophilic leukaemia (RBL-2H3) cells (IC50 = 5.6 microM), and inhibited [14C]anandamide hydrolysis by both N18TG2 and RBL-2H3 intact cells without affecting [14C]anandamide uptake. While AEG behaved as a competitive inhibitor and was hydrolyzed to arachidonic acid (AA) by FAAH preparations, AA-5-HT was resistant to FAAH-catalyzed hydrolysis and behaved as a tight-binding, albeit non-covalent, mixed inhibitor. AA-5-HT did not interfere with cPLA2-mediated, ionomycin or antigen-induced release of [3H]AA from RBL-2H3 cells, nor with cPLA2 activity in cell-free experiments. Finally, AA-5-HT did not activate CB1 cannabinoid receptors since it acted as a very weak ligand in in vitro binding assays, and, at 10-15 mg/kg body weight, it was not active in the 'open field', 'hot plate' and rectal hypothermia tests carried out in mice. Conversely AEG behaved as a cannabimimetic substance in these tests as well as in the 'ring' immobility test where AA-5

  4. 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

  5. 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

  6. 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.

  7. Regulation of Meiotic Recombination

    SciTech Connect

    Gregory p. Copenhaver

    2011-11-09

    Meiotic recombination results in the heritable rearrangement of DNA, primarily through reciprocal exchange between homologous chromosome or gene conversion. In plants these events are critical for ensuring proper chromosome segregation, facilitating DNA repair and providing a basis for genetic diversity. Understanding this fundamental biological mechanism will directly facilitate trait mapping, conventional plant breeding, and development of genetic engineering techniques that will help support the responsible production and conversion of renewable resources for fuels, chemicals, and the conservation of energy (1-3). Substantial progress has been made in understanding the basal recombination machinery, much of which is conserved in organisms as diverse as yeast, plants and mammals (4, 5). Significantly less is known about the factors that regulate how often and where that basal machinery acts on higher eukaryotic chromosomes. One important mechanism for regulating the frequency and distribution of meiotic recombination is crossover interference - or the ability of one recombination event to influence nearby events. The MUS81 gene is thought to play an important role in regulating the influence of interference on crossing over. The immediate goals of this project are to use reverse genetics to identify mutants in two putative MUS81 homologs in the model plant Arabidopsis thaliana, characterize those mutants and initiate a novel forward genetic screen for additional regulators of meiotic recombination. The long-term goal of the project is to understand how meiotic recombination is regulated in higher eukaryotes with an emphasis on the molecular basis of crossover interference. The ability to monitor recombination in all four meiotic products (tetrad analysis) has been a powerful tool in the arsenal of yeast geneticists. Previously, the qrt mutant of Arabidopsis, which causes the four pollen products of male meiosis to remain attached, was developed as a facile system

  8. Isolation and characterization of 9-lipoxygenase and epoxide hydrolase 2 genes: Insight into lactone biosynthesis in mango fruit (Mangifera indica L.).

    PubMed

    Deshpande, Ashish B; Chidley, Hemangi G; Oak, Pranjali S; Pujari, Keshav H; Giri, Ashok P; Gupta, Vidya S

    2017-06-01

    Uniqueness and diversity of mango flavour across various cultivars are well known. Among various flavour metabolites lactones form an important class of aroma volatiles in certain mango varieties due to their ripening specific appearance and lower odour detection threshold. In spite of their biological and biochemical importance, lactone biosynthetic pathway in plants remains elusive. Present study encompasses quantitative real-time analysis of 9-lipoxygenase (Mi9LOX), epoxide hydrolase 2 (MiEH2), peroxygenase, hydroperoxide lyase and acyl-CoA-oxidase genes during various developmental and ripening stages in fruit of Alphonso, Pairi and Kent cultivars with high, low and no lactone content and explains their variable lactone content. Study also covers isolation, recombinant protein characterization and transient over-expression of Mi9LOX and MiEH2 genes in mango fruits. Recombinant Mi9LOX utilized linoleic and linolenic acids, while MiEH2 utilized aromatic and fatty acid epoxides as their respective substrates depicting their role in fatty acid metabolism. Significant increase in concentration of δ-valerolactone and δ-decalactone upon Mi9LOX over-expression and that of δ-valerolactone, γ-hexalactone and δ-hexalactone upon MiEH2 over-expression further suggested probable involvement of these genes in lactone biosynthesis in mango.

  9. A Versatile Family 3 Glycoside Hydrolase from Bifidobacterium adolescentis Hydrolyzes β-Glucosides of the Fusarium Mycotoxins Deoxynivalenol, Nivalenol, and HT-2 Toxin in Cereal Matrices

    PubMed Central

    Varga, Elisabeth; Malachova, Alexandra; Nguyen, Nhung Thi; Lorenz, Cindy; Haltrich, Dietmar; Berthiller, Franz; Adam, Gerhard

    2015-01-01

    Glycosylation plays a central role in plant defense against xenobiotics, including mycotoxins. Glucoconjugates of Fusarium toxins, such as deoxynivalenol-3-O-β-d-glucoside (DON-3G), often cooccur with their parental toxins in cereal-based food and feed. To date, only limited information exists on the occurrence of glucosylated mycotoxins and their toxicological relevance. Due to a lack of analytical standards and the requirement of high-end analytical instrumentation for their direct determination, hydrolytic cleavage of β-glucosides followed by analysis of the released parental toxins has been proposed as an indirect determination approach. This study compares the abilities of several fungal and recombinant bacterial β-glucosidases to hydrolyze the model analyte DON-3G. Furthermore, substrate specificities of two fungal and two bacterial (Lactobacillus brevis and Bifidobacterium adolescentis) glycoside hydrolase family 3 β-glucosidases were evaluated on a broader range of substrates. The purified recombinant enzyme from B. adolescentis (BaBgl) displayed high flexibility in substrate specificity and exerted the highest hydrolytic activity toward 3-O-β-d-glucosides of the trichothecenes deoxynivalenol (DON), nivalenol, and HT-2 toxin. A Km of 5.4 mM and a Vmax of 16 μmol min−1 mg−1 were determined with DON-3G. Due to low product inhibition (DON and glucose) and sufficient activity in several extracts of cereal matrices, this enzyme has the potential to be used for indirect analyses of trichothecene-β-glucosides in cereal samples. PMID:25979885

  10. Potent Natural Soluble Epoxide Hydrolase Inhibitors from Pentadiplandra brazzeana Baillon: Synthesis, Quantification, and Measurement of Biological Activities In Vitro and In Vivo

    PubMed Central

    Kitamura, Seiya; Morisseau, Christophe; Inceoglu, Bora; Kamita, Shizuo G.; De Nicola, Gina R.; Nyegue, Maximilienne; Hammock, Bruce D.

    2015-01-01

    We describe here three urea-based soluble epoxide hydrolase (sEH) inhibitors from the root of the plant Pentadiplandra brazzeana. The concentration of these ureas in the root was quantified by LC-MS/MS, showing that 1, 3-bis (4-methoxybenzyl) urea (MMU) is the most abundant (42.3 μg/g dry root weight). All of the ureas were chemically synthesized, and their inhibitory activity toward recombinant human and recombinant rat sEH was measured. The most potent compound, MMU, showed an IC50 of 92 nM via fluorescent assay and a Ki of 54 nM via radioactivity-based assay on human sEH. MMU effectively reduced inflammatory pain in a rat nociceptive pain assay. These compounds are among the most potent sEH inhibitors derived from natural sources. Moreover, inhibition of sEH by these compounds may mechanistically explain some of the therapeutic effects of P. brazzeana. PMID:25659109

  11. Engineering cellulosic bioreactors by template assisted DNA shuffling and in vitro recombination (TADSir).

    PubMed

    Davis, Leroy K

    2014-10-01

    The current study focuses on development of a bioreactor engineering strategy based on exploitation of the Arabidopsis thaliana genome. Chimeric A. thaliana glycosyl hydrolase (GH) gene libraries were assembled using a novel directed evolution strategy (TADSir: template assisted DNA shuffling and in vitro recombination) that promotes DNA recombination by reassembly of DNA fragments on unique gene templates. TADSir was modeled using a set of algorithms designed to simulate DNA interactions based on nearest neighbor base stacking interactions and Gibb's free energy differences between helical coil and folded DNA states. The algorithms allow for target gene prediction and for in silica analysis of chimeric gene library composition. Further, the study investigated utilization of A. thaliana GH sequence space for bioreactor design by evolving 20 A. thaliana genes representing the GH1, GH3, GH5, GH9 and GH10 gene families. Notably, TADSir achieved streamlined engineering of Saccharomyces cerevisiae and spinach mesophyll protoplast bioreactors capable of processing CM cellulose, Avicel and xylan.

  12. Discovery of novel leukotriene A4 hydrolase inhibitors based on piperidine and piperazine scaffolds.

    PubMed

    Sandanayaka, Vincent; Mamat, Bjorn; Bhagat, Nikhil; Bedell, Louis; Halldorsdottir, Gudrun; Sigthorsdottir, Heida; Andrésson, Thorkell; Kiselyov, Alex; Gurney, Mark; Singh, Jasbir

    2010-05-01

    Novel piperidine and piperazine derivatives have been designed and tested as inhibitors of LTA(4) hydrolase (LTA(4)H). Most potent compounds showed good potency in both enzymatic and functional human whole blood assay. Crystallography studies further confirmed observed structure-activity relationship and LTA(4)H binding mode for analogs from the piperidine series.

  13. 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.

  14. Purification and properties of D(-)-3-hydroxybutyrate-dimer hydrolase from Zoogloea ramigera I-16-M.

    PubMed

    Tanaka, Y; Saito, T; Fukui, T; Tanio, T; Tomita, K

    1981-08-01

    D(-)-3-Hydroxybutyrate-dimer hydrolase from Zoogloea ramigera I-16-M was purified 7000-fold to electrophoretic homogeneity. The molecular weight of the purified enzyme was 28 000 as determined by Sephadex G-100 gel filtration, and 30 000 as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The isoelectric point was at pH 5.7. The pH optimum for the enzyme reaction was 8.0. The dimer hydrolase was stereospecific for D(-)-3-[D(-)-3-hydroxybutyryloxy]butyric acid (DD-dimer) but also hydrolyzed D(-)-3-[L(+)-3-hydroxybutyryloxy]butyric acid (DL-dimmer) and L(+)-3-[D(-)-3-hydroxybutyryloxy]butyric acid (LD-dimer) at reduced rates. However, the enzyme did not attack L(+)-3-[L(+)-3-hydroxybutyryloxy]butyric acid (LL-dimer) at all. In addition, the purified hydrolase hydrolyzed several oligomeric esters of D(-)-3-hydroxybutyric acid (DDD-dimer, DDDD-tetramer and DDDDD-pentamer) faster than DD-dimer. Time course experiments with these oligomers and analysis of hydrolytic products of DDD-tetramer methyl ester with the hydrolase indicated that the enzyme attached these substrates from the free hydroxyl terminus releasing monomer units one at a time.

  15. 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...

  16. O-hydroxyacetamide carbamates as a highly potent and selective class of endocannabinoid hydrolase inhibitors.

    PubMed

    Niphakis, Micah J; Johnson, Douglas S; Ballard, T Eric; Stiff, Cory; Cravatt, Benjamin F

    2012-05-16

    The two major endocannabinoid transmitters, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are degraded by distinct enzymes in the nervous system, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. FAAH and MAGL inhibitors cause elevations in brain AEA and 2-AG levels, respectively, and reduce pain, anxiety, and depression in rodents without causing the full spectrum of psychotropic behavioral effects observed with direct cannabinoid receptor-1 (CB1) agonists. These findings have inspired the development of several classes of endocannabinoid hydrolase inhibitors, most of which have been optimized to show specificity for either FAAH or MAGL or, in certain cases, equipotent activity for both enzymes. Here, we investigate an unusual class of O-hydroxyacetamide carbamate inhibitors and find that individual compounds from this class can serve as selective FAAH or dual FAAH/MAGL inhibitors in vivo across a dose range (0.125-12.5 mg kg(-1)) suitable for behavioral studies. Competitive and click chemistry activity-based protein profiling confirmed that the O-hydroxyacetamide carbamate SA-57 is remarkably selective for FAAH and MAGL in vivo, targeting only one other enzyme in brain, the additional 2-AG hydrolase ABHD6. These data designate O-hydroxyacetamide carbamates as a versatile chemotype for creating endocannabinoid hydrolase inhibitors that display excellent in vivo activity and tunable selectivity for FAAH-anandamide versus MAGL (and ABHD6)-2-AG pathways.

  17. O-Hydroxyacetamide Carbamates as a Highly Potent and Selective Class of Endocannabinoid Hydrolase Inhibitors

    PubMed Central

    2011-01-01

    The two major endocannabinoid transmitters, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are degraded by distinct enzymes in the nervous system, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. FAAH and MAGL inhibitors cause elevations in brain AEA and 2-AG levels, respectively, and reduce pain, anxiety, and depression in rodents without causing the full spectrum of psychotropic behavioral effects observed with direct cannabinoid receptor-1 (CB1) agonists. These findings have inspired the development of several classes of endocannabinoid hydrolase inhibitors, most of which have been optimized to show specificity for either FAAH or MAGL or, in certain cases, equipotent activity for both enzymes. Here, we investigate an unusual class of O-hydroxyacetamide carbamate inhibitors and find that individual compounds from this class can serve as selective FAAH or dual FAAH/MAGL inhibitors in vivo across a dose range (0.125–12.5 mg kg–1) suitable for behavioral studies. Competitive and click chemistry activity-based protein profiling confirmed that the O-hydroxyacetamide carbamate SA-57 is remarkably selective for FAAH and MAGL in vivo, targeting only one other enzyme in brain, the additional 2-AG hydrolase ABHD6. These data designate O-hydroxyacetamide carbamates as a versatile chemotype for creating endocannabinoid hydrolase inhibitors that display excellent in vivo activity and tunable selectivity for FAAH-anandamide versus MAGL (and ABHD6)-2-AG pathways. PMID:22860211

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

    USDA-ARS?s Scientific Manuscript database

    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...

  19. Lipid composition and acid hydrolase content of lamellar granules of fetal rat epidermis.

    PubMed

    Freinkel, R K; Traczyk, T N

    1985-10-01

    Lipids and acid hydrolases have been characterized in a subcellular fraction, enriched with lamellar granules (LG), derived from fetal rat epidermis. This fraction contains 23% glycosyl ceramides and ceramides, 15% free sterols, and 34% phospholipids. The lipid/protein ratio is 2.0. The sterols and sphingolipids were present in proportions similar to those previously reported in stratum corneum. These findings provide direct biochemical evidence for the widely accepted hypothesis that stratum corneum lipids are derived from exocytosis of lamellar granules into the intercellular space. The LG fraction was enriched in certain acid hydrolases including glucosidase, acid phosphatase, phospholipases A, and sphingomyelinase; other acid hydrolases, i.e., amino-glycosidases, glactosidase and aryl sulfatase (pH 5.5), and steroid sulfatase were not preferentially localized in this fraction. By modulation of phospholipids, glycolipids, and proteins in the membrane regions of stratum corneum, the acid hydrolases of LG may play a role relevant to the function and desquamation of stratum corneum.

  20. 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
    ...

  1. 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

  2. Ligand bound structures of a glycosyl hydrolase family 30 glucuronoxylan xylanohydrolase

    Treesearch

    Franz St. Johns; Jason C. Hurlbert; John D. Rice; James F. Preston; Edwin. Pozharski

    2011-01-01

    Xylanases of glycosyl hydrolase family 30 (GH30) have been shown to cleave β-1,4 linkages of 4-O-methylglucuronoxylan (MeGXn) as directed by the position along the xylan chain of an α-1,2-linked 4-O-methylglucuronate (MeGA) moiety. Complete hydrolysis of MeGXn by...

  3. 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...

  4. 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
    ...

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

    PubMed Central

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

    2011-01-01

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

  6. 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

  7. 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.

  8. 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

  9. 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.

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

    USDA-ARS?s Scientific Manuscript database

    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....

  11. Deconjugated Bile Salts Produced by Extracellular Bile-Salt Hydrolase-Like Activities from the Probiotic Lactobacillus johnsonii La1 Inhibit Giardia duodenalis In vitro Growth.

    PubMed

    Travers, Marie-Agnès; Sow, Cissé; Zirah, Séverine; Deregnaucourt, Christiane; Chaouch, Soraya; Queiroz, Rayner M L; Charneau, Sébastien; Allain, Thibault; Florent, Isabelle; Grellier, Philippe

    2016-01-01

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

  12. Deconjugated Bile Salts Produced by Extracellular Bile-Salt Hydrolase-Like Activities from the Probiotic Lactobacillus johnsonii La1 Inhibit Giardia duodenalis In vitro Growth

    PubMed Central

    Travers, Marie-Agnès; Sow, Cissé; Zirah, Séverine; Deregnaucourt, Christiane; Chaouch, Soraya; Queiroz, Rayner M. L.; Charneau, Sébastien; Allain, Thibault; Florent, Isabelle; Grellier, Philippe

    2016-01-01

    Giardiasis, currently considered a neglected disease, is caused by the intestinal protozoan parasite Giardia duodenalis and is widely spread in human as well as domestic and wild animals. The lack of appropriate medications and the spread of resistant parasite strains urgently call for the development of novel therapeutic strategies. Host microbiota or certain probiotic strains have the capacity to provide some protection against giardiasis. By combining biological and biochemical approaches, we have been able to decipher a molecular mechanism used by the probiotic strain Lactobacillus johnsonii La1 to prevent Giardia growth in vitro. We provide evidence that the supernatant of this strain contains active principle(s) not directly toxic to Giardia but able to convert non-toxic components of bile into components highly toxic to Giardia. By using bile acid profiling, these components were identified as deconjugated bile-salts. A bacterial bile-salt-hydrolase of commercial origin was able to mimic the properties of the supernatant. Mass spectrometric analysis of the bacterial supernatant identified two of the three bile-salt-hydrolases encoded in the genome of this probiotic strain. These observations document a possible mechanism by which L. johnsonii La1, by secreting, or releasing BSH-like activity(ies) in the vicinity of replicating Giardia in an environment where bile is present and abundant, can fight this parasite. This discovery has both fundamental and applied outcomes to fight giardiasis, based on local delivery of deconjugated bile salts, enzyme deconjugation of bile components, or natural or recombinant probiotic strains that secrete or release such deconjugating activities in a compartment where both bile salts and Giardia are present. PMID:27729900

  13. The Activation of Phytophthora Effector Avr3b by Plant Cyclophilin is Required for the Nudix Hydrolase Activity of Avr3b

    PubMed Central

    Kong, Guanghui; Zhao, Yao; Jing, Maofeng; Huang, Jie; Yang, Jin; Xia, Yeqiang; Kong, Liang; Ye, Wenwu; Xiong, Qin; Qiao, Yongli; Dong, Suomeng; Ma, Wenbo; Wang, Yuanchao

    2015-01-01

    Plant pathogens secrete an arsenal of effector proteins to impair host immunity. Some effectors possess enzymatic activities that can modify their host targets. Previously, we demonstrated that a Phytophthora sojae RXLR effector Avr3b acts as a Nudix hydrolase when expressed in planta; and this enzymatic activity is required for full virulence of P. sojae strain P6497 in soybean (Glycine max). Interestingly, recombinant Avr3b produced by E. coli does not have the hydrolase activity unless it was incubated with plant protein extracts. Here, we report the activation of Avr3b by a prolyl-peptidyl isomerase (PPIase), cyclophilin, in plant cells. Avr3b directly interacts with soybean cyclophilin GmCYP1, which activates the hydrolase activity of Avr3b in a PPIase activity-dependent manner. Avr3b contains a putative Glycine-Proline (GP) motif; which is known to confer cyclophilin-binding in other protein substrates. Substitution of the Proline (P132) in the putative GP motif impaired the interaction of Avr3b with GmCYP1; as a result, the mutant Avr3bP132A can no longer be activated by GmCYP1, and is also unable to promote Phytophthora infection. Avr3b elicits hypersensitive response (HR) in soybean cultivars producing the resistance protein Rps3b, but Avr3bP132A lost its ability to trigger HR. Furthermore, silencing of GmCYP1 rendered reduced cell death triggered by Avr3b, suggesting that GmCYP1-mediated Avr3b maturation is also required for Rps3b recognition. Finally, cyclophilins of Nicotiana benthamiana can also interact with Avr3b and activate its enzymatic activity. Overall, our results demonstrate that cyclophilin is a “helper” that activates the enzymatic activity of Avr3b after it is delivered into plant cells; as such, cyclophilin is required for the avirulence and virulence functions of Avr3b. PMID:26317500

  14. [Construction of multifunctional genetically engineered pesticides-degrading bacteria by homologous recombination].

    PubMed

    Jiang, Jian-Dong; Gu, Li-Feng; Sun, Ji-Quan; Dai, Xian-Zhu; Wen, Yang; Li, Shun-Peng

    2005-11-01

    Construction of multifunctional pesticides-degrading genetically engineered microorganisms (GEMs) is increasing important in the bioremediation of various pesticides contaminants in environment. However, construction of genetically stable GEMs without any exogenous antibiotic resistance is thought to be one of the bottlenecks in GEMs construction. In this article, homologous recombination vectors with the recipient's 16S rDNA as homologous recombination directing sequence (HRDS) and sacB gene as double crossover recombinants positive selective marker were firstly constructed. The methyl parathion hydroalse gene (mpd) was inserted into the 16S rDNA site of the carbofuran degrading strain Sphingomonas sp. CDS-1 by homologous recombination single crossover in the level of about 3.7 x 10-(7) - 6.8 x 10(-7). Multifunctional pesticides-degrading GEMs with one or two mpd genes inserted into the chromosome without any antibiotic marker were successfully constructed. The homologous recombination events were confirmed by PCR and southern blot methods. The obtained GEMs were genetically stable and could degrade methyl parathion and carbofuran simultaneously. The insertion of mpd gene into rrn site did not have any significant effect on recipient' s physiological and original degrading characteristics. The methyl parathion hydrolase (MPH) was expressed at a relatively high level in the recombinants and the recombinant MPH specific activity in cell lysate was higher than that of original bacterium (DLL-1) in every growth phase tested. The highest recombinant MPH specific activity was 6.22 mu/tg. In this article, we describe a first attempt to use rRNA-encoding regions of Sphingomonas strains as target site for expression of exogenous MPH, and constructed multifunctional pesticides degrading GEMs, which are genetically stable and promising for developing bioremediation strategies for the decontamination of pesticides polluted soils.

  15. 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

  16. 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.

  17. Post-synthetic modification of plant cell walls by expression of microbial hydrolases in the apoplast.

    PubMed

    Pogorelko, Gennady; Fursova, Oksana; Lin, Ming; Pyle, Eric; Jass, Johanna; Zabotina, Olga A

    2011-11-01

    The systematic creation of defined cell wall modifications in the model plant Arabidopsis thaliana by expression of microbial hydrolases with known specific activities is a promising approach to examine the impacts of cell wall composition and structure on both plant fitness and cell wall recalcitrance. Moreover, this approach allows the direct evaluation in living plants of hydrolase specificity, which can differ from in vitro specificity. To express genes encoding microbial hydrolases in A. thaliana, and target the hydrolases to the apoplast compartment, we constructed an expression cassette composed of the Cauliflower Mosaic Virus 35S RNA promoter, the A. thaliana β-expansin signal peptide, and the fluorescent marker protein YFP. Using this construct we successfully introduced into Colombia-0 plants three Aspergillus nidulans hydrolases, β-xylosidase/α-arabinosidase, feruloyl esterase, acetylxylan esterase, and a Xanthomonas oryzae putative a-L: -arabinofuranosidase. Fusion with YFP permitted quick and easy screening of transformants, detection of apoplastic localization, and protein size confirmation. Compared to wild-type Col-0, all transgenic lines showed a significant increase in the corresponding hydrolytic activity in the apoplast and changes in cell wall composition. Examination of hydrolytic activity in the transgenic plants also showed, for the first time, that the X. oryzae gene indeed encoded an enzyme with α-L: -arabinofuranosidase activity. None of the transgenic plants showed a visible phenotype; however, the induced compositional changes increased the degradability of biomass from plants expressing feruloyl esterase and β-xylosidase/α-arabinosidase. Our results demonstrate the viability of creating a set of transgenic A. thaliana plants with modified cell walls to use as a toolset for investigation of how cell wall composition contributes to recalcitrance and affects plant fitness.

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

    SciTech Connect

    Lundeen, S.G.; Savage, D.C. )

    1990-08-01

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

  19. Supplementing with Non-Glycoside Hydrolase Proteins Enhances Enzymatic Deconstruction of Plant Biomass

    PubMed Central

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

    2012-01-01

    The glycoside hydrolases (GH) of Caldicellulosiruptor bescii are thermophilic enzymes, and therefore they can hydrolyze plant cell wall polysaccharides at high temperatures. Analyses of two C. bescii glycoside hydrolases, CbCelA-TM1 and CbXyn10A with cellulase and endoxylanase activity, respectively, demonstrated that each enzyme is highly thermostable under static incubation at 70°C. Both enzymes, however, rapidly lost their enzymatic activities when incubated at 70°C with end-over-end shaking. Since crowding conditions, even at low protein concentrations, seem to influence enzymatic properties, three non-glycoside hydrolase proteins were tested for their capacity to stabilize the thermophilic proteins at high temperatures. The three proteins investigated were a small heat shock protein CbHsp18 from C. bescii, a histone MkHistone1 from Methanopyrus kandleri, and bovine RNase A, from a commercial source. Fascinatingly, each of these proteins increased the thermostability of the glycoside hydrolases at 70°C during end-over-end shaking incubation, and this property translated into increases in hydrolysis of several substrates including the bioenergy feedstock Miscanthus. Furthermore, MkHistone1 and RNase A also altered the initial products released from the cello-oligosaccharide cellopentaose during hydrolysis with the cellodextrinase CbCdx1A, which further demonstrated the capacity of the three non-GH proteins to influence hydrolysis of substrates by the thermophilic glycoside hydrolases. The non-GH proteins used in the present report were small proteins derived from each of the three lineages of life, and therefore expand the space from which different polypeptides can be tested for their influence on plant cell wall hydrolysis, a critical step in the emerging biofuel industry. PMID:22952777

  20. Recombinant renewable polyclonal antibodies.

    PubMed

    Ferrara, Fortunato; D'Angelo, Sara; Gaiotto, Tiziano; Naranjo, Leslie; Tian, Hongzhao; Gräslund, Susanne; Dobrovetsky, Elena; Hraber, Peter; Lund-Johansen, Fridtjof; Saragozza, Silvia; Sblattero, Daniele; Kiss, Csaba; Bradbury, Andrew R M

    2015-01-01

    Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products.

  1. The dissociative recombination of ?

    NASA Astrophysics Data System (ADS)

    Laubé, S.; Lehfaoui, L.; Rowe, B. R.; Mitchell, J. B. A.

    1998-09-01

    The dissociative recombination rate coefficient for 0953-4075/31/18/016/img2 has been measured at 300 K using a flowing afterglow Langmuir probe-mass spectrometer apparatus. A value of 0953-4075/31/18/016/img3 has been found.

  2. Introduction to dissociative recombination

    NASA Technical Reports Server (NTRS)

    Guberman, Steven L.; Mitchell, J. Brian A.

    1989-01-01

    Dissociative recombination (DR) of molecular ions with electrons has important consequences in many areas of physical science. Ab-initio calculations coupled with resonant scattering theory and multichannel quantum defect studies have produced detailed results illuminating the role of ion vibrational excitation, the quantum yields of the DR products, and the role of Rydberg states. The theoretical and experimental results are discussed.

  3. Recombinant renewable polyclonal antibodies

    PubMed Central

    Ferrara, Fortunato; D’Angelo, Sara; Gaiotto, Tiziano; Naranjo, Leslie; Tian, Hongzhao; Gräslund, Susanne; Dobrovetsky, Elena; Hraber, Peter; Lund-Johansen, Fridtjof; Saragozza, Silvia; Sblattero, Daniele; Kiss, Csaba; Bradbury, Andrew RM

    2015-01-01

    Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products. PMID:25530082

  4. Recombinant DNA for Teachers.

    ERIC Educational Resources Information Center

    Duvall, James G., III

    1992-01-01

    A science teacher describes his experience at a workshop to learn to teach the Cold Spring Harbor DNA Science Laboratory Protocols. These protocols lead students through processes for taking E. coli cells and transforming them into a new antibiotic resistant strain. The workshop featured discussions of the role of DNA recombinant technology in…

  5. Recombineering linear BACs.

    PubMed

    Chen, Qingwen; Narayanan, Kumaran

    2015-01-01

    Recombineering is a powerful genetic engineering technique based on homologous recombination that can be used to accurately modify DNA independent of its sequence or size. One novel application of recombineering is the assembly of linear BACs in E. coli that can replicate autonomously as linear plasmids. A circular BAC is inserted with a short telomeric sequence from phage N15, which is subsequently cut and rejoined by the phage protelomerase enzyme to generate a linear BAC with terminal hairpin telomeres. Telomere-capped linear BACs are protected against exonuclease attack both in vitro and in vivo in E. coli cells and can replicate stably. Here we describe step-by-step protocols to linearize any BAC clone by recombineering, including inserting and screening for presence of the N15 telomeric sequence, linearizing BACs in vivo in E. coli, extracting linear BACs, and verifying the presence of hairpin telomere structures. Linear BACs may be useful for functional expression of genomic loci in cells, maintenance of linear viral genomes in their natural conformation, and for constructing innovative artificial chromosome structures for applications in mammalian and plant cells.

  6. Recombinant DNA for Teachers.

    ERIC Educational Resources Information Center

    Duvall, James G., III

    1992-01-01

    A science teacher describes his experience at a workshop to learn to teach the Cold Spring Harbor DNA Science Laboratory Protocols. These protocols lead students through processes for taking E. coli cells and transforming them into a new antibiotic resistant strain. The workshop featured discussions of the role of DNA recombinant technology in…

  7. 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.

  8. Cel5I, a SLH-Containing Glycoside Hydrolase: Characterization and Investigation on Its Role in Ruminiclostridium cellulolyticum

    PubMed Central

    Franche, Nathalie; Tardif, Chantal; Ravachol, Julie; Harchouni, Seddik; Ferdinand, Pierre-Henri; Borne, Romain; Fierobe, Henri-Pierre; Perret, Stéphanie

    2016-01-01

    Ruminiclostridium cellulolyticum (Clostridium cellulolyticum) is a mesophilic cellulolytic anaerobic bacterium that produces a multi-enzymatic system composed of cellulosomes and non-cellulosomal enzymes to degrade plant cell wall polysaccharides. We characterized one of the non-cellulosomal enzymes, Cel5I, composed of a Family-5 Glycoside Hydrolase catalytic module (GH5), a tandem of Family-17 and -28 Carbohydrate Binding Modules (CBM), and three S-layer homologous (SLH) modules, where the latter are expected to anchor the protein on the cell surface. Cel5I is the only putative endoglucanase targeting the cell surface as well as the only putative protein in R. cellulolyticum containing CBM17 and/or CBM28 modules. We characterized different recombinant structural variants from Cel5I. We showed that Cel5I has an affinity for insoluble cellulosic substrates through its CBMs, that it is the most active endoglucanase on crystalline cellulose of R. cellulolyticum characterized to date and mostly localized in the cell envelope of R. cellulolyticum. Its role in vivo was analyzed using a R. cellulolyticum cel5I mutant strain. Absence of Cel5I in the cell envelope did not lead to a significant variation of the phenotype compared to the wild type strain. Neither in terms of cell binding to cellulose, nor for its growth on crystalline cellulose, thus indicating that the protein has a rather subtle role in tested conditions. Cel5I might be more important in a natural environment, at low concentration of degradable glucose polymers, where its role might be to generate higher concentration of short cellodextrins close to the cell surface, facilitating their uptake or for signalization purpose. PMID:27501457

  9. Cloning and identification of novel hydrolase genes from a dairy cow rumen metagenomic library and characterization of a cellulase gene

    PubMed Central

    2012-01-01

    Background Interest in cellulose degrading enzymes has increased in recent years due to the expansion of the cellulosic biofuel industry. The rumen is a highly adapted environment for the degradation of cellulose and a promising source of enzymes for industrial use. To identify cellulase enzymes that may be of such use we have undertaken a functional metagenomic screen to identify cellulase enzymes from the bacterial community in the rumen of a grass-hay fed dairy cow. Results Twenty five clones specifying cellulose activity were identified. Subcloning and sequence analysis of a subset of these hydrolase-positive clones identified 10 endoglucanase genes. Preliminary characterization of the encoded cellulases was carried out using crude extracts of each of the subclones. Zymogram analysis using carboxymethylcellulose as a substrate showed a single positive band for each subclone, confirming that only one functional cellulase gene was present in each. One cellulase gene, designated Cel14b22, was expressed at a high level in Escherichia coli and purified for further characterization. The purified recombinant enzyme showed optimal activity at pH 6.0 and 50°C. It was stable over a broad pH range, from pH 4.0 to 10.0. The activity was significantly enhanced by Mn2+ and dramatically reduced by Fe3+ or Cu2+. The enzyme hydrolyzed a wide range of beta-1,3-, and beta-1,4-linked polysaccharides, with varying activities. Activities toward microcrystalline cellulose and filter paper were relatively high, while the highest activity was toward Oat Gum. Conclusion The present study shows that a functional metagenomic approach can be used to isolate previously uncharacterized cellulases from the rumen environment. PMID:23062472

  10. Characterization and crystal structure of a thermostable glycoside hydrolase family 45 1,4-β-endoglucanase from Thielavia terrestris.

    PubMed

    Gao, Jian; Huang, Jian-Wen; Li, Qian; Liu, Weidong; Ko, Tzu-Ping; Zheng, Yingying; Xiao, Xiansha; Kuo, Chih-Jung; Chen, Chun-Chi; Guo, Rey-Ting

    2017-04-01

    1,4-β-Endoglucanase is one of the most important biocatalysts in modern industries. Here, a glycoside hydrolase (GH) family 45 endoglucanase from thermophilic fungus Theilavia terrestris (TtCel45A) was expressed in Pichia pastoris. The recombinant protein shows optimal activity at 60°C, pH 4-5. The enzyme exhibits extraordinary thermostability that more than 80% activity was detected after heating at 80°C for 2.5h. The high resolution crystal structures of apo-form enzyme and that in complex with cellobiose and cellotetraose were solved to 1.36-1.58Å. The protein folds into two overall regions: one is a six-stranded β-barrel, and the other one consists of several extended loops. Between the two regions lies the substrate-binding channel, which is an open cleft spanning across the protein surface. A continuous substrate-binding cleft from subsite -4 to +3 were clearly identified in the complex structures. Notably, the flexible V-VI loop ((113)Gly-(114)Gly-(115)Asp-(116)Leu-(117)Gly-(118)Ser) is found to open in the presence of -1 sugar, with D115 and L116 swung away to yield a space to accommodate the catalytic acid D122 and the (2,5)B boat conformation of -1 sugar during transition state. Collectively, we characterized the enzyme properties of P. pastoris-expressed TtCel45A and solved high-resolution crystal structures of the enzyme. These results are of great interests in industrial applications and provide new insights into the fundamental understanding of enzyme catalytic mechanism of GH45 endoglucanases. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Occurrence of glycosidically conjugated 1-phenylethanol and its hydrolase β-primeverosidase in tea (Camellia sinensis) flowers.

    PubMed

    Zhou, Ying; Dong, Fang; Kunimasa, Aiko; Zhang, Yuqian; Cheng, Sihua; Lu, Jiamin; Zhang, Ling; Murata, Ariaki; Mayer, Frank; Fleischmann, Peter; Watanabe, Naoharu; Yang, Ziyin

    2014-08-13

    A previous study found that 1-phenylethanol (1PE) was a major endogenous volatile compound in tea (Camellia sinensis) flowers and can be transformed to glycosically conjugated 1PE (1PE-Gly). However, occurrences of 1PE-Gly in plants remain unknown. In this study, four 1PE-Glys have been isolated from tea flowers. Three of them were determined as (R)-1PE β-d-glucopyranoside ((R)-1PE-Glu), (S)-1PE-Glu, and (S)-1PE β-primeveroside ((S)-1PE-Pri), respectively, on the basis of NMR, MS, LC-MS, and GC-MS evidence. The other one was identified as (R)-1PE-Pri on the basis of LC-MS and GC-MS data. Moreover, these 1PE-Glys were chemically synthesized as the authentic standards to further confirm their occurrences in tea flowers. 1PE-Glu had a higher molar concentration than 1PE-Pri in each floral stage and organ. The ratio of (R) to (S) differed between 1PE-Glu and 1PE-Pri. In addition, a 1PE-Gly hydrolase β-primeverosidase recombinant protein produced in Escherichia coli exhibited high hydrolysis activity toward (R)-1PE-Pri. However, β-primeverosidase transcript level was not highly expressed in the anther part, which accumulated the highest contents of 1PE-Gly and 1PE. This suggests that 1PE-Gly may not be easily hydrolyzed to liberate 1PE in tea flowers. This study provides evidence of occurrences of 1PE-Glys in plants for the first time.

  12. Three Bacillus cereus bacteriophage endolysins are unrelated but reveal high homology to cell wall hydrolases from different bacilli.

    PubMed

    Loessner, M J; Maier, S K; Daubek-Puza, H; Wendlinger, G; Scherer, S

    1997-05-01

    The ply genes encoding the endolysin proteins from Bacillus cereus phages Bastille, TP21, and 12826 were identified, cloned, and sequenced. The endolysins could be overproduced in Escherichia coli (up to 20% of total cellular protein), and the recombinant proteins were purified by a two-step chromatographical procedure. All three enzymes induced rapid and specific lysis of viable cells of several Bacillus species, with highest activity on B. cereus and B. thuringiensis. Ply12 and Ply21 were experimentally shown to be N-acetylmuramoyl-L-alanine amidases (EC 3.5.1.28). No apparent holin genes were found adjacent to the ply genes. However, Ply21 may be endowed with a signal peptide which could play a role in timing of cell lysis by the cytoplasmic phage endolysin. The individual lytic enzymes (PlyBa, 41.1 kDa; Ply21, 29.5 kDa, Ply12, 27.7 kDa) show remarkable heterogeneity, i.e., their amino acid sequences reveal only little homology. The N-terminal part of Ply21 was found to be almost identical to the catalytic domains of a Bacillus sp. cell wall hydrolase (CwlSP) and an autolysin of B. subtilis (CwlA). The C terminus of PlyBa contains a 77-amino-acid sequence repeat which is also homologous to the binding domain of CwlSP. Ply12 shows homology to the major autolysins from B. subtilis and E. coli. Comparison with database sequences indicated a modular organization of the phage lysis proteins where the enzymatic activity is located in the N-terminal region and the C-termini are responsible for specific recognition and binding of Bacillus peptidoglycan. We speculate that the close relationship of the phage enzymes and cell wall autolysins is based upon horizontal gene transfer among different Bacillus phages and their hosts.

  13. High genetic diversity and different distributions of glycosyl hydrolase family 10 and 11 xylanases in the goat rumen.

    PubMed

    Wang, Guozeng; Luo, Huiying; Meng, Kun; Wang, Yaru; Huang, Huoqing; Shi, Pengjun; Pan, Xia; Yang, Peilong; Diao, Qiyu; Zhang, Hongfu; Yao, Bin

    2011-02-03

    The rumen harbors a complex microbial ecosystem for efficient hydrolysis of plant polysaccharides which are the main constituent of the diet. Xylanase is crucial for hemicellulose hydrolysis and plays an important role in the plant cell wall degradation. Xylanases of ruminal strains were widely studied, but few studies have focused on their diversity in rumen microenvironment. We explored the genetic diversity of xylanases belonging to two major glycosyl hydrolase families (GH 10 and 11) in goat rumen contents by analyzing the amplicons generated with two degenerate primer sets. Fifty-two distinct GH 10 and 35 GH 11 xylanase gene fragments (similarity <95%) were retrieved, and most had low identities with known sequences. Based on phylogenetic analysis, all GH 10 xylanase sequences fell into seven clusters, and 88.5% of them were related to xylanases from Bacteroidetes. Five clusters of GH 11 xylanase sequences were identified. Of these, 85.7% were related to xylanases from Firmicutes, and 14.3% were related to those of rumen fungi. Two full-length xylanase genes (one for each family) were directly cloned and expressed in Escherichia coli. Both the recombinant enzymes showed substantial xylanase activity, and were purified and characterized. Combined with the results of sheep rumen, Bacteroidetes and Firmicutes are the two major phyla of xylan-degrading microorganisms in rumen, which is distinct from the representatives of other environments such as soil and termite hindgut, suggesting that xylan-degrading microorganisms are environment specific. The numerous new xylanase genes suggested the functional diversity of xylanase in the rumen microenvironment which may have great potential applications in industry and agriculture. The phylogenetic diversity and different distributions of xylanase genes will help us understand their roles in plant cell wall degradation in the rumen microenvironment.

  14. Functional Diversity of Four Glycoside Hydrolase Family 3 Enzymes from the Rumen Bacterium Prevotella bryantii B14 ▿ †

    PubMed Central

    Dodd, Dylan; Kiyonari, Shinichi; Mackie, Roderick I.; Cann, Isaac K. O.

    2010-01-01

    Prevotella bryantii B14 is a member of the phylum Bacteroidetes and contributes to the degradation of hemicellulose in the rumen. The genome of P. bryantii harbors four genes predicted to encode glycoside hydrolase (GH) family 3 (GH3) enzymes. To evaluate whether these genes encode enzymes with redundant biological functions, each gene was cloned and expressed in Escherichia coli. Biochemical analysis of the recombinant proteins revealed that the enzymes exhibit different substrate specificities. One gene encoded a cellodextrinase (CdxA), and three genes encoded β-xylosidase enzymes (Xyl3A, Xyl3B, and Xyl3C) with different specificities for either para-nitrophenyl (pNP)-linked substrates or substituted xylooligosaccharides. To identify the amino acid residues that contribute to catalysis and substrate specificity within this family of enzymes, the roles of conserved residues (R177, K214, H215, M251, and D286) in Xyl3B were probed by site-directed mutagenesis. Each mutation led to a severely decreased catalytic efficiency without a change in the overall structure of the mutant enzymes. Through amino acid sequence alignments, an amino acid residue (E115) that, when mutated to aspartic acid, resulted in a 14-fold decrease in the kcat/Km for pNP-β-d-xylopyranoside (pNPX) with a concurrent 1.1-fold increase in the kcat/Km for pNP-β-d-glucopyranoside (pNPG) was identified. Amino acid residue E115 may therefore contribute to the discrimination between β-xylosides and β-glucosides. Our results demonstrate that each of the four GH3 enzymes has evolved to perform a specific role in lignopolysaccharide hydrolysis and provide insight into the role of active-site residues in catalysis and substrate specificity for GH3 enzymes. PMID:20190048

  15. Functional diversity of four glycoside hydrolase family 3 enzymes from the rumen bacterium Prevotella bryantii B14.

    PubMed

    Dodd, Dylan; Kiyonari, Shinichi; Mackie, Roderick I; Cann, Isaac K O

    2010-05-01

    Prevotella bryantii B(1)4 is a member of the phylum Bacteroidetes and contributes to the degradation of hemicellulose in the rumen. The genome of P. bryantii harbors four genes predicted to encode glycoside hydrolase (GH) family 3 (GH3) enzymes. To evaluate whether these genes encode enzymes with redundant biological functions, each gene was cloned and expressed in Escherichia coli. Biochemical analysis of the recombinant proteins revealed that the enzymes exhibit different substrate specificities. One gene encoded a cellodextrinase (CdxA), and three genes encoded beta-xylosidase enzymes (Xyl3A, Xyl3B, and Xyl3C) with different specificities for either para-nitrophenyl (pNP)-linked substrates or substituted xylooligosaccharides. To identify the amino acid residues that contribute to catalysis and substrate specificity within this family of enzymes, the roles of conserved residues (R177, K214, H215, M251, and D286) in Xyl3B were probed by site-directed mutagenesis. Each mutation led to a severely decreased catalytic efficiency without a change in the overall structure of the mutant enzymes. Through amino acid sequence alignments, an amino acid residue (E115) that, when mutated to aspartic acid, resulted in a 14-fold decrease in the k(cat)/K(m) for pNP-beta-d-xylopyranoside (pNPX) with a concurrent 1.1-fold increase in the k(cat)/K(m) for pNP-beta-d-glucopyranoside (pNPG) was identified. Amino acid residue E115 may therefore contribute to the discrimination between beta-xylosides and beta-glucosides. Our results demonstrate that each of the four GH3 enzymes has evolved to perform a specific role in lignopolysaccharide hydrolysis and provide insight into the role of active-site residues in catalysis and substrate specificity for GH3 enzymes.

  16. A Shinella β-N-acetylglucosaminidase of glycoside hydrolase family 20 displays novel biochemical and molecular characteristics.

    PubMed

    Zhou, Junpei; Song, Zhifeng; Zhang, Rui; Chen, Caihong; Wu, Qian; Li, Junjun; Tang, Xianghua; Xu, Bo; Ding, Junmei; Han, Nanyu; Huang, Zunxi

    2017-07-01

    β-N-Acetylglucosaminidases (GlcNAcases) are important for many biological functions and industrial applications. In this study, a glycoside hydrolase family 20 GlcNAcase from Shinella sp. JB10 was expressed in Escherichia coli BL21 (DE3). Compared to many GlcNAcases, the purified recombinant enzyme (rJB10Nag) exhibited a higher specificity activity (538.8 µmol min(-1) mg(-1)) or V max (1030.0 ± 82.1 µmol min(-1) mg(-1)) toward p-nitrophenyl β-N-acetylglucosaminide and N,N'-diacetylchitobiose (specificity activity of 35.4 µmol min(-1) mg(-1)) and a higher N-acetylglucosaminide tolerance (approximately 50% activity in 70.0 mM N-acetylglucosaminide). The degree of synergy on enzymatic degradation of chitin by a commercial chitinase and rJB10Nag was as high as 2.35. The enzyme was tolerant to most salts, especially 3.0-15.0% (w/v) NaCl and KCl. These biochemical characteristics make the JB10 GlcNAcase a candidate for use in many potential applications, including processing marine materials and the bioconversion of chitin waste. Furthermore, the enzyme has the highest proportions of alanine (16.5%), glycine (10.5%), and random coils (48.8%) with the lowest proportion of α-helices (24.9%) among experimentally characterized GH 20 GlcNAcases from other organisms.

  17. Genomic homologous recombination in planta.

    PubMed Central

    Gal, S; Pisan, B; Hohn, T; Grimsley, N; Hohn, B

    1991-01-01

    A system for monitoring intrachromosomal homologous recombination in whole plants is described. A multimer of cauliflower mosaic virus (CaMV) sequences, arranged such that CaMV could only be produced by recombination, was integrated into Brassica napus nuclear DNA. This set-up allowed scoring of recombination events by the appearance of viral symptoms. The repeated homologous regions were derived from two different strains of CaMV so that different recombinant viruses (i.e. different recombination events) could be distinguished. In most of the transgenic plants, a single major virus species was detected. About half of the transgenic plants contained viruses of the same type, suggesting a hotspot for recombination. The remainder of the plants contained viruses with cross-over sites distributed throughout the rest of the homologous sequence. Sequence analysis of two recombinant molecules suggest that mismatch repair is linked to the recombination process. Images PMID:2026150

  18. Isolation and Characterization of Two Persimmon Xyloglucan Endotransglycosylase/Hydrolase (XTH) Genes That Have Divergent Functions in Cell Wall Modification and Fruit Postharvest Softening

    PubMed Central

    Han, Ye; Ban, Qiuyan; Hou, Yali; Meng, Kun; Suo, Jiangtao; Rao, Jingping

    2016-01-01

    Fruit cell wall modification is the primary factor affecting fruit softening. Xyloglucan endotransglycosylase/hydrolase (XTH), a cell wall-modifying enzyme, is involved in fruit softening. In this study, two novel XTH genes (DkXTH6 and DkXTH7) were identified from persimmon fruit. Transcriptional profiles of both of the two genes were analyzed in different tissues of persimmon, and in response to multiple hormonal and environmental treatments [gibberellic acid (GA3), abscisic acid (ABA), propylene, and low temperature]. Expression of DkXTH6 was positively up-regulated during ethylene production and by propylene and ABA treatments, and suppressed by GA3 and cold treatment. In contrast, DkXTH7 exhibited its highest transcript levels in GA3-treated fruit and cold-treated fruit, which had higher fruit firmness. We found that DkXTH6 protein was localized in cell wall by its signal peptide, while cytoplasmic DkXTH7 protein contained no signal peptide. When expressed in vitro, the recombinant proteins of both DkXTH6 and DkXTH7 exhibited strict xyloglucan endotransglycosylase (XET) activity but no xyloglucan endohydrolase (XEH) activity. The recombinant protein of DkXTH6 showed a higher affinity with small acceptor molecules than the recombinant DkXTH7. Taken together with their opposing expression patterns and subcellular localizations, these results suggested that DkXTH6 might take part in cell wall restructuring and DkXTH7 was likely to be involved in cell wall assembly, indicating their special roles in persimmon fruit softening. PMID:27242828

  19. Site directed recombination

    DOEpatents

    Jurka, Jerzy W.

    1997-01-01

    Enhanced homologous recombination is obtained by employing a consensus sequence which has been found to be associated with integration of repeat sequences, such as Alu and ID. The consensus sequence or sequence having a single transition mutation determines one site of a double break which allows for high efficiency of integration at the site. By introducing single or double stranded DNA having the consensus sequence flanking region joined to a sequence of interest, one can reproducibly direct integration of the sequence of interest at one or a limited number of sites. In this way, specific sites can be identified and homologous recombination achieved at the site by employing a second flanking sequence associated with a sequence proximal to the 3'-nick.

  20. The putative α/β-hydrolases of Dietzia cinnamea P4 strain as potential enzymes for biocatalytic applications.

    PubMed

    Procópio, Luciano; Macrae, Andrew; van Elsas, Jan Dirk; Seldin, Lucy

    2013-03-01

    The draft genome of the soil actinomycete Dietzia cinnamea P4 reveals a versatile group of α/β-hydrolase fold enzymes. Phylogenetic and comparative sequence analyses were used to classify the α/β-hydrolases of strain P4 into six different groups: (i) lipases, (ii) esterases, (iii) epoxide hydrolases, (iv) haloacid dehalogenases, (v) C-C breaking enzymes and (vi) serine peptidases. The high number of lipases/esterases (41) and epoxide hydrolase enzymes (14) present in the relatively small (3.6 Mb) P4 genome is unusual; it is likely to be linked to the survival of strain P4 in its natural environment. Strain P4 is thus equipped with a large number of genes which would appear to confer survivability in harsh hot tropical soil. As such, this highly resilient soil bacterial strain provides an interesting genome for enzyme mining for applications in the field of biotransformations of polymeric compounds.

  1. 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.

  2. A novel enantioselective epoxide hydrolase for (R)-phenyl glycidyl ether to generate (R)-3-phenoxy-1,2-propanediol.

    PubMed

    Wu, Shijin; Shen, Jiajia; Zhou, Xiaoyun; Chen, Jianmeng

    2007-10-01

    Bacillus sp. Z018, a novel strain producing epoxide hydrolase, was isolated from soil. The epoxide hydrolase catalyzed the stereospecific hydrolysis of (R)-phenyl glycidyl ether to generate (R)-3-phenoxy-1,2-propanediol. Epoxide hydrolase from Bacillus sp. Z018 was inducible, and (R)-phenyl glycidyl ether was able to act as an inducer. The fermentation conditions for epoxide hydrolase were 35 degrees C, pH 7.5 with glucose and NH(4)Cl as the best carbon and nitrogen source, respectively. Under optimized conditions, the biotransformation yield of 45.8% and the enantiomeric excess of 96.3% were obtained for the product (R)-3-phenoxy-1,2-propanediol.

  3. The recombination epoch revisited

    NASA Technical Reports Server (NTRS)

    Krolik, Julian H.

    1989-01-01

    Previous studies of cosmological recombination have shown that this process produces as a by-product a highly superthermal population of Ly-alpha photons which retard completion of recombination. Cosmological redshifting was thought to determine the frequency distribution of the photons, while two-photon decay of hydrogen's 2s state was thought to control their numbers. It is shown here that frequency diffusion due to photon scattering dominate the cosmological redshift in the frequency range near line center which fixes the ratio of ground state to excited state population, while incoherent scattering into the far-red damping wing effectively destroys Ly-alpha photons as a rate which is competitive with two-photon decay. The former effect tends to hold back recombination, while the latter tends to accelerate it; the net results depends on cosmological parameters, particularly the combination Omega(b) h/sq rt (2q0), where Omega(b) is the fraction of the critical density provided by baryons.

  4. Recombinant human milk proteins.

    PubMed

    Lönnerdal, Bo

    2006-01-01

    Human milk provides proteins that benefit newborn infants. They not only provide amino acids, but also facilitate the absorption of nutrients, stimulate growth and development of the intestine, modulate immune function, and aid in the digestion of other nutrients. Breastfed infants have a lower prevalence of infections than formula-fed infants. Since many women in industrialized countries choose not to breastfeed, and an increasing proportion of women in developing countries are advised not to breastfeed because of the risk of HIV transmission, incorporation of recombinant human milk proteins into infant foods is likely to be beneficial. We are expressing human milk proteins known to have anti-infective activity in rice. Since rice is a normal constituent of the diet of infants and children, limited purification of the proteins is required. Lactoferrin has antimicrobial and iron-binding activities. Lysozyme is an enzyme that is bactericidal and also acts synergistically with lactoferrin. These recombinant proteins have biological activities identical to their native counterparts. They are equally resistant to heat processing, which is necessary for food applications, and to acid and proteolytic enzymes which are needed to maintain their biological activity in the gastrointestinal tract of infants. These recombinant human milk proteins may be incorporated into infant formulas, baby foods and complementary foods, and used with the goal to reduce infectious diseases.

  5. Cell biology of mitotic recombination.

    PubMed

    Lisby, Michael; Rothstein, Rodney

    2015-03-02

    Homologous recombination provides high-fidelity DNA repair throughout all domains of life. Live cell fluorescence microscopy offers the opportunity to image individual recombination events in real time providing insight into the in vivo biochemistry of the involved proteins and DNA molecules as well as the cellular organization of the process of homologous recombination. Herein we review the cell biological aspects of mitotic homologous recombination with a focus on Saccharomyces cerevisiae and mammalian cells, but will also draw on findings from other experimental systems. Key topics of this review include the stoichiometry and dynamics of recombination complexes in vivo, the choreography of assembly and disassembly of recombination proteins at sites of DNA damage, the mobilization of damaged DNA during homology search, and the functional compartmentalization of the nucleus with respect to capacity of homologous recombination.

  6. Cell Biology of Mitotic Recombination

    PubMed Central

    Lisby, Michael; Rothstein, Rodney

    2015-01-01

    Homologous recombination provides high-fidelity DNA repair throughout all domains of life. Live cell fluorescence microscopy offers the opportunity to image individual recombination events in real time providing insight into the in vivo biochemistry of the involved proteins and DNA molecules as well as the cellular organization of the process of homologous recombination. Herein we review the cell biological aspects of mitotic homologous recombination with a focus on Saccharomyces cerevisiae and mammalian cells, but will also draw on findings from other experimental systems. Key topics of this review include the stoichiometry and dynamics of recombination complexes in vivo, the choreography of assembly and disassembly of recombination proteins at sites of DNA damage, the mobilization of damaged DNA during homology search, and the functional compartmentalization of the nucleus with respect to capacity of homologous recombination. PMID:25731763

  7. A Simple Assay Demonstrating the Effect of Rehydration on the Orsellinate Depside Hydrolase Activity of Evernia prunastri.

    PubMed

    González, A; Vicente, C; Estrella Legaz, M

    1984-09-01

    A new, simple assay of orsellinate depside hydrolase (EC. 3.1.1.40) by high performance liquid chromatography is described. Enzymatic hydrolysis of evernic acid produces equimolar amounts of both orsellinic and everninic acids. Evernia prunastri thallus has a pre-existent, partially inactive hydrolase, which is activated after rehydration of the thallus. Copyright © 1984 Gustav Fisher Verlag, Stuttgart. Published by Elsevier GmbH.. All rights reserved.

  8. 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

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

    PubMed

    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.

  10. Proteins with an alpha/beta hydrolase fold: Relationships between subfamilies in an ever-growing superfamily.

    PubMed

    Lenfant, Nicolas; Hotelier, Thierry; Bourne, Yves; Marchot, Pascale; Chatonnet, Arnaud

    2013-03-25

    Alpha/beta hydrolases function as hydrolases, lyases, transferases, hormone precursors or transporters, chaperones or routers of other proteins. The amount of structural and functional available data related to this protein superfamily expands exponentially, as does the number of proteins classified as alpha/beta hydrolases despite poor sequence similarity and lack of experimental data. However the superfamily can be rationally divided according to sequence or structural homologies, leading to subfamilies of proteins with potentially similar functions. Since the discovery of proteins homologous to cholinesterases but devoid of enzymatic activity (e.g., the neuroligins), divergent functions have been ascribed to members of other subfamilies (e.g., lipases, dipeptidylaminopeptidase IV, etc.). To study the potentially moonlighting properties of alpha/beta hydrolases, the ESTHER database (for ESTerase and alpha/beta Hydrolase Enzymes and Relatives; http://bioweb.ensam.inra.fr/esther), which collects, organizes and disseminates structural and functional information related to alpha/beta hydrolases, has been updated with new tools and the web server interface has been upgraded. A new Overall Table along with a new Tree based on HMM models has been included to tentatively group subfamilies. These tools provide starting points for phylogenetic studies aimed at pinpointing the origin of duplications leading to paralogous genes (e.g., acetylcholinesterase versus butyrylcholinesterase, or neuroligin versus carboxylesterase). Another of our goals is to implement new tools to distinguish catalytically active enzymes from non-catalytic proteins in poorly studied or annotated subfamilies. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  11. 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°.

  12. Molecular characterization of aspartylglucosaminidase, a lysosomal hydrolase upregulated during strobilation in the moon jellyfish, Aurelia aurita.

    PubMed

    Tsujita, Natsumi; Kuwahara, Hiroyuki; Koyama, Hiroki; Yanaka, Noriyuki; Arakawa, Kenji; Kuniyoshi, Hisato

    2017-05-01

    The life cycle of the moon jellyfish, Aurelia aurita, alternates between a benthic asexual polyp stage and a planktonic sexual medusa (jellyfish) stage. Transition from polyp to medusa is called strobilation. To investigate the molecular mechanisms of strobilation, we screened for genes that are upregulated during strobilation using the differential display method and we identified aspartylglucosaminidase (AGA), which encodes a lysosomal hydrolase. Similar to AGAs from other species, Aurelia AGA possessed an N-terminal signal peptide and potential N-glycosylation sites. The genomic region of Aurelia AGA was approximately 9.8 kb in length and contained 12 exons and 11 introns. Quantitative RT-PCR analysis revealed that AGA expression increased during strobilation, and was then decreased in medusae. To inhibit AGA function, we administered the lysosomal acidification inhibitors, chloroquine or bafilomycin A1, to animals during strobilation. Both inhibitors disturbed medusa morphogenesis at the oral end, suggesting involvement of lysosomal hydrolases in strobilation.

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

    PubMed

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

    2016-01-18

    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.

  14. Organophosphorus hydrolase: a multifaceted plant genetic marker which is selectable, scorable, and quantifiable in whole seed.

    PubMed

    Pinkerton, T Scott; Wild, James R; Howard, John A

    2012-01-01

    Organophosphorus hydrolase (OPH, EC 3.1.8.1) provides a novel function as an alternative genetic marker system for use in many types of plant transformations. OPH is a high-capacity hydrolase with multiple organophosphorus substrates, many of which are neurotoxins and thus used extensively as pesticides. This spectrum of organophosphates includes compounds that are phytotoxic as well as those that are hydrolyzed to products that are easily detected visually without significant disruption of plant health. This dichotomy gives OPH the features of both a selectable marker as well as that of a scorable marker system, and these characteristics have been tested at several stages during the plant transformation and regeneration process. Finally, it is possible to quantify hydrolytic activity in the seed without interfering with its subsequent growth and regeneration.

  15. Production of monospecific antiserum to a cytosolic epoxide hydrolase from human liver.

    PubMed

    Qato, M K; Reinmund, S G; Guenthner, T M

    1990-01-15

    A method for the purification to apparent homogeneity of cytosolic trans-stilbene oxide hydrolase from human liver is presented. The method employed ion exchange and gel filtration chromatography. From 50 g of human liver, 4.9 mg of homogenous enzyme protein was obtained. Although the enzyme had lost much of its catalytic activity during purification, it was nevertheless suitable for the preparation of antibodies to the enzyme. Only one immunogenic species was present in the antigen preparation, but some antibodies that were cross-reactive to sites on catalase were present in the antiserum. These catalase-specific antibodies were removed by immunoaffinity chromatography, and an IgG fraction that is monospecific to the cytosolic epoxide hydrolase was obtained. The usefulness of antibodies to this enzyme in immunoblotting experiments, following either sodium dodecyl sulfate-polyacrylamide gel electrophoresis or isoelectric focussing, as well as in enzyme-linked immunosorbent assays, is demonstrated.

  16. 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

  17. Physical comparison of parathion hydrolase plasmids from Pseudomonas diminuta and Flavobacterium sp.

    PubMed

    Mulbry, W W; Kearney, P C; Nelson, J O; Karns, J S

    1987-09-01

    Restriction maps of two plasmids encoding parathion hydrolase have been determined. pPDL2 is a 39-kb plasmid harbored by Flavobacterium sp. (ATCC 27551), while pCMS1 is a 70-kb plasmid found in Pseudomonas diminuta (strain MG). Both plasmids previously have been shown to share homologous parathion hydrolase genes (termed opd for organophosphate degradation) as judged by DNA-DNA hybridization and restriction mapping. In the present study, we conducted DNA hybridization experiments using each of nine PstI restriction fragments from pCMS1 as probes against Flavobacterium plasmid DNA. The opd genes of both plasmids are located within a highly conserved region of approximately 5.1 kb. This region of homology extends approximately 2.6 kb upstream and 1.7 kb downstream from the opd genes. No homology between the two plasmids is evident outside of this region.

  18. Synthesis of bicyclic N-arylmethyl-substituted iminoribitol derivatives as selective nucleoside hydrolase inhibitors.

    PubMed

    Berg, Maya; Bal, Gunther; Goeminne, Annelies; Van der Veken, Pieter; Versées, Wim; Steyaert, Jan; Haemers, Achiel; Augustyns, Koen

    2009-02-01

    The purine metabolism of Trypanosoma and Leishmania spp. provides a good target in the search for new selective drugs. Bicyclic N-arylmethyl-substituted iminoribitols were developed as inhibitors of T. vivax nucleoside hydrolase, a key enzyme of the purine salvage pathway. The obtained results and structure-activity data confirmed our model for inhibitor binding with a hydrogen bond between a nitrogen atom of the nucleobase mimetic and the protonated Asp40 from the enzyme. This interaction depends on an optimal pK(a) value, which can be influenced by the electronic properties of the substituents. These compounds are potent, selective inhibitors of nucleoside hydrolase and are inactive toward human nucleoside phosphorylase.

  19. Effects of proteins and polynucleotides on the activity of various hydrolases

    PubMed Central

    Palmieri, M. J.; Koldovský, O.

    1972-01-01

    The effect of various macromolecules on the activity of several hydrolases was studied. Dilution of partially purified acid β-galactosidase from ileal mucosa of suckling rats resulted in a decrease of specific activity. The relationship between specific activity and dilution of the enzyme suggests a dissociation of the enzyme. This could be prevented by addition of several proteins tested. However, addition of DNA to the assay mixture for acid β-galactosidase caused an inhibition. This inhibition could be prevented by addition of proteins. Other polynucleotides and tRNA also exert an inhibitory effect that is prevented by albumin, but nucleotides have no effect. This inhibition occurs maximally at a low pH (3.0–4.0); no inhibition is observed at pH5.5. A similar pH-dependent inhibition by DNA was also found with various other acid hydrolases. PMID:5076227

  20. Heterologous Expression of Glycosyl Hydrolases in planta: A New Departure for Biofuels

    SciTech Connect

    Taylor II, L. E.; Dai, Z.; Decker, S. R.; Brunecky, R.; Adney, W. S.; Ding, S. Y.; Himmel M. E.

    2008-01-01

    The concept of expressing non-plant glycosyl hydrolase genes in plant tissue is nearly two decades old, yet relatively little work in this field has been reported. However, resurgent interest in technologies aimed at enabling processes that convert biomass to sugars and fuels has turned attention toward this intuitive solution. There are several challenges facing researchers in this field, including the development of better and more specifically targeted delivery systems for hydrolytic genes, the successful folding and post-translational modification of heterologous proteins and the development of cost-effective process strategies utilizing these transformed plants. The integration of these concepts, from the improvement of biomass production and conversion characteristics to the heterologous production of glycosyl hydrolases in a high yielding bioenergy crop, holds considerable promise for improving the lignocellulosic conversion of biomass to ethanol and subsequently to fuels.

  1. Heterologous Expression of Glycosyl Hydrolases in planta: A New Departure for Biofuels

    SciTech Connect

    Taylor, Larry E.; Dai, Ziyu; Decker, Stephen R.; Brunecky, Roman; Adney, William S.; Ding, Shi-You; Himmel, Michael E.

    2008-08-01

    The concept of expressing non-plant glycosyl hydrolase genes in plant tissue is less than two decades old, and yet little work in this field has been reported. However, the resurgent interest in technologies aimed at enabling biomass to sugars/fuels processes have again pointed to this intuitive solution. Research challenges in this regard include developing better and more specifically targeted delivery systems for hydrolytic genes, achieving successful folding and post-translational modification of heterologous proteins, and developing cost effective process strategies utilizing these transformed plants. Integration of these concepts, from the improvement of biomass production and conversion characteristics to heterologous production of glycosyl hydrolases in a high yielding bioenergy crop, holds considerable promise for improving the lignocellulosic biomass to ethanol/fuels process.

  2. Tolerance of β-diketone hydrolases as representatives of the crotonase superfamily towards organic solvents.

    PubMed

    Siirola, Elina; Grischek, Barbara; Clay, Dorina; Frank, Annika; Grogan, Gideon; Kroutil, Wolfgang

    2011-12-01

    Crotonase superfamily enzymes catalyze a wide variety of reactions, including hydrolytic C-C bond cleavage in symmetrical β-diketones by 6-oxo camphor hydrolase (OCH) from Rhodococcus sp. The organic solvent tolerance and temperature stability of OCH and its structurally related ortholog Anabaena β-diketone hydrolase have been investigated. Both enzymes showed excellent tolerance toward organic solvents; for instance, even in the presence of 80% (v/v) THF or dioxane, OCH was still active. In most solvent mixtures, except methanol, the stereospecificity was conserved (>99% e.e. of product), hence neither the type of solvent nor its concentration appeared to have an effect on the stereoselectivity of the enzyme. Attempts to correlate the observed activities with log P, functional solvent group or denaturing capacity (DC) of the solvent were only successful in the case of DC for water miscible solvents. This study represents the first investigation of organic solvent stability for members of the crotonase superfamily.

  3. 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

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

    PubMed

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

    1998-01-01

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

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

    PubMed Central

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

    2008-01-01

    We describe a new enzymatic functionality for the surface layer (S-layer) of Lactobacillus acidophilus ATCC 4356, namely, an endopeptidase activity against the cell wall of Salmonella enterica serovar Newport, assayed via zymograms and identified by Western blotting. Based on amino acid sequence comparisons, the hydrolase activity was predicted to be located at the C terminus. Subsequent cloning and expression of the C-terminal domain in Bacillus subtilis resulted in the functional verification of the enzymatic activity. PMID:18931300

  6. Measuring Enzyme Kinetics of Glycoside Hydrolases Using the 3,5-Dinitrosalicylic Acid Assay.

    PubMed

    McKee, Lauren S

    2017-01-01

    Use of the 3,5-dinitrosalicylic acid reagent allows the simple and rapid quantification of reducing sugars. The method can be used for analysis of biological samples or in the characterization of enzyme reactions. Presented here is an application of the method in measuring the kinetics of a glycoside hydrolase reaction, including the optimization of the DNSA reagent, and the production of a standard curve of absorbance and sugar concentration.

  7. 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.

  8. Orientation Dependence in Homologous Recombination

    PubMed Central

    Yamamoto, K.; Takahashi, N.; Fujitani, Y.; Yoshikura, H.; Kobayashi, I.

    1996-01-01

    Homologous recombination was investigated in Escherichia coli with two plasmids, each carrying the homologous region (two defective neo genes, one with an amino-end deletion and the other with a carboxyl-end deletion) in either direct or inverted orientation. Recombination efficiency was measured in recBC sbcBC and recBC sbcA strains in three ways. First, we measured the frequency of cells carrying neo(+) recombinant plasmids in stationary phase. Recombination between direct repeats was much more frequent than between inverted repeats in the recBC sbcBC strain but was equally frequent in the two substrates in the recBC sbcA strain. Second, the fluctuation test was used to exclude bias by a rate difference between the recombinant and parental plasmids and led to the same conclusion. Third, direct selection for recombinants just after transformation with or without substrate double-strand breaks yielded essentially the same results. Double-strand breaks elevated recombination in both the strains and in both substrates. These results are consistant with our previous findings that the major route of recombination in recBC sbcBC strains generates only one recombinant DNA from two DNAs and in recBC sbcA strains generates two recombinant DNAs from two DNAs. PMID:8722759

  9. Regulated proteolysis of a cross-link-specific peptidoglycan hydrolase contributes to bacterial morphogenesis.

    PubMed

    Singh, Santosh Kumar; Parveen, Sadiya; SaiSree, L; Reddy, Manjula

    2015-09-01

    Bacterial growth and morphogenesis are intimately coupled to expansion of peptidoglycan (PG), an extensively cross-linked macromolecule that forms a protective mesh-like sacculus around the cytoplasmic membrane. Growth of the PG sacculus is a dynamic event requiring the concerted action of hydrolases that cleave the cross-links for insertion of new material and synthases that catalyze cross-link formation; however, the factors that regulate PG expansion during bacterial growth are poorly understood. Here, we show that the PG hydrolase MepS (formerly Spr), which is specific to cleavage of cross-links during PG expansion in Escherichia coli, is modulated by proteolysis. Using combined genetic, molecular, and biochemical approaches, we demonstrate that MepS is rapidly degraded by a proteolytic system comprising an outer membrane lipoprotein of unknown function, NlpI, and a periplasmic protease, Prc (or Tsp). In summary, our results indicate that the NlpI-Prc system contributes to growth and enlargement of the PG sacculus by modulating the cellular levels of the cross-link-cleaving hydrolase MepS. Overall, this study signifies the importance of PG cross-link cleavage and its regulation in bacterial cell wall biogenesis.

  10. A new group of glycoside hydrolase family 13 α-amylases with an aberrant catalytic triad

    PubMed Central

    Sarian, Fean D.; Janeček, Štefan; Pijning, Tjaard; Ihsanawati; Nurachman, Zeily; Radjasa, Ocky K.; Dijkhuizen, Lubbert; Natalia, Dessy; van der Maarel, Marc J. E. C.

    2017-01-01

    α-Amylases are glycoside hydrolase enzymes that act on the α(1→4) glycosidic linkages in glycogen, starch, and related α-glucans, and are ubiquitously present in Nature. Most α-amylases have been classified in glycoside hydrolase family 13 with a typical (β/α)8-barrel containing two aspartic acid and one glutamic acid residue that play an essential role in catalysis. An atypical α-amylase (BmaN1) with only two of the three invariant catalytic residues present was isolated from Bacillus megaterium strain NL3, a bacterial isolate from a sea anemone of Kakaban landlocked marine lake, Derawan Island, Indonesia. In BmaN1 the third residue, the aspartic acid that acts as the transition state stabilizer, was replaced by a histidine. Three-dimensional structure modeling of the BmaN1 amino acid sequence confirmed the aberrant catalytic triad. Glucose and maltose were found as products of the action of the novel α-amylase on soluble starch, demonstrating that it is active in spite of the peculiar catalytic triad. This novel BmaN1 α-amylase is part of a group of α-amylases that all have this atypical catalytic triad, consisting of aspartic acid, glutamic acid and histidine. Phylogenetic analysis showed that this group of α-amylases comprises a new subfamily of the glycoside hydrolase family 13. PMID:28287181

  11. 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

  12. 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

  13. Thermal unfolding of nucleoside hydrolases from the hyperthermophilic archaeon Sulfolobus solfataricus: role of disulfide bonds.

    PubMed

    Porcelli, Marina; De Leo, Ester; Del Vecchio, Pompea; Fuccio, Francesca; Cacciapuoti, Giovanna

    2012-03-01

    Nucleoside hydrolases are metalloproteins that hydrolyze the N-glycosidic bond of β-ribonucleosides, forming the free purine/pyrimidine base and ribose. We report the stability of the two hyperthermophilic enzymes Sulfolobus solfataricus pyrimidine-specific nucleoside hydrolase (SsCU-NH) and Sulfolobus solfataricus purine-specific inosineadenosine- guanosine nucleoside hydrolase (SsIAG-NH) against the denaturing action of temperature and guanidine hydrochloride by means of circular dichroism and fluorescence spectroscopy. The guanidine hydrochloride-induced unfolding is reversible for both enzymes as demonstrated by the analysis of the refolding process by activity assays and fluorescence measurements. The evidence that the denaturation of SsIAG-NH carried out in the presence of reducing agents proved to be reversible indicates that the presence of disulfide bonds interferes with the refolding process of this enzyme. Both enzymes are highly thermostable and no thermal unfolding transition can be obtained up to 108°C. SsIAG-NH is thermally denatured under reducing conditions (T(m)=93°C) demonstrating the contribution of disulfide bridges to enzyme thermostability.

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

    DOE PAGES

    Chen, Mo; Bu, Lintao; Alahuhta, Markus; ...

    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

  15. A new group of glycoside hydrolase family 13 α-amylases with an aberrant catalytic triad.

    PubMed

    Sarian, Fean D; Janeček, Štefan; Pijning, Tjaard; Ihsanawati; Nurachman, Zeily; Radjasa, Ocky K; Dijkhuizen, Lubbert; Natalia, Dessy; van der Maarel, Marc J E C

    2017-03-13

    α-Amylases are glycoside hydrolase enzymes that act on the α(1→4) glycosidic linkages in glycogen, starch, and related α-glucans, and are ubiquitously present in Nature. Most α-amylases have been classified in glycoside hydrolase family 13 with a typical (β/α)8-barrel containing two aspartic acid and one glutamic acid residue that play an essential role in catalysis. An atypical α-amylase (BmaN1) with only two of the three invariant catalytic residues present was isolated from Bacillus megaterium strain NL3, a bacterial isolate from a sea anemone of Kakaban landlocked marine lake, Derawan Island, Indonesia. In BmaN1 the third residue, the aspartic acid that acts as the transition state stabilizer, was replaced by a histidine. Three-dimensional structure modeling of the BmaN1 amino acid sequence confirmed the aberrant catalytic triad. Glucose and maltose were found as products of the action of the novel α-amylase on soluble starch, demonstrating that it is active in spite of the peculiar catalytic triad. This novel BmaN1 α-amylase is part of a group of α-amylases that all have this atypical catalytic triad, consisting of aspartic acid, glutamic acid and histidine. Phylogenetic analysis showed that this group of α-amylases comprises a new subfamily of the glycoside hydrolase family 13.

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

    PubMed

    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.

  17. Ubiquitin dimers control the hydrolase activity of UCH-L3.

    PubMed

    Setsuie, Rieko; Sakurai, Mikako; Sakaguchi, Yuriko; Wada, Keiji

    2009-01-01

    Ubiquitin (Ub) carboxy terminal hydrolase (UCH)-L1 and UCH-L3 are two of the deubiquitinating enzymes expressed in the brain. Both gad mice, which lack UCH-L1 expression and Uchl3 knockout mice exhibit neurodegeneration, although at distinct areas. These phenotypes indicate the importance of UCH-L1 and UCH-L3 in the regulation of the central nervous system. However, molecular substrates and the molecular regulators of UCH-L1 and UCH-L3 remain poorly identified. Here we show that Ub dimers interact non-covalently with UCH-L3 in vitro and in cells. These interactions were not observed with UCH-L1 in cells. In vitro, K48-linked Ub dimers pronouncedly inhibited the hydrolase activity of UCH-L3, while mono-Ub, a previously identified interacting protein, inhibited the hydrolase activity of UCH-L1. These results indicate that mono-Ub and Ub dimers may regulate the enzymatic functions of UCH-L1 and UCH-L3, respectively, in vivo.

  18. Production of polyclonal anti-dUCH (Drosophila ubiquitin carboxyl-terminal hydrolase) antibodies.

    PubMed

    Tram, Nguyen Thi Quynh; Trang, Nguyen Thi Thu; Thao, Dang Thi Phuong; Thuoc, Tran Linh

    2013-04-01

    Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), which is a member of the ubiquitin carboxyl-terminal hydrolase (UCH) family, is highly expressed in neurons. In vitro, UCH- L1 exhibits both ubiquitin hydrolase and ligase activity. Many studies have suggested that UCH-L1 is involved in the pathogenesis of Parkinson's disease and some different human cancer diseases, but its role in a living system is still unclear. Recently, Drosophila melanogaster has been shown to be a compatible model for studying human diseases. To investigate the role of UCH-L1 in a living system, the UCH-L1 homologous protein in Drosophila melanogaster (dUCH) is used for analyzing the role of the protein's function in transgenic Drosophila. Here, we used DNA molecular techniques to clone, express, and purify dUCH protein from Escherichia coli. The purified dUCH protein was injected into a rabbit to produce an anti-dUCH antibody, which was shown to have high specificity and sensitivity to the dUCH protein. The affinity of the antibody is 1:320,000 at 7.81 ng/μL antigen concentration. The 1:40,000 dilution-produced antibodies can detect antigen at a low concentration of 0.98 ng/μL. Success in producing this antibody provides good material for further experiments in the study of the role of UCH-L1 by a Drosophila model.

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

    PubMed Central

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

    2015-01-01

    ABSTRACT Most bacterial cells are enclosed in a single macromolecule of the cell wall polymer, peptidoglycan, which is required for shape determination and maintenance of viability, while peptidoglycan biosynthesis is an important antibiotic target. It is hypothesized that cellular enlargement requires regional expansion of the cell wall through coordinated insertion and hydrolysis of peptidoglycan. Here, a group of (apparent glucosaminidase) peptidoglycan hydrolases are identified that are together required for cell enlargement and correct cellular morphology of Staphylococcus aureus, demonstrating the overall importance of this enzyme activity. These are Atl, SagA, ScaH, and SagB. The major advance here is the explanation of the observed morphological defects in terms of the mechanical and biochemical properties of peptidoglycan. It was shown that cells lacking groups of these hydrolases have increased surface stiffness and, in the absence of SagB, substantially increased glycan chain length. This indicates that, beyond their established roles (for example in cell separation), some hydrolases enable cellular enlargement by making peptidoglycan easier to stretch, providing the first direct evidence demonstrating that cellular enlargement occurs via modulation of the mechanical properties of peptidoglycan. PMID:26220963

  20. 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

  1. LytF, a Novel Competence-Regulated Murein Hydrolase in the Genus Streptococcus

    PubMed Central

    Berg, Kari Helene; Ohnstad, Hilde Solheim

    2012-01-01

    Streptococcus pneumoniae and probably most other members of the genus Streptococcus are competent for natural genetic transformation. During the competent state, S. pneumoniae produces a murein hydrolase, CbpD, that kills and lyses noncompetent pneumococci and closely related species. Previous studies have shown that CbpD is essential for efficient transfer of genomic DNA from noncompetent to competent cells in vitro. Consequently, it has been proposed that CbpD together with the cognate immunity protein ComM constitutes a DNA acquisition mechanism that enables competent pneumococci to capture homologous DNA from closely related streptococci sharing the same habitat. Although genes encoding CbpD homologs or CbpD-related proteins are present in many different streptococcal species, the genomes of a number of streptococci do not encode CbpD-type proteins. In the present study we show that the genomes of nearly all species lacking CbpD encode an unrelated competence-regulated murein hydrolase termed LytF. Using Streptococcus gordonii as a model system, we obtained evidence indicating that LytF is a functional analogue of CbpD. In sum, our results show that a murein hydrolase gene is part of the competence regulon of most or all streptococcal species, demonstrating that these muralytic enzymes constitute an essential part of the streptococcal natural transformation system. PMID:22123253

  2. Exported Epoxide Hydrolases Modulate Erythrocyte Vasoactive Lipids during Plasmodium falciparum Infection

    PubMed Central

    Dalmia, Varun K.

    2016-01-01

    ABSTRACT Erythrocytes are reservoirs of important epoxide-containing lipid signaling molecules, including epoxyeicosatrienoic acids (EETs). EETs function as vasodilators and anti-inflammatory modulators in the bloodstream. Bioactive EETs are hydrolyzed to less active diols (dihydroxyeicosatrienoic acids) by epoxide hydrolases (EHs). The malaria parasite Plasmodium falciparum infects host red blood cells (RBCs) and exports hundreds of proteins into the RBC compartment. In this study, we show that two parasite epoxide hydrolases, P. falciparum epoxide hydrolases 1 (PfEH1) and 2 (PfEH2), both with noncanonical serine nucleophiles, are exported to the periphery of infected RBCs. PfEH1 and PfEH2 were successfully expressed in Escherichia coli, and they hydrolyzed physiologically relevant erythrocyte EETs. Mutations in active site residues of PfEH1 ablated the ability of the enzyme to hydrolyze an epoxide substrate. Overexpression of PfEH1 or PfEH2 in parasite-infected RBCs resulted in a significant alteration in the epoxide fatty acids stored in RBC phospholipids. We hypothesize that the parasite disruption of epoxide-containing signaling lipids leads to perturbed vascular function, creating favorable conditions for binding and sequestration of infected RBCs to the microvascular endothelium. PMID:27795395

  3. Diadenosine tetraphosphate hydrolase is part of the transcriptional regulation network in immunologically activated mast cells.

    PubMed

    Carmi-Levy, Irit; Yannay-Cohen, Nurit; Kay, Gillian; Razin, Ehud; Nechushtan, Hovav

    2008-09-01

    We previously discovered that microphthalmia transcription factor (MITF) and upstream stimulatory factor 2 (USF2) each forms a complex with its inhibitor histidine triad nucleotide-binding 1 (Hint-1) and with lysyl-tRNA synthetase (LysRS). Moreover, we showed that the dinucleotide diadenosine tetraphosphate (Ap(4)A), previously shown to be synthesized by LysRS, binds to Hint-1, and as a result the transcription factors are released from their suppression. Thus, transcriptional activity is regulated by Ap(4)A, suggesting that Ap(4)A is a second messenger in this context. For Ap(4)A to be unambiguously established as a second messenger, several criteria have to be fulfilled, including the presence of a metabolizing enzyme. Since several enzymes are able to hydrolyze Ap(4)A, we provided here evidence that the "Nudix" type 2 gene product, Ap(4)A hydrolase, is responsible for Ap(4)A degradation following the immunological activation of mast cells. The knockdown of Ap(4)A hydrolase modulated Ap(4)A accumulation, resulting in changes in the expression of MITF and USF2 target genes. Moreover, our observations demonstrated that the involvement of Ap(4)A hydrolase in gene regulation is not a phenomenon exclusive to mast cells but can also be found in cardiac cells activated with the beta-agonist isoproterenol. Thus, we have provided concrete evidence establishing Ap(4)A as a second messenger in the regulation of gene expression.

  4. Hydrolase activity in the venom of the pupal endoparasitic wasp, Pimpla hypochondriaca.

    PubMed

    Dani, M P; Edwards, J P; Richards, E H

    2005-07-01

    Venom from the pupal endoparasitoid, Pimpla hypochondriaca has previously been shown to contain a mixture of biologically active molecules. Currently, P. hypochondriaca venom was examined for the presence of hydrolase activity. Six hydrolases were consistently detected using the API ZYM semiquantitative colourimetric kit. The main hydrolases detected were; acid phosphatase, beta-glucosidase, esterase, beta-galactosidase, esterase lipase, and lipase. The most rapid and intense colour reaction was detected for acid phosphatase. The pH optimum and the specific activity of venom acid phosphatase was determined using p-nitrophenol phosphate as a substrate and were 4.8 and 0.47 nmol p-nitrophenol/min/microg of venom protein, respectively. The acid phosphatase activity was inhibited in a dose dependent manner by sodium fluoride (IC(50) 4.2 x 10(-4) M), and by cocktail inhibitor 2 (CI 2). P. hypochondriaca venom has previously been shown to display potent cytotoxic activity towards Lacanobia oleracea haemocytes maintained in vitro. The contribution of acid phosphatase in venom to this cytotoxic activity was investigated by titrating venom against CI 2 prior to the addition of L. oleracea haemocytes. The results suggest that, despite the relatively high levels of acid phosphatase activity in venom, venom acid phosphatase plays no role in the antihaemocytic activity of P. hypochondriaca venom in vitro.

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

    NASA Astrophysics Data System (ADS)

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

    2012-05-01

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

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

    PubMed Central

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

    2017-01-01

    Cyanuric acid hydrolases are of industrial importance because of their use in aquatic recreational facilities to remove cyanuric acid, a stabilizer for the chlorine. Degradation of excess cyanuric acid is necessary to maintain chlorine disinfection in the waters. Cyanuric acid hydrolase opens the cyanuric acid ring hydrolytically and subsequent decarboxylation produces carbon dioxide and biuret. In the present study, we report the X-ray structure of TrzD, a cyanuric acid hydrolase from Acidovorax citrulli. The crystal structure at 2.19 Å resolution shows a large displacement of the catalytic lysine (Lys163) in domain 2 away from the active site core, whereas the two other active site lysines from the two other domains are not able to move. The lysine displacement is proposed here to open up a channel for product release. Consistent with that, the structure also showed two molecules of the co-product, carbon dioxide, one in the active site and another trapped in the proposed exit channel. Previous data indicated that the domain 2 lysine residue plays a role in activating an adjacent serine residue carrying out nucleophilic attack, opening the cyanuric acid ring, and the mobile lysine guides products through the exit channel. PMID:28345631

  7. Recombinant Collagenlike Proteins

    NASA Technical Reports Server (NTRS)

    Fertala, Andzej

    2007-01-01

    A group of collagenlike recombinant proteins containing high densities of biologically active sites has been invented. The method used to express these proteins is similar to a method of expressing recombinant procollagens and collagens described in U. S. Patent 5,593,859, "Synthesis of human procollagens and collagens in recombinant DNA systems." Customized collagenous proteins are needed for biomedical applications. In particular, fibrillar collagens are attractive for production of matrices needed for tissue engineering and drug delivery. Prior to this invention, there was no way of producing customized collagenous proteins for these and other applications. Heretofore, collagenous proteins have been produced by use of such biological systems as yeasts, bacteria, and transgenic animals and plants. These products are normal collagens that can also be extracted from such sources as tendons, bones, and hides. These products cannot be made to consist only of biologically active, specific amino acid sequences that may be needed for specific applications. Prior to this invention, it had been established that fibrillar collagens consist of domains that are responsible for such processes as interaction with cells, binding of growth factors, and interaction with a number of structural proteins present in the extracellular matrix. A normal collagen consists of a sequence of domains that can be represented by a corresponding sequence of labels, e.g., D1D2D3D4. A collagenlike protein of the present invention contains regions of collagen II that contain multiples of a single domain (e.g., D1D1D1D1 or D4D4D4D4) chosen for its specific biological activity. By virtue of the multiplicity of the chosen domain, the density of sites having that specific biological activity is greater than it is in a normal collagen. A collagenlike protein according to this invention can thus be made to have properties that are necessary for tissue engineering.

  8. Making recombinant extracellular matrix proteins.

    PubMed

    Ruggiero, Florence; Koch, Manuel

    2008-05-01

    A variety of approaches to understand extracellular matrix protein structure and function require production of recombinant proteins. Moreover, the expression of heterologous extracellular matrix proteins, in particular collagens, using the recombinant technology is of major interest to the biomedical industry. Although extracellular matrix proteins are large, modular and often multimeric, most of them have been successfully produced in various expression systems. This review provides important factors, including the design of the construct, the cloning strategies, the expression vectors, the transfection method and the host cell systems, to consider in choosing a reliable and cost-effective way to make recombinant extracellular matrix proteins. Advantages and drawbacks of each system have been appraised. Protocols that may ease efficient recombinant production of extracellular matrix are described. Emphasis is placed on the recombinant collagen production. Members of the collagen superfamily exhibit specific structural features and generally require complex post-translational modifications to retain full biological activity that make more arduous their recombinant production.

  9. Expression of recombinant antibodies.

    PubMed

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with "human-like" post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications.

  10. Dissociative recombination of HCl+

    NASA Astrophysics Data System (ADS)

    Larson, Åsa; Fonseca dos Santos, Samantha; E. Orel, Ann

    2017-08-01

    The dissociative recombination of HCl+, including both the direct and indirect mechanisms, is studied. For the direct process, the relevant electronic states are calculated ab initio by combining electron scattering calculations to obtain resonance positions and autoionization widths with multi-reference configuration interaction calculations of the ion and Rydberg states. The cross section for the direct dissociation along electronic resonant states is computed by solution of the time-dependent Schrödinger equation. For the indirect process, an upper bound value for the cross section is obtained using a vibrational frame transformation of the elements of the scattering matrix at energies just above the ionization threshold. Vibrational excitations of the ionic core from the ground vibrational state, v = 0 , to the first three excited vibrational states, v = 1 , v = 2 , and v = 3 , are considered. Autoionization is neglected and the effect of the spin-orbit splitting of the ionic potential energy upon the indirect dissociative recombination cross section is considered. The calculated cross sections are compared to measurements.

  11. Recombinant electric storage battery

    SciTech Connect

    Flicker, R.P.; Fenstermacher, S.

    1989-10-10

    This patent describes a recombinant storage battery. It comprises: a plurality of positive plates containing about 2 to 4 percent of antimony based upon the total weight of the alloy and positive active material, and essentially antimony free negative plates in a closed case; a fibrous sheet plate separator between adjacent ones of the plates, and a body of an electrolyte to which the sheet separators are inert absorbed by each of the separators and maintained in contact with each of the adjacent ones of the plates. Each of the separator sheets comprising first fibers which impart to the sheet a given absorbency greater than 90 percent relative to the electrolyte and second fibers which impart to the sheet a different absorbency less than 80 percent relative to the electrolyte. The first and second fibers being present in such proportions that each of the sheet separators has an absorbency with respect to the electrolyte of from 75 to 95 percent and the second fibers being present in such proportions that the battery has a recombination rate adequate to compensate for gassing.

  12. Expression of Recombinant Antibodies

    PubMed Central

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with “human-like” post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications. PMID:23908655

  13. Prediction of drug-drug interactions with carbamazepine-10,11-epoxide using a new in vitro assay for epoxide hydrolase inhibition.

    PubMed

    Rosa, Maria; Bonnaillie, Pierre; Chanteux, Hugues

    2016-12-01

    1. Carbamazepine is an antiepileptic drug which is metabolized by CYP3A4 into carbamazepine-10,11-epoxide. This metabolite is then detoxified by epoxide hydrolase. As carbamazepine-10,11-epoxide has been associated with neurotoxicity, it is critical to identify whether a new antiepileptic drug has the potential to inhibit epoxide hydrolase and therefore increase carbamazepine-10,11-epoxide plasma levels. 2. In this study, an in vitro assay was developed to evaluate epoxide hydrolase activity by using carbamazepine-10,11-epoxide as probe substrate. The ability of this assay to predict drug-drug interactions (DDI) at the epoxide hydrolase level was also investigated. 3. To this aim, known inhibitors of epoxide hydrolase for which in vivo data are available were used. Firstly, carbamazepine-10,11-epoxide hydrolase activity was determined in liver microsomes, cytosol and hepatocytes. Thereafter, the IC50 of epoxide hydrolase inhibitors (progabide, valproic acid, valpromide and valnoctamide) was determined in liver microsomes and hepatocytes. Finally, prediction of AUC increase was performed using the in vitro data generated. 4. Interestingly, epoxide hydrolase activity was found to be much higher in human hepatocytes compared to liver microsomes/cytosol. Even though assessed on a limited number of compounds, this study demonstrated that the use of hepatocytes seems to be a more relevant model to assess and predict DDI at the epoxide hydrolase level.

  14. Biochemical properties of a novel glycoside hydrolase family 1 β-glucosidase (PtBglu1) from Paecilomyces thermophila expressed in Pichia pastoris.

    PubMed

    Yang, Shaoqing; Hua, Chengwei; Yan, Qiaojuan; Li, Yinan; Jiang, Zhengqiang

    2013-01-30

    A novel β-glucosidase gene (PtBglu1) from the thermophilic fungus, Paecilomyces thermophila, was cloned and expressed in Pichia pastoris. PtBglu1 contained an open reading frame of 1440-bp nucleotides and encoded a protein of 479 amino acids which showed significant similarity to other fungal β-glucosidases from glycoside hydrolase (GH) family 1. The recombinant β-glucosidase (PtBglu1) was secreted at high level of 190.2 U mL(-1) in high cell density fermentor (5L). PtBglu1 was purified to homogeneity, and was found to be a glycoprotein with molecular mass of 56.7 kDa. The purified PtBglu1 showed optimum catalytic activity at pH 6.0 and 55 °C. The enzyme exhibited broad substrate specificity with highest activity toward pNP-β-D-glucopyranoside, followed by pNP-β-D-galactopyranoside and cellobiose. The K(m) values for pNP-β-D-glucopyranoside, cellobiose, gentiobiose and salicin were 0.55 mM, 1.0 mM, 1.74 mM and 6.85 mM, respectively. These properties make PtBglu1 a potential candidate for various industrial applications. Copyright © 2012 Elsevier Ltd. All rights reserved.

  15. Development of a Novel Optical Biosensor for Detection of Organophoshorus Pesticides Based on Methyl Parathion Hydrolase Immobilized by Metal-Chelate Affinity

    PubMed Central

    Lan, Wensheng; Chen, Guoping; Cui, Feng; Tan, Feng; Liu, Ran; Yushupujiang, Maolidan

    2012-01-01

    We have developed a novel optical biosensor device using recombinant methyl parathion hydrolase (MPH) enzyme immobilized on agarose by metal-chelate affinity to detect organophosphorus (OP) compounds with a nitrophenyl group. The biosensor principle is based on the optical measurement of the product of OP catalysis by MPH (p-nitrophenol). Briefly, MPH containing six sequential histidines (6× His tag) at its N-terminal was bound to nitrilotriacetic acid (NTA) agarose with Ni ions, resulting in the flexible immobilization of the bio-reaction platform. The optical biosensing system consisted of two light-emitting diodes (LEDs) and one photodiode. The LED that emitted light at the wavelength of the maximum absorption for p-nitrophenol served as the signal light, while the other LED that showed no absorbance served as the reference light. The optical sensing system detected absorbance that was linearly correlated to methyl parathion (MP) concentration and the detection limit was estimated to be 4 μM. Sensor hysteresis was investigated and the results showed that at lower concentration range of MP the difference got from the opposite process curves was very small. With its easy immobilization of enzymes and simple design in structure, the system has the potential for development into a practical portable detector for field applications. PMID:23012501

  16. Legionella pneumophila Secretes an Endoglucanase That Belongs to the Family-5 of Glycosyl Hydrolases and Is Dependent upon Type II Secretion

    PubMed Central

    Pearce, Meghan M.; Cianciotto, Nicholas P.

    2009-01-01

    Examination of cell-free culture supernatants revealed that Legionella pneumophila strains secrete an endoglucanase activity. L. pneumophila lspF mutants were deficient for this activity, indicating that the endoglucanase is secreted by the bacterium’s type II protein secretion system. Inactivation of celA, encoding a member of the family-5 of glycosyl hydrolases, abolished the endoglucanase activity in L. pneumophila culture supernatants. The cloned celA gene conferred activity upon recombinant Escherichia coli. Thus, CelA is the major secreted endoglucanase of L. pneumophila. Mutants inactivated for celA grew normally in protozoa and macrophage, indicating that CelA is not required for the intracellular phase of L. pneumophila. The CelA endoglucanase is one of at least 25 proteins secreted by the type II system of L. pneumophila and the seventeenth type of enzyme effector associated with this pathway. Only a subset of the other Legionella species tested expressed secreted endoglucanase activity, suggesting that the type II secretion output differs among the different legionellae. Overall, this study represents the first documentation of an endoglucanase (EC 3.2.1.4) being produced by a strain of Legionella. PMID:19817866

  17. Identification of glycosyl hydrolases from a metagenomic library of microflora in sugarcane bagasse collection site and their cooperative action on cellulose degradation.

    PubMed

    Kanokratana, Pattanop; Eurwilaichitr, Lily; Pootanakit, Kusol; Champreda, Verawat

    2015-04-01

    Lignocellulose decomposition is a natural process involving the cooperative action of various glycosyl hydrolases (GH) on plant cell wall components. In this study, a metagenomic library was constructed to capture the genetic diversity of microbes inhabiting an industrial bagasse collection site. A variety of putative genes encoding GH families 2, 3, 5, 9, 11, and 16 were identified using activity-based screening, which showed low to moderate homology to various cellulases and hemicellulases. The recombinant GH9 endoglucanase (Cel9) and GH11 endo-xylanase (Xyn11) were thermophilic with optimal activity between 75°C and 80°C and the maximal activity at slightly acidic to neutral pH range. The enzymes exhibited cooperative activity with Trichoderma reesei cellulase on the degradation of lignocellulosic substrates. Mixture design showed positive interactions among the enzyme components. The optimal combination was determined to be 41.4% Celluclast, 18.0% Cel9, and 40.6% Xyn11 with the predicted relative reducing sugar of 658% when compared to Celluclast alone on hydrolysis of alkaline-pretreated bagasse. The work demonstrates the potential of lignocellulolytic enzymes from a novel uncultured microbial resource for enhancing efficiency of biomass-degrading enzyme systems for bio-industries.

  18. Molecular cloning, overexpression, and enzymatic characterization of glycosyl hydrolase family 16 β-Agarase from marine bacterium Saccharophagus sp. AG21 in Escherichia coli.

    PubMed

    Lee, Youngdeuk; Oh, Chulhong; De Zoysa, Mahanama; Kim, Hyowon; Wickramaarachchi, Wickramaarachchige Don Niroshana; Whang, Ilson; Kang, Do-Hyung; Lee, Jehee

    2013-01-01

    An agar-degrading bacterium was isolated from red seaweed (Gelidium amansii) on a natural seawater agar plate, and identified as Saccharophagus sp. AG21. The β-agarase gene from Saccharophagus sp. AG21 (agy1) was screened by long and accurate (LA)-PCR. The predicted sequence has a 1,908 bp open reading frame encoding 636 amino acids (aa), and includes a glycosyl hydrolase family 16 (GH16) β-agarase module and two carbohydrate binding modules of family 6 (CBM6). The deduced aa sequence showed 93.7% and 84.9% similarity to β-agarase of Saccharophagus degradans and Microbulbifer agarilyticus, respectively. The mature agy1 was cloned and overexpressed as a His-tagged recombinant β-agarase (rAgy1) in Escherichia coli, and had a predicted molecular mass of 69 kDa and an isoelectric point of 4.5. rAgy1 showed optimum activity at 55oC and pH 7.6, and had a specific activity of 85 U/mg. The rAgy1 activity was enhanced by FeSO4 (40%), KCl (34%), and NaCl (34%), compared with the control. The newly identified rAgy1 is a β-agarase, which acts to degrade agarose to neoagarotetraose (NA4) and neoagarohexaose (NA6) and may be useful for applications in the cosmetics, food, bioethanol, and reagent industries.

  19. DkXTH8, a novel xyloglucan endotransglucosylase/hydrolase in persimmon, alters cell wall structure and promotes leaf senescence and fruit postharvest softening

    PubMed Central

    Han, Ye; Ban, Qiuyan; Li, Hua; Hou, Yali; Jin, Mijing; Han, Shoukun; Rao, Jingping

    2016-01-01

    Fruit softening is mainly associated with cell wall structural modifications, and members of the xyloglucan endotransglucosylase/hydrolase (XTH) family are key enzymes involved in cleaving and re-joining xyloglucan in the cell wall. In this work, we isolated a new XTH gene, DkXTH8, from persimmon fruit. Transcriptional profiling revealed that DkXTH8 peaked during dramatic fruit softening, and expression of DkXTH8 was stimulated by propylene and abscisic acid but suppressed by gibberellic acid and 1-MCP. Transient expression assays in onion epidermal cells indicated direct localization of DkXTH8 to the cell wall via its signal peptide. When expressed in vitro, the recombinant DkXTH8 protein exhibited strict xyloglucan endotransglycosylase activity, whereas no xyloglucan endohydrolase activity was observed. Furthermore, overexpression of DkXTH8 resulted in increased leaf senescence coupled with higher electrolyte leakage in Arabidopsis and faster fruit ripening and softening rates in tomato. Most importantly, transgenic plants overexpressing DkXTH8 displayed more irregular and twisted cells due to cell wall restructuring, resulting in wider interstitial spaces with less compact cells. We suggest that DkXTH8 expression causes cells to be easily destroyed, increases membrane permeability and cell peroxidation, and accelerates leaf senescence and fruit softening in transgenic plants. PMID:27966647

  20. Elucidation of exo-beta-D-glucosaminidase activity of a family 9 glycoside hydrolase (PBPRA0520) from Photobacterium profundum SS9.

    PubMed

    Honda, Yuji; Shimaya, Nozomi; Ishisaki, Kana; Ebihara, Mitsuru; Taniguchi, Hajime

    2011-04-01

    A glycoside hydrolase (GH) gene from Photobacterium profundum SS9 (PBPRA0520) belonging to GH family 9 was expressed in Escherichia coli. The protein was expressed with the intact N-terminal sequence, suggesting that it is an intracellular enzyme. The recombinant protein showed hydrolytic activity toward chitobiose [(GlcN)(2)] and cellobiose (CG(2)) in various disaccharides. This protein also released 4-nitrophenol (PNP) from both 4-nitrophenyl-β-D-glucosaminide (GlcN-PNP) and 4-nitrophenyl-β-D-glucoside (Glc-PNP). The hydrolytic pattern observed in chitooligosaccharides and cellooligosaccharides suggested that the reaction proceeded from the nonreducing end in an exo-type manner. Time-dependent (1)H-nuclear magnetic resonance (NMR) analysis of the anomeric form of the enzymatic reaction products indicated that the protein is an inverting enzyme. k(cat)/K(m) of (GlcN)(2) hydrolysis was 14 times greater than that of CG(2) hydrolysis. These results suggested that the protein is an exo-β-D-glucosaminidase (EC 3.2.1.165) rather than a glucan 1,4-β-D-glucosidase (EC 3.2.1.74). Based on the results, we suggest that the function of conserved GH9 proteins in the chitin catabolic operon is to cleave a (GlcN)(2)-phosphate derivative by hydrolysis during intracellular chitooligosaccharide catabolism in Vibrionaceae.

  1. Development of a novel optical biosensor for detection of organophosphorus pesticides based on methyl parathion hydrolase immobilized by metal-chelate affinity.

    PubMed

    Lan, Wensheng; Chen, Guoping; Cui, Feng; Tan, Feng; Liu, Ran; Yushupujiang, Maolidan

    2012-01-01

    We have developed a novel optical biosensor device using recombinant methyl parathion hydrolase (MPH) enzyme immobilized on agarose by metal-chelate affinity to detect organophosphorus (OP) compounds with a nitrophenyl group. The biosensor principle is based on the optical measurement of the product of OP catalysis by MPH (p-nitrophenol). Briefly, MPH containing six sequential histidines (6 × His tag) at its N-terminal was bound to nitrilotriacetic acid (NTA) agarose with Ni ions, resulting in the flexible immobilization of the bio-reaction platform. The optical biosensing system consisted of two light-emitting diodes (LEDs) and one photodiode. The LED that emitted light at the wavelength of the maximum absorption for p-nitrophenol served as the signal light, while the other LED that showed no absorbance served as the reference light. The optical sensing system detected absorbance that was linearly correlated to methyl parathion (MP) concentration and the detection limit was estimated to be 4 μM. Sensor hysteresis was investigated and the results showed that at lower concentration range of MP the difference got from the opposite process curves was very small. With its easy immobilization of enzymes and simple design in structure, the system has the potential for development into a practical portable detector for field applications.

  2. Substrate specificity and gene expression of two Penicillium chrysogenum α-L-arabinofuranosidases (AFQ1 and AFS1) belonging to glycoside hydrolase families 51 and 54.

    PubMed

    Sakamoto, Tatsuji; Inui, Misako; Yasui, Kana; Hosokawa, Sachiko; Ihara, Hideshi

    2013-02-01

    We previously isolated two α-L-arabinofuranosidases (ABFs), termed AFQ1 and AFS1, from the culture filtrate of Penicillium chrysogenum 31B. afq1 and afs1 complementary DNAs encoding AFQ1 and AFS1 were isolated by in vitro cloning. The deduced amino acid sequences of AFQ1 and AFS1 are highly similar to those of Penicillium purpurogenum ABF 2 and ABF 1, respectively, which belong to glycoside hydrolase (GH) families 51 and 54, respectively. Pfam analysis revealed an "Alpha-L-AF_C" domain in AFQ1 and "ArabFuran-catal" and "AbfB" domains in AFS1. Semi-quantitative RT-PCR analysis indicated that the afq1 gene was constitutively expressed in P. chrysogenum 31B at a low level, although the expression was slightly induced with arabinose, arabinitol, arabinan, and arabinoxylan. In contrast, expression of the afs1 gene was strongly expressed by the above four carbohydrates and less strongly induced by galactan. Recombinant enzymes (rAFQ1 and rAFS1) expressed in Escherichia coli were active against both p-nitrophenyl α-L-arabinofuranoside and polysaccharides with different specificities. (1)H-NMR analysis revealed that rAFS1 degraded arabinofuranosyl side chains that were both singly and doubly linked to the backbones of arabinoxylan and L-arabinan. On the other hand, rAFQ1 preferentially released arabinose linked to C-3 of single-substituted xylose or arabinose residues in the two polysaccharides.

  3. Optimization of synergism of a recombinant auxiliary activity 9 from Chaetomium globosum with cellulase in cellulose hydrolysis.

    PubMed

    Kim, In Jung; Nam, Ki Hyun; Yun, Eun Ju; Kim, Sooah; Youn, Hak Jin; Lee, Hee Jin; Choi, In-Geol; Kim, Kyoung Heon

    2015-10-01

    Auxiliary activity family 9 (AA9, formerly known as glycoside hydrolase family 61 or polysaccharide monooxygenase) is a group of fungal proteins that were recently found to have a significant synergism with cellulase in cellulose hydrolysis via the oxidative cleavage of glycosidic bonds of cellulose chains. In this study, we report the active expression of a recombinant fungal AA9 from Chaetomium globosum (CgAA9) in a bacterial host, Escherichia coli, and the optimization of its synergistic activity in cellulose hydrolysis by using cellulase. The recombinant CgAA9 (0.9 mg/g cellulose) exhibited 1.7-fold synergism in the hydrolysis of Avicel when incubated with 0.9 filter paper units of Celluclast 1.5 L/g cellulose. The first study of the active expression of AA9 using a bacterial host and its synergistic optimization could be useful for the industrial application of AA9 for the saccharification of lignocellulose.

  4. A Versatile Family 3 Glycoside Hydrolase from Bifidobacterium adolescentis Hydrolyzes β-Glucosides of the Fusarium Mycotoxins Deoxynivalenol, Nivalenol, and HT-2 Toxin in Cereal Matrices.

    PubMed

    Michlmayr, Herbert; Varga, Elisabeth; Malachova, Alexandra; Nguyen, Nhung Thi; Lorenz, Cindy; Haltrich, Dietmar; Berthiller, Franz; Adam, Gerhard

    2015-08-01

    Glycosylation plays a central role in plant defense against xenobiotics, including mycotoxins. Glucoconjugates of Fusarium toxins, such as deoxynivalenol-3-O-β-d-glucoside (DON-3G), often cooccur with their parental toxins in cereal-based food and feed. To date, only limited information exists on the occurrence of glucosylated mycotoxins and their toxicological relevance. Due to a lack of analytical standards and the requirement of high-end analytical instrumentation for their direct determination, hydrolytic cleavage of β-glucosides followed by analysis of the released parental toxins has been proposed as an indirect determination approach. This study compares the abilities of several fungal and recombinant bacterial β-glucosidases to hydrolyze the model analyte DON-3G. Furthermore, substrate specificities of two fungal and two bacterial (Lactobacillus brevis and Bifidobacterium adolescentis) glycoside hydrolase family 3 β-glucosidases were evaluated on a broader range of substrates. The purified recombinant enzyme from B. adolescentis (BaBgl) displayed high flexibility in substrate specificity and exerted the highest hydrolytic activity toward 3-O-β-d-glucosides of the trichothecenes deoxynivalenol (DON), nivalenol, and HT-2 toxin. A Km of 5.4 mM and a Vmax of 16 μmol min(-1) mg(-1) were determined with DON-3G. Due to low product inhibition (DON and glucose) and sufficient activity in several extracts of cereal matrices, this enzyme has the potential to be used for indirect analyses of trichothecene-β-glucosides in cereal samples. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  5. Recombineering: A Homologous Recombination-Based Method of Genetic Engineering

    PubMed Central

    Sharan, Shyam K.; Thomason, Lynn C.; Kuznetsov, Sergey G.; Court, Donald L.

    2009-01-01

    Recombineering is an efficient method of in vivo genetic engineering applicable to chromosomal as well as episomal replicons in E. coli. This method circumvents the need for most standard in vitro cloning techniques. Recombineering allows construction of DNA molecules with precise junctions without constraints being imposed by restriction enzyme site location. Bacteriophage homologous recombination proteins catalyze these recombineering reactions using double- and single-strand linear DNA substrates, so-called targeting constructs, introduced by electroporation. Gene knockouts, deletions and point mutations are readily made, gene tags can be inserted, and regions of bacterial artificial chromosomes (BACs) or the E. coli genome can be subcloned by gene retrieval using recombineering. Most of these constructs can be made within about a week's time. PMID:19180090

  6. Primordial magnetogenesis before recombination

    NASA Astrophysics Data System (ADS)

    Fabre, Ophélia; Shankaranarayanan, S.

    2016-04-01

    The origin of large magnetic fields in the Universe remains currently unknown. We investigate here a mechanism before recombination based on known physics. The source of the vorticity is due to the changes in the photon distribution function caused by the fluctuations in the background photons. We show that the magnetic field generated in the MHD limit, due to the Coulomb scattering, is of the order 10-49 G on a coherence scale of 10 kpc. We explicitly show that the magnetic fields generated from this process are sustainable and are not erased by resistive diffusion. We compare the results with current observations and discuss the implications. Our seed magnetic fields are generated on small scales whereas the main mechanisms studied in the literature are on scale bigger than 1 Mpc. However, compared to more exotic theories generating seed magnetic fields on similar scales, the strength of our fields are generally smaller.

  7. Demystified...recombinant antibodies.

    PubMed

    Smith, K A; Nelson, P N; Warren, P; Astley, S J; Murray, P G; Greenman, J

    2004-09-01

    Recombinant antibodies are important tools for biomedical research and are increasingly being used as clinical diagnostic/therapeutic reagents. In this article, a background to humanized antibodies is given, together with details of the generation of antibody fragments--for example, single chain Fv fragments. Phage antibody fragments are fast becoming popular and can be generated by simple established methods of affinity enrichment from libraries derived from immune cells. Phage display methodology can also be used for the affinity enrichment of existing antibody fragments to provide a reagent with a higher affinity. Here, phage antibodies are demystified to provide a greater understanding of the potential of these reagents and to engage clinicians and biomedical scientists alike to think about potential applications in pathology and clinical settings.

  8. Recombinant glucose uptake system

    DOEpatents

    Ingrahm, Lonnie O.; Snoep, Jacob L.; Arfman, Nico

    1997-01-01

    Recombinant organisms are disclosed that contain a pathway for glucose uptake other than the pathway normally utilized by the host cell. In particular, the host cell is one in which glucose transport into the cell normally is coupled to PEP production. This host cell is transformed so that it uses an alternative pathway for glucose transport that is not coupled to PEP production. In a preferred embodiment, the host cell is a bacterium other than Z. mobilis that has been transformed to contain the glf and glk genes of Z. mobilis. By uncoupling glucose transport into the cell from PEP utilization, more PEP is produced for synthesis of products of commercial importance from a given quantity of biomass supplied to the host cells.

  9. The recombination of genetic material

    SciTech Connect

    Low, K.B.

    1988-01-01

    Genetic recombination is the major mechanism by which new arrangements of genetic elements are produced in all living organisms, from the simplest bacterial viruses to humans. This volume presents an overview of the types of recombination found in prokaryotes and eukaryotes.

  10. Defining sequence space and reaction products within the cyanuric acid hydrolase (AtzD)/barbiturase protein family.

    PubMed

    Seffernick, Jennifer L; Erickson, Jasmine S; Cameron, Stephan M; Cho, Seunghee; Dodge, Anthony G; Richman, Jack E; Sadowsky, Michael J; Wackett, Lawrence P

    2012-09-01

    Cyanuric acid hydrolases (AtzD) and barbiturases are homologous, found almost exclusively in bacteria, and comprise a rare protein family with no discernible linkage to other protein families or an X-ray structural class. There has been confusion in the literature and in genome projects regarding the reaction products, the assignment of individual sequences as either cyanuric acid hydrolases or barbiturases, and spurious connection of this family to another protein family. The present study has addressed those issues. First, the published enzyme reaction products of cyanuric acid hydrolase are incorrectly identified as biuret and carbon dioxide. The current study employed (13)C nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry to show that cyanuric acid hydrolase releases carboxybiuret, which spontaneously decarboxylates to biuret. This is significant because it revealed that homologous cyanuric acid hydrolases and barbiturases catalyze completely analogous reactions. Second, enzymes that had been annotated incorrectly in genome projects have been reassigned here by bioinformatics, gene cloning, and protein characterization studies. Third, the AtzD/barbiturase family has previously been suggested to consist of members of the amidohydrolase superfamily, a large class of metallohydrolases. Bioinformatics and the lack of bound metals both argue against a connection to the amidohydrolase superfamily. Lastly, steady-state kinetic measurements and observations of protein stability suggested that the AtzD/barbiturase family might be an undistinguished protein family that has undergone some resurgence with the recent introduction of industrial s-triazine compounds such as atrazine and melamine into the environment.

  11. Enzymic hydrolysis of barley and other β-glucans by a β-(1→4)-glucan hydrolase

    PubMed Central

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

    1966-01-01

    1. A barley glucan with 68% of β-(1→4)-linkages and 32% of β-(1→3)-linkages was exhaustively hydrolysed with an Aspergillus niger β-(1→4)-glucan 4-glucanohydrolase (EC 3.2.1.4) (Clarke & Stone, 1965b). The hydrolysis products were separated and estimated. 2. The lower-molecular-weight products were identified as: glucose, 1·4%; cellobiose, 11·9%; 32-O-β-glucosylcellobiose, 45·0%; a tetrasaccharide(s), which was a substituted cellobiose, 16·4%. A series of unidentified higher-molecular-weight products (26·5%) were also found. 3. The identity of the products suggests that the A. niger β-(1→4)-glucan hydrolase hydrolyses β-glucosidic linkages joining 4-O-substituted glucose residues. 4. When an enzyme fraction containing the β-(1→4)-glucan hydrolase and an exo-β-(1→3)-glucan hydrolase was used, the same products were found, but the higher-molecular-weight products were observed to have only a transient existence in the hydrolysate and were virtually absent after prolonged incubation. It is suggested that these oligosaccharides are resistant to attack by β-(1→4)-glucan hydrolase but are partially hydrolysed by the exo-β-(1→3)-glucan hydrolase and therefore possess one or more (1→3)-linked glucose residues at their non-reducing end. PMID:4290404

  12. Auger recombination in sodium iodide

    NASA Astrophysics Data System (ADS)

    McAllister, Andrew; Kioupakis, Emmanouil; Åberg, Daniel; Schleife, André

    2014-03-01

    Scintillators are an important tool used to detect high energy radiation - both in the interest of national security and in medicine. However, scintillator detectors currently suffer from lower energy resolutions than expected from basic counting statistics. This has been attributed to non-proportional light yield compared to incoming radiation, but the specific mechanism for this non-proportionality has not been identified. Auger recombination is a non-radiative process that could be contributing to the non-proportionality of scintillating materials. Auger recombination comes in two types - direct and phonon-assisted. We have used first-principles calculations to study Auger recombination in sodium iodide, a well characterized scintillating material. Our findings indicate that phonon-assisted Auger recombination is stronger in sodium iodide than direct Auger recombination. Computational resources provided by LLNL and NERSC. Funding provided by NA-22.

  13. Bimolecular Recombination in Organic Photovoltaics

    NASA Astrophysics Data System (ADS)

    Lakhwani, Girish; Rao, Akshay; Friend, Richard H.

    2014-04-01

    The recombination of electrons and holes is a major loss mechanism in photovoltaic devices that controls their performance. We review scientific literature on bimolecular recombination (BR) in bulk heterojunction organic photovoltaic devices to bring forward existing ideas on the origin and nature of BR and highlight both experimental and theoretical work done to quantify its extent. For these systems, Langevin theory fails to explain BR, and recombination dynamics turns out to be dependent on mobility, temperature, electric field, charge carrier concentration, and trapped charges. Relationships among the photocurrent, open-circuit voltage, fill factor, and morphology are discussed. Finally, we highlight the recent emergence of a molecular-level picture of recombination, taking into account the spin and delocalization of charges. Together with the macroscopic picture of recombination, these new insights allow for a comprehensive understanding of BR and provide design principles for future materials and devices.

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

    PubMed

    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

    2016-05-01

    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.

  15. 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

  16. Gene expression of 5-lipoxygenase and LTA4 hydrolase in renal tissue of nephrotic syndrome patients

    PubMed Central

    Menegatti, E; Roccatello, D; Fadden, K; Piccoli, G; De Rosa, G; Sena, L M; Rifai, A

    1999-01-01

    Leukotrienes (LT) of the 5-lipoxygenase pathway constitute a class of potent biological lipid mediators of inflammation implicated in the pathogenesis of different models of experimental glomerulonephritis. The key enzyme, 5-lipoxygenase (5-LO), catalyses oxygenation of arachidonic acid to generate the primary leukotriene LTA4. This LT, in turn, serves as a substrate for either LTA4 hydrolase, to form the potent chemoattractant LTB4, or LTC4 synthase, to produce the powerful vasoconstrictor LTC4. To investigate the potential role of LT in the pathogenesis of human glomerulonephritis with nephrotic syndrome, we examined the gene expression of 5-LO and LTA4 hydrolase in renal tissue of 21 adult patients with nephrotic syndrome and 11 controls. The patients consisted of 11 cases of membranous nephropathy (MN), seven focal and segmental glomerulosclerosis (FSGS), two non-IgA mesangial glomerulonephritis and one minimal change disease. Total RNA purified from renal tissue was reverse transcribed into cDNA and amplified with specific primers in a polymerase chain reaction (RT-PCR). Eight patients' renal tissue, four MN and four FSGS, co-expressed 5-LO and LTA4 hydrolase. In situ hybridization analysis revealed 5-LO expression and distribution limited to the interstitial cells surrounding the peritubular capillaries. Comparative clinical and immunohistological data showed that these eight patients had impaired renal function and interstitial changes that significantly correlated with 5-LO expression. These findings suggest that leukotrienes may play an important role in the pathogenesis of MN and FSGS. These results are also relevant to elucidating the pathophysiologic mechanisms which underlie progression to renal failure in these diseases. PMID:10337029

  17. Polyglycine hydrolases secreted by Pleosporineae fungi that target the linker region of plant class IV chitinases.

    PubMed

    Naumann, Todd A; Wicklow, Donald T; Price, Neil P J

    2014-06-01

    Cmps (chitinase-modifying proteins) are fungal proteases that truncate plant class IV chitinases by cleaving near their N-termini. We previously described Fv-cmp, a fungalysin protease that cleaves a conserved glycine-cysteine bond within the hevein domain. In the present paper we describe a new type of cmp, polyglycine hydrolases, as proteases that selectively cleave glycine-glycine peptide bonds within the polyglycine linker of plant class IV chitinases. Polyglycine hydrolases were purified from Cochliobolus carbonum (syn. Bipolaris zeicola; Bz-cmp) and Epicoccum sorghi (syn. Phoma sorghina; Es-cmp) and were shown to cleave three different maize class IV chitinase substrates. The proteolytic cleavage sites were assessed by SDS/PAGE and MALDI-TOF-MS and indicated the cleavage of multiple peptide bonds within the polyglycine linker regions. Site-directed mutagenesis was used to produce mutants of maize ChitB chitinase in which two serine residues in its linker were systematically modified to glycine. Serine to glycine changes in the ChitB linker resulted in higher susceptibility to truncation by Bz-cmp and altered substrate specificity for Bz-cmp and Es-cmp, such that different glycine-glycine peptide bonds were cleaved. Removal of the hevein domain led to loss of Es-cmp activity, indicating that interactions outside of the active site are important for recognition. Our findings demonstrate that plant class IV chitinases with polyglycine linkers are targeted for truncation by selective polyglycine hydrolases that are secreted by plant pathogenic fungi. This novel proteolysis of polyglycine motifs is previously unreported, but the specificity is similar to that of bacterial lysostaphin proteases, which cleave pentaglycine cross-links from peptidoglycan.

  18. Substrate ambiguity among the nudix hydrolases: biologically significant, evolutionary remnant, or both?

    PubMed

    McLennan, Alexander G

    2013-02-01

    Many members of the nudix hydrolase family exhibit considerable substrate multispecificity and ambiguity, which raises significant issues when assessing their functions in vivo and gives rise to errors in database annotation. Several display low antimutator activity when expressed in bacterial tester strains as well as some degree of activity in vitro towards mutagenic, oxidized nucleotides such as 8-oxo-dGTP. However, many of these show greater activity towards other nucleotides such as ADP-ribose or diadenosine tetraphosphate (Ap(4)A). The antimutator activities have tended to gain prominence in the literature, whereas they may in fact represent the residual activity of an ancestral antimutator enzyme that has become secondary to the more recently evolved major activity after gene duplication. Whether any meaningful antimutagenic function has also been retained in vivo requires very careful assessment. Then again, other examples of substrate ambiguity may indicate as yet unexplored regulatory systems. For example, bacterial Ap(4)A hydrolases also efficiently remove pyrophosphate from the 5' termini of mRNAs, suggesting a potential role for Ap(4)A in the control of bacterial mRNA turnover, while the ability of some eukaryotic mRNA decapping enzymes to degrade IDP and dIDP or diphosphoinositol polyphosphates (DIPs) may also be indicative of new regulatory networks in RNA metabolism. DIP phosphohydrolases also degrade diadenosine polyphosphates and inorganic polyphosphates, suggesting further avenues for investigation. This article uses these and other examples to highlight the need for a greater awareness of the possible significance of substrate ambiguity among the nudix hydrolases as well as the need to exert caution when interpreting incomplete analyses.

  19. 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.

  20. A real-time fluorogenic assay for the visualization of glycoside hydrolase activity in planta.

    PubMed

    Ibatullin, Farid M; Banasiak, Alicja; Baumann, Martin J; Greffe, Lionel; Takahashi, Junko; Mellerowicz, Ewa J; Brumer, Harry

    2009-12-01

    There currently exists a diverse array of molecular probes for the in situ localization of polysaccharides, nucleic acids, and proteins in plant cells, including reporter enzyme strategies (e.g. protein-glucuronidase fusions). In contrast, however, there is a paucity of methods for the direct analysis of endogenous glycoside hydrolases and transglycosidases responsible for cell wall remodeling. To exemplify the potential of fluorogenic resorufin glycosides to address this issue, a resorufin beta-glycoside of a xylogluco-oligosaccharide (XXXG-beta-Res) was synthesized as a specific substrate for in planta analysis of XEH activity. The resorufin aglycone is particularly distinguished for high sensitivity in muro assays due to a low pK(a) (5.8) and large extinction coefficient (epsilon 62,000 M(-1) cm(-1)), long-wavelength fluorescence (excitation 571 nm/emission 585 nm), and high quantum yield (0.74) of the corresponding anion. In vitro analyses demonstrated that XXXG-beta-Res is hydrolyzed by the archetypal plant XEH, nasturtium (Tropaeolum majus) NXG1, with classical Michaelis-Menten substrate saturation kinetics and a linear dependence on both enzyme concentration and incubation time. Further, XEH activity could be visualized in real time by observing the localized increase in fluorescence in germinating nasturtium seeds and Arabidopsis (Arabidopsis thaliana) inflorescent stems by confocal microscopy. Importantly, this new in situ XEH assay provides an essential complement to the in situ xyloglucan endotransglycosylase assay, thus allowing delineation of the disparate activities encoded by xyloglucan endotransglycosylase/hydrolase genes directly in plant tissues. The observation that XXXG-beta-Res is also hydrolyzed by diverse microbial XEHs indicates that this substrate, and resorufin glycosides in general, may find broad applicability for the analysis of wall restructuring by polysaccharide hydrolases during morphogenesis and plant-microbe interactions.

  1. Carboxyl ester hydrolases production and growth of a halophilic archaeon, Halobacterium sp. NRC-1.

    PubMed

    Camacho, Rosa María; Mateos-Díaz, Juan Carlos; Diaz-Montaño, Dulce María; González-Reynoso, Orfil; Córdova, Jesús

    2010-01-01

    The capability of Halobacterium sp. NRC-1 to synthesize carboxyl ester hydrolases was investigated, and the effect of physicochemical conditions on the growth rate and production of esterases was evaluated. The haloarchaeon synthesized a carboxyl ester hydrolase, confirming the genomic prediction. This enzymatic activity was intracellularly produced as a growth-associated metabolite. Esterase activity was assayed using different p-nitrophenyl-esters and triacyl-glycerides, which showed a preference for hydrolyzing tributyrin. The archaeal growth rate and esterase production were significantly influenced by the pH and the NaCl concentration. An interaction effect between temperature and NaCl was also seen. The maximal growth rate and esterase production found for Halobacterium sp. NRC-1 were 0.136 h(-1) (at 4.2 M NaCl, pH 6 and 44 degrees C) and 1.64 U/l (at 4.6 M NaCl, pH 6 and 30 degrees C), respectively. Furthermore, the effects of NaCl concentration, pH and temperature on enzyme activity were studied. Two maximal esterase activities were elucidated from the intracellular crude extract when it was incubated at different NaCl concentrations (1 M and 5 M) and at different pHs (6 and 7.5). This is the first report that shows experimentally the synthesis of carboxyl ester hydrolases by Halobacterium sp. NRC-1. This enzyme was found to be extremely halophilic (5 M NaCl) and thermophilic (80 degrees C), making it very interesting for future investigations in non-aqueous biocatalysis.

  2. A novel and enantioselective epoxide hydrolase from Aspergillus brasiliensis CCT 1435: purification and characterization.

    PubMed

    Beloti, Lilian L; Costa, Bruna Z; Toledo, Marcelo A S; Santos, Clelton A; Crucello, Aline; Fávaro, Marianna T P; Santiago, André S; Mendes, Juliano S; Marsaioli, Anita J; Souza, Anete P

    2013-10-01

    A novel epoxide hydrolase from Aspergillus brasiliensis CCT1435 (AbEH) was cloned and overexpressed in Escherichia coli cells with a 6xHis-tag and purified by nickel affinity chromatography. Gel filtration analysis and circular dichroism measurements indicated that this novel AbEH is a homodimer in aqueous solution and contains the typical secondary structure of an α/β hydrolase fold. The activity of AbEH was initially assessed using the fluorogenic probe O-(3,4-epoxybutyl) umbelliferone and was active in a broad range of pH (6-9) and temperature (25-45°C); showing optimum performance at pH 6.0 and 30°C. The Michaelis constant (KM) and maximum rate (Vmax) values were 495μM and 0.24μM/s, respectively. Racemic styrene oxide (SO) was used as a substrate to assess the AbEH activity and enantioselectivity, and 66% of the SO was hydrolyzed after only 5min of reaction, with the remaining (S)-SO ee exceeding 99% in a typical kinetic resolution behavior. The AbEH-catalyzed hydrolysis of SO was also evaluated in a biphasic system of water:isooctane; (R)-diol in 84% ee and unreacted (S)-SO in 36% ee were produced, with 43% conversion in 24h, indicating a discrete enantioconvergent behavior for AbEH. This novel epoxide hydrolase has biotechnological potential for the preparation of enantiopure epoxides or vicinal diols.

  3. Fatty acid amide hydrolase: an emerging therapeutic target in the endocannabinoid system.

    PubMed

    Cravatt, Benjamin F; Lichtman, Aron H

    2003-08-01

    The medicinal properties of exogenous cannabinoids have been recognized for centuries and can largely be attributed to the activation in the nervous system of a single G-protein-coupled receptor, CB1. However, the beneficial properties of cannabinoids, which include relief of pain and spasticity, are counterbalanced by adverse effects such as cognitive and motor dysfunction. The recent discoveries of anandamide, a natural lipid ligand for CB1, and an enzyme, fatty acid amide hydrolase (FAAH), that terminates anandamide signaling have inspired pharmacological strategies to augment endogenous cannabinoid ('endocannabinoid') activity with FAAH inhibitors, which might exhibit superior selectivity in their elicited behavioral effects compared with direct CB1 agonists.

  4. Remodeling Natural Products: Chemistry and Serine Hydrolase Activity of a Rocaglate-Derived β-Lactone

    PubMed Central

    2015-01-01

    Flavaglines are a class of natural products with potent insecticidal and anticancer activities. β-Lactones are a privileged structural motif found in both therapeutic agents and chemical probes. Herein, we report the synthesis, unexpected light-driven di-epimerization, and activity-based protein profiling of a novel rocaglate-derived β-lactone. In addition to in vitro inhibition of the serine hydrolases ABHD10 and ACOT1/2, the most potent β-lactone enantiomer was also found to inhibit these enzymes, as well as the serine peptidases CTSA and SCPEP1, in PC3 cells. PMID:24447064

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

    PubMed Central

    2013-01-01

    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. [11C]-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

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

    DOE PAGES

    Yarbrough, John M.; Mittal, Ashutosh; Mansfield, Elisabeth; ...

    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.

  7. Enantioselective hydrolysis of racemic epichlorohydrin using an epoxide hydrolase from Novosphingobium aromaticivorans.

    PubMed

    Woo, Jung-Hee; Hwang, Young-Ok; Kang, Ji-Hyun; Lee, Hyun Sook; Kim, Sang-Jin; Kang, Sung Gyun

    2010-09-01

    Previously we reported that an epoxide hydrolase (EHase) from Novosphingobium aromaticivorans could preferentially hydrolyze (R)-styrene oxide. In this study, we demonstrate that the purified NEH could be also effective in chiral resolution of racemic epichlorohydrin (ECH). Particularly, the purified NEH showed excellent hydrolyzing activity toward ECH to complete the reaction at a short period of incubation time. Enantiopure (S)-ECH could be obtained with a high enantiopurity of more than 99.99% enantiomeric excess (ee) and yield of 20.7% (theoretical, 50%). The chiral resolution of the purified NEH toward ECH was not susceptible to substrate inhibition by 500 mM racemic ECH.

  8. Some hydrolase activities from the tick Hyalomma lusitanicum koch, 1844 (Ixodoidea: Ixodida).

    PubMed

    Giménez-Pardo, C; Martínez-Grueiro, M M

    2008-12-01

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

  9. Acyl hydrolases from trans-AT polyketide synthases target acetyl units on acyl carrier proteins.

    PubMed

    Jenner, Matthew; Afonso, Jose P; Kohlhaas, Christoph; Karbaum, Petra; Frank, Sarah; Piel, Jörn; Oldham, Neil J

    2016-04-18

    Acyl hydrolase (AH) domains are a common feature of trans-AT PKSs. They have been hypothesised to perform a proofreading function by removing acyl chains from stalled sites. This study determines the substrate tolerance of the AH PedC for a range of acyl-ACPs. Clear preference towards short, linear acyl-ACPs is shown, with acetyl-ACP the best substrate. These results imply a more targeted housekeeping role for PedC: namely the removal of unwanted acetyl groups from ACP domains caused by erroneous transfer of acetyl-CoA, or possibly by decarboxylation of malonyl-ACP.

  10. [Research advances on ubiquitin C-terminal hydrolase in oncogenesis and progression].

    PubMed

    Yu, Juan; Chen, Wei-lin

    2015-03-01

    By regulating the ubiquitination and deubiquitination of key proteins, ubiquitin-proteasome system mediates a variety of cellular activities. Ubiquitin C-terminal hydrolase (UCH) is a deubiquitinating enzyme which can remove ubiquitin chains at the end of ubiquited proteins. The abnormal expression of UCH has been found in a variety of tumor tissues, indicating that it participates in the process of tumor development. Here we review the characteristics of UCH members and current understanding about the role of UCH in tumor development, and the potential target for cancer treatment.

  11. N-aryl 2-aryloxyacetamides as a new class of fatty acid amide hydrolase (FAAH) inhibitors.

    PubMed

    Sunduru, Naresh; Svensson, Mona; Cipriano, Mariateresa; Marwaha, Sania; Andersson, C David; Svensson, Richard; Fowler, Christopher J; Elofsson, Mikael

    2017-12-01

    Fatty acid amide hydrolase (FAAH) is a promising target for the development of drugs to treat neurological diseases. In search of new FAAH inhibitors, we identified 2-(4-cyclohexylphenoxy)-N-(3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)acetamide, 4g, with an IC50 of 2.6 µM as a chemical starting point for the development of potent FAAH inhibitors. Preliminary hit-to-lead optimisation resulted in 2-(4-phenylphenoxy)-N-(3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)acetamide, 4i, with an IC50 of 0.35 µM.

  12. Aryl Piperazinyl Ureas as Inhibitors of Fatty Acid Amide Hydrolase (FAAH) in Rat, Dog, and Primate.

    PubMed

    Keith, John M; Apodaca, Rich; Tichenor, Mark; Xiao, Wei; Jones, William; Pierce, Joan; Seierstad, Mark; Palmer, James; Webb, Michael; Karbarz, Mark; Scott, Brian; Wilson, Sandy; Luo, Lin; Wennerholm, Michelle; Chang, Leon; Brown, Sean; Rizzolio, Michele; Rynberg, Raymond; Chaplan, Sandra; Breitenbucher, J Guy

    2012-10-11

    A series of aryl piperazinyl ureas that act as covalent inhibitors of fatty acid amide hydrolase (FAAH) is described. A potent and selective (does not inhibit FAAH-2) member of this class, JNJ-40355003, was found to elevate the plasma levels of three fatty acid amides: anandamide, oleoyl ethanolamide, and palmitoyl ethanolamide, in the rat, dog, and cynomolgous monkey. The elevation of the levels of these lipids in the plasma of monkeys suggests that FAAH-2 may not play a significant role in regulating plasma levels of fatty acid ethanolamides in primates.

  13. 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.

  14. Characterization of organophosphorus hydrolases and the genetic manipulation of the phosphotriesterase from Pseudomonas diminuta.

    PubMed

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

    1993-06-01

    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 Flavobacterium sp. ATCC 27551, and the heterologous expression, post-translational modification, and genetic engineering studies undertaken with this enzyme are described.

  15. Synthesis and biochemical evaluation of guanidino-alkyl-ribitol derivatives as nucleoside hydrolase inhibitors.

    PubMed

    Goeminne, A; McNaughton, M; Bal, G; Surpateanu, G; Van Der Veken, P; De Prol, S; Versées, W; Steyaert, J; Haemers, A; Augustyns, K

    2008-02-01

    Nucleoside hydrolase (NH) is a key enzyme in the purine salvage pathway. The purine specificity of the IAG-NH from Trypanosoma vivax is at least in part due to cation-pi-stacking interactions. Guanidinium ions can be involved in cation-pi-stacking interactions, therefore a series of guanidino-alkyl-ribitol derivatives were synthesized in order to examine the binding affinity of these compounds towards the target enzyme. The compounds show moderate to good inhibiting activity towards the IAG-NH from T. vivax.

  16. 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.

  17. Neutron diffraction analysis of Pseudomonas aeruginosa peptidyl-tRNA hydrolase 1.

    PubMed

    McFeeters, Hana; Vandavasi, Venu Gopal; Weiss, Kevin L; Coates, Leighton; McFeeters, Robert L

    2016-03-01

    Perdeuterated peptidyl-tRNA hydrolase 1 from Pseudomonas aeruginosa was crystallized for structural analysis using neutron diffraction. Crystals of perdeuterated protein were grown to 0.15 mm(3) in size using batch crystallization in 22.5% polyethylene glycol 4000, 100 mM Tris pH 7.5, 10%(v/v) isopropyl alcohol with a 20-molar excess of trilysine as an additive. Neutron diffraction data were collected from a crystal at room temperature using the MaNDi single-crystal diffractometer at Oak Ridge National Laboratory.

  18. Assay of phospholipases C and D in presence of other lipid hydrolases

    SciTech Connect

    Hostetler, K.Y.; Gardner, M.F.; Aldern, K.A.

    1991-01-01

    The activity of a phospholipase C or phospholipase D may be assessed by measuring the radioactivity or phosphate released into the aqueous phase of a lipid extract. However, in crude enzyme fractions, this type of analysis may not be possible due to formation of water-soluble metabolites by other enzymatic reactions, as demonstrated here with a crude lysosomal enzyme fraction. In such instances, analysis of both water-soluble and lipid-soluble metabolites, at various times of incubation, may still provide clear identification of phospholipases C or D, even when a variety of lipases and other hydrolases are present.

  19. 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.

  20. Effect of aloe vera leaf gel extract on membrane bound phosphatases and lysosomal hydrolases in rats with streptozotocin diabetes.

    PubMed

    Rajasekaran, S; Sriram, N; Arulselvan, P; Subramanian, S

    2007-03-01

    Diabetes mellitus is known to promote deterioration of membrane function and impair intra cellular metabolism in the organism. The aim of the present study was to examine the effect of the ethanolic extract from Aloe vera leaf gel on membrane bound phosphatases and lysosomal hydrolases in the liver and kidney of streptozotocin (STZ)-induced diabetic rats. The rats treated with STZ showed significant alterations in the activities of membrane bound phosphatases and lysosomal hydrolases in the liver and kidney. Oral administration of Aloe vera gel extract at a dose of 300 mg/kg body weight/day to STZ-induced diabetic rats for a period of 21 days significantly restored the alterations in enzymes activity to near normalcy. These results were compared with glibenclamide, a reference drug. Thus, the present study confirms that Aloe vera gel extract possesses a significant beneficial effect on membrane bound phosphatases and lysosomal hydrolases.

  1. Delayed recombination and standard rulers

    SciTech Connect

    De Bernardis, Francesco; Melchiorri, Alessandro; Bean, Rachel; Galli, Silvia; Silk, Joseph I.; Verde, Licia

    2009-02-15

    Measurements of baryonic acoustic oscillations (BAOs) in galaxy surveys have been recognized as a powerful tool for constraining dark energy. However, this method relies on the knowledge of the size of the acoustic horizon at recombination derived from cosmic microwave background (CMB) anisotropy measurements. This estimate is typically derived assuming a standard recombination scheme; additional radiation sources can delay recombination altering the cosmic ionization history and the cosmological inferences drawn from CMB and BAO data. In this paper we quantify the effect of delayed recombination on the determination of dark energy parameters from future BAO surveys such as the Baryon Oscillation Spectroscopic Survey and the Wide-Field Multi-Object Spectrograph. We find the impact to be small but still not negligible. In particular, if recombination is nonstandard (to a level still allowed by CMB data), but this is ignored, future surveys may incorrectly suggest the presence of a redshift-dependent dark energy component. On the other hand, in the case of delayed recombination, adding to the analysis one extra parameter describing deviations from standard recombination does not significantly degrade the error bars on dark energy parameters and yields unbiased estimates. This is due to the CMB-BAO complementarity.

  2. Distinct rat hepatic microsomal epoxide hydrolases catalyze the hydration of cholesterol 5,6 alpha-oxide and certain xenobiotic alkene and arene oxides.

    PubMed

    Levin, W; Michaud, D P; Thomas, P E; Jerina, D M

    1983-02-01

    Metabolism of cholesterol 5,6 alpha-oxide to the 5,6-glycol is catalyzed by a rat liver microsomal epoxide hydrolase that is distinct from the microsomal epoxide hydrolase that metabolizes a wide range of xenobiotic alkene and arene oxides. The two enzymes are antigenically distinct, and the purified microsomal epoxide hydrolase that metabolizes xenobiotic oxides does not catalyze the hydration of cholesterol 5,6 alpha-oxide. In vivo treatment of rats with inducers of microsomal epoxide hydrolase does not enhance the activity of cholesterol 5,6 alpha-oxide hydrolase and, in some cases, actually depresses enzyme activity in the resultant microsomal preparations. Octene 1,2-oxide and benz[a]anthracene 5,6-oxide, both good substrates for xenobiotic epoxide hydrolase, are not competitive inhibitors of cholesterol oxide hydration by rat liver microsomes. The above results establish the existence of a liver microsomal epoxide hydrolase that is under different regulatory control and that appears to have a different substrate specificity than the well-characterized microsomal epoxide hydrolase involved in the metabolism of a widely diverse group of alkene and arene oxides.

  3. Qualitative analysis of the fluorophosphonate-based chemical probes using the serine hydrolases from mouse liver and poly-3-hydroxybutyrate depolymerase (PhaZ) from Bacillus thuringiensis.

    PubMed

    Huang, Yi-Long; Chung, Tsai-Wen; Chang, Chia-Mao; Chen, Chih-Hau; Liao, Chen-Chung; Tsay, Yeou-Guang; Shaw, Gwo-Chyuan; Liaw, Shwu-Huey; Sun, Chung-Ming; Lin, Chao-Hsiung

    2012-11-01

    The serine hydrolase family consists of more than 200 members and is one of the largest enzyme families in the human genome. Although up to 50 % of this family remains unannotated, there are increasing evidences that activities of certain serine hydrolases are associated with diseases like cancer neoplasia, invasiveness, etc. By now, several activity-based chemical probes have been developed and are applied to profile the global activity of serine hydrolases in diverse proteomes. In this study, two fluorophosphonate (FP)-based chemical probes were synthesized. Further examination of their abilities to label and pull down serine hydrolases was conducted. In addition, the poly-3-hydroxybutyrate depolymerase (PhaZ) from Bacillus thuringiensis was demonstrated as an appropriate standard serine hydrolase, which can be applied to measure the labeling ability and pull-down efficiency of FP-based probes. Furthermore, mass spectrometry (MS) was used to identify the serine residue that covalently bonded to the active probes. Finally, these FP-based probes were shown capable of establishing the serine hydrolase profiles in diverse mouse tissues; the serine hydrolases pulled down from mouse liver organ were further identified by MS. In summary, our study provides an adequate method to evaluate the reactivity of FP-based probes targeting serine hydrolases.

  4. Co-purification of microsomal epoxide hydrolase with the warfarin-sensitive vitamin K1 oxide reductase of the vitamin K cycle.

    PubMed

    Guenthner, T M; Cai, D; Wallin, R

    1998-01-15

    Vitamin K1 oxide reductase activity has been partially purified from rat liver microsomes. A three-step procedure produced a preparation in which warfarin-sensitive vitamin K1 oxide reductase activity was 118-fold enriched over the activity in intact rat liver microsomes. A major component of the multi-protein mixture was identified as a 50 kDa protein that strongly cross-reacts with antiserum prepared against homogeneous rat liver microsomal epoxide hydrolase. The reductase preparation also had a high level or epoxide hydrolase activity against two xenobiotic epoxide substrates. The K(m) values for hydrolysis by the reductase preparation were similar to those for homogeneous microsomal epoxide hydrolase itself, and the specific hydrolase activities of the reductase preparation were 25-35% of the specific activities measured for the homogeneous hydrolase preparation. Antibodies prepared against homogeneous microsomal epoxide hydrolase inhibited up to 80% of reductase activity of the reductase preparation. Homogeneous microsomal epoxide hydrolase had no vitamin K1 oxide reductase activity. This evidence suggests that microsomal epoxide hydrolase, or a protein that is very similar to it, is a major functional component of a multi-protein complex that is responsible for vitamin K1 oxide reduction in rat liver microsomes.

  5. 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

  6. Genetic surface-display of methyl parathion hydrolase on Yarrowia lipolytica for removal of methyl parathion in water.

    PubMed

    Wang, Xing-Xing; Chi, Zhe; Ru, Shao-Guo; Chi, Zhen-Ming

    2012-09-01

    In this study, the mph gene encoding methyl parathion hydrolase from Pseudomonas sp. WBC-3 was expressed in Yarrowia lipolytica and the expressed methyl parathion hydrolase was displayed on cell surface of Y. lipolytica. The activity of methyl parathion hydrolase displayed on the yeast cells of the transformant Z51 was 59.5 U mg⁻¹ of cell dry cells (450.6 U per mL of the culture) in the presence of 5.0 mM of Co²⁺. The displayed methyl parathion hydrolase had the optimal pH of 9.5 and the optimal temperature of 40 °C, respectively and was stable in the pH range of 4.5-11 and up to 40 °C. The displayed methyl parathion hydrolase was also stimulated by Co²⁺, Cu²⁺, Ni²⁺ and Mn²⁺, and was not affected by Fe²⁺, Fe³⁺, Na⁺, K⁺, Ca²⁺ and Zn²⁺, but was inhibited by other cations tested. Under the optimal conditions (OD(600 nm) = 2.6, the substrate concentration = 100 mg L⁻¹ and 40 °C), 90.8 % of methyl parathion was hydrolyzed within 30 min. Under the similar conditions, 98.7, 97.0, 96.5 and 94.4 % of methyl parathion in tap water (pH 9.5), tap water (pH 6.8), seawater (pH 9.5) and natural seawater (pH 8.2) were hydrolyzed, respectively, suggesting that the methyl parathion hydrolase displayed on the yeast cells can effectively remove methyl parathion in water.

  7. 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.

  8. 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

  9. Testing for recombinant erythropoietin.

    PubMed

    Delanghe, Joris R; Bollen, Mathieu; Beullens, Monique

    2008-03-01

    Erythropoietin (Epo) is a glycoprotein hormone that promotes the production of red blood cells. Recombinant human Epo (rhEpo) is illicitly used to improve performance in endurance sports. Doping in sports is discouraged by the screening of athletes for rhEpo. Both direct tests (indicating the presence of exogeneous Epo isoforms) and indirect tests (indicating hematological changes induced by exogenous Epo administration) can be used for Epo detection. At present, the test adopted by the World Anti Doping Agency is based on a combination of isoelectric focusing and double immunoblotting, and distinguishes between endogenous and rhEpo. However, the adopted monoclonal anti-Epo antibodies are not monospecific. Therefore, the test can occasionally lead to the false-positive detection of rhEpo (epoetin-beta) in post-exercise, protein-rich urine, or in case of contamination of the sample with microorganisms. An improved preanalytical care may counteract a lot of these problems. Adaptation of the criteria may be helpful to further refine direct Epo testing. Indirect tests have the disadvantage that they require blood instead of urine samples, but they can be applied to detect a broader range of performance improving techniques which are illicitly used in sports.

  10. Recombinant monoclonal antibody technology.

    PubMed

    Siegel, D L

    2002-01-01

    With the development of murine hybridoma technology over a quarter century ago, the ability to produce large quantities of well-characterized monoclonal antibody preparations revolutionized diagnostic and therapeutic medicine. For many applications in transfusion medicine, however, the production of serological reagents in mice has certain biological limitations relating to the difficulty in obtaining murine monoclonal antibodies specific for many human blood group antigens. Furthermore, for therapeutic purposes, the efficacy of murine-derived immunoglobulin preparations is limited by the induction of anti-mouse immune responses. Technical difficulties inherent in human hybridoma formation have led to novel molecular approaches that facilitate the isolation and production of human antibodies without the need for B-cell transformation, tissue culture, or even immunized individuals. These technologies, referred to as 'repertoire cloning' or 'Fab/phage display', involve the rapid cloning of immunoglobulin gene segments to create immune libraries from which antibodies with desired specificities can be selected. The use of such recombinant methods in transfusion medicine is anticipated to play an important role in the development and production of renewable supplies of low-cost reagents for diagnostic and therapeutic applications.

  11. 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°.

  12. 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.

  13. Hydrolysis of the 2',3'-allylic epoxides of allylbenzene, estragole, eugenol, and safrole by both microsomal and cytosolic epoxide hydrolases.

    PubMed

    Luo, G; Qato, M K; Guenthner, T M

    1992-01-01

    2',3'-Allylic epoxide derivatives of allylbenzene and its analogs estragole, eugenol, and safrole were synthesized, and their enzymatic conversion to dihydrodiols by cytosolic and microsomal epoxide hydrolases was examined. All four epoxides were good substrates for both epoxide hydrolases, with Michaelis constants in the low micromolar range. Two putatively selective inhibitors of cytosolic and microsomal epoxide hydrolases, trichloropropylene oxide and nordihydroguaiaretic acid, were used to inhibit the hydrolysis of these allylic epoxides. Minimal selectivity toward either hydrolase was seen with either inhibitor, suggesting that the "selectivity" of these inhibitors is highly substrate-dependent. The susceptibilities of these epoxides to rapid hydrolysis by both epoxide hydrolases may explain their low genotoxic potencies in vivo.

  14. Recombineering: genetic engineering in bacteria using homologous recombination.

    PubMed

    Thomason, Lynn C; Sawitzke, James A; Li, Xintian; Costantino, Nina; Court, Donald L

    2014-04-14

    The bacterial chromosome and bacterial plasmids can be engineered in vivo by homologous recombination using PCR products and synthetic oligonucleotides as substrates. This is possible because bacteriophage-encoded recombination proteins efficiently recombine sequences with homologies as short as 35 to 50 bases. Recombineering allows DNA sequences to be inserted or deleted without regard to location of restriction sites. This unit first describes preparation of electrocompetent cells expressing the recombineering functions and their transformation with dsDNA or ssDNA. It then presents support protocols that describe several two-step selection/counter-selection methods of making genetic alterations without leaving any unwanted changes in the targeted DNA, and a method for retrieving onto a plasmid a genetic marker (cloning by retrieval) from the Escherichia coli chromosome or a co-electroporated DNA fragment. Additional protocols describe methods to screen for unselected mutations, removal of the defective prophage from recombineering strains, and other useful techniques. Copyright © 2014 John Wiley & Sons, Inc.

  15. Inhibition of recombinant human carboxylesterase 1 and 2 and monoacylglycerol lipase by chlorpyrifos oxon, paraoxon and methyl paraoxon

    SciTech Connect

    Crow, J. Allen; Bittles, Victoria; Herring, Katye L.; Borazjani, Abdolsamad; Potter, Philip M.; Ross, Matthew K.

    2012-01-01

    Oxons are the bioactivated metabolites of organophosphorus insecticides formed via cytochrome P450 monooxygenase-catalyzed desulfuration of the parent compound. Oxons react covalently with the active site serine residue of serine hydrolases, thereby inactivating the enzyme. A number of serine hydrolases other than acetylcholinesterase, the canonical target of oxons, have been reported to react with and be inhibited by oxons. These off-target serine hydrolases include carboxylesterase 1 (CES1), CES2, and monoacylglycerol lipase. Carboxylesterases (CES, EC 3.1.1.1) metabolize a number of xenobiotic and endobiotic compounds containing ester, amide, and thioester bonds and are important in the metabolism of many pharmaceuticals. Monoglyceride lipase (MGL, EC 3.1.1.23) hydrolyzes monoglycerides including the endocannabinoid, 2-arachidonoylglycerol (2-AG). The physiological consequences and toxicity related to the inhibition of off-target serine hydrolases by oxons due to chronic, low level environmental exposures are poorly understood. Here, we determined the potency of inhibition (IC{sub 50} values; 15 min preincubation, enzyme and inhibitor) of recombinant CES1, CES2, and MGL by chlorpyrifos oxon, paraoxon and methyl paraoxon. The order of potency for these three oxons with CES1, CES2, and MGL was chlorpyrifos oxon > paraoxon > methyl paraoxon, although the difference in potency for chlorpyrifos oxon with CES1 and CES2 did not reach statistical significance. We also determined the bimolecular rate constants (k{sub inact}/K{sub I}) for the covalent reaction of chlorpyrifos oxon, paraoxon and methyl paraoxon with CES1 and CES2. Consistent with the results for the IC{sub 50} values, the order of reactivity for each of the three oxons with CES1 and CES2 was chlorpyrifos oxon > paraoxon > methyl paraoxon. The bimolecular rate constant for the reaction of chlorpyrifos oxon with MGL was also determined and was less than the values determined for chlorpyrifos oxon with CES1

  16. 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

  17. Ubiquitin hydrolase Dub3 promotes oncogenic transformation by stabilizing Cdc25A.

    PubMed

    Pereg, Yaron; Liu, Bob Y; O'Rourke, Karen M; Sagolla, Meredith; Dey, Anwesha; Komuves, Laszlo; French, Dorothy M; Dixit, Vishva M

    2010-04-01

    The dual specificity (Tyr/Thr) phosphatase Cdc25A activates cyclin-dependent kinases (Cdks) to promote cell-cycle progression and has significant oncogenic potential. Cdc25A protein levels are regulated tightly in normal tissues, but many human cancers overexpress Cdc25A. The underlying mechanism for overexpression has been enigmatic. Here we show that Cdc25A is stabilized by the ubiquitin hydrolase Dub3. Upon binding Cdc25A, Dub3 removes the polyubiquitin modifications that mark Cdc25A for proteasomal degradation. Dub3 knockdown in cells increased Cdc25A ubiquitylation and degradation, resulting in reduced Cdk/Cyclin activity and arrest at G1/S and G2/M phases of the cell cycle. In contrast, acute Dub3 overexpression produced a signature response to oncogene induction: cells accumulated in S and G2 because of replication stress, and activated a DNA damage response. Dub3 also transformed NIH-3T3 cells and cooperated with activated H-Ras to promote growth in soft agar. Importantly, we show that Dub3 overexpression is responsible for an abnormally high level of Cdc25A in a subset of human breast cancers. Moreover, Dub3 knockdown significantly retarded the growth of breast tumour xenografts in nude mice. As a major regulator of Cdc25A, Dub3 is an example of a transforming ubiquitin hydrolase that subverts a key component of the cell cycle machinery.

  18. 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.

  19. 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.

  20. Hepatic Retinyl Ester Hydrolases and the Mobilization of Retinyl Ester Stores.

    PubMed

    Grumet, Lukas; Taschler, Ulrike; Lass, Achim

    2016-12-27

    For mammals, vitamin A (retinol and metabolites) is an essential micronutrient that is required for the maintenance of life. Mammals cannot synthesize vitamin A but have to obtain it from their diet. Resorbed dietary vitamin A is stored in large quantities in the form of retinyl esters (REs) in cytosolic lipid droplets of cells to ensure a constant supply of the body. The largest quantities of REs are stored in the liver, comprising around 80% of the body's total vitamin A content. These hepatic vitamin A stores are known to be mobilized under times of insufficient dietary vitamin A intake but also under pathological conditions such as chronic alcohol consumption and different forms of liver diseases. The mobilization of REs requires the activity of RE hydrolases. It is astounding that despite their physiological significance little is known about their identities as well as about factors or stimuli which lead to their activation and consequently to the mobilization of hepatic RE stores. In this review, we focus on the recent advances for the understanding of hepatic RE hydrolases and discuss pathological conditions which lead to the mobilization of hepatic RE stores.

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

    PubMed

    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

    2015-11-05

    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.

  2. 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.

  3. Beta-glucuronidase of family-2 glycosyl hydrolase: a missing member in plants.

    PubMed

    Arul, Loganathan; Benita, George; Sudhakar, Duraialagaraja; Thayumanavan, Balsamy; Balasubramanian, Ponnusamy

    2008-01-01

    Glycosyl hydrolases hydrolyze the glycosidic bond in carbohydrates or between a carbohydrate and a non-carbohydrate moiety. beta-glucuronidase (GUS) is classified under two glycosyl hydrolase families (2 and 79) and the family-2 beta-glucuronidase is reported in a wide range of organisms, but not in plants. The family-79 endo-beta-glucuronidase (heparanase) is reported in microorganisms, vertebrates and plants. The E. coli family-2 beta-glucuronidase (uidA) had been successfully devised as a reporter gene in plant transformation on the basis that plants do not have homologous GUS activity. On the contrary, histochemical staining with X-Gluc was reported in wild type (non-transgenic) plants. Data shows that, family-2 beta-glucuronidase homologous sequence is not found in plants. Further, beta-glucuronidases of family-2 and 79 lack appreciable sequence similarity. However, the catalytic site residues, glutamic acid and tyrosine of the family-2 beta-glucuronidase are found to be conserved in family-79 beta-glucuronidase of plants. This led to propose that the GUS staining reported in wild type plants is largely because of the broad substrate specificity of family-79 beta-glucuronidase on X-Gluc and not due to the family-2 beta-glucuronidase, as the latter has been found to be missing in plants.

  4. Alteration of the mutagenicity 3,3'-dichlorobenzidine by modifiers of rat hepatic epoxide hydrolase activity

    SciTech Connect

    Iba, M.M.

    1986-03-05

    The involvement of arene oxides in the activation of benzidines was assessed by examining the effect of (I) the epoxide hydrolase inhibitor trichloropropylene oxide (TCPO), (II) purified rat liver microsomal (P) epoxide hydrolase (EH), and (III) pretreatment of rats with phenobarbital (PB) on hepatic Sg- or P-catalyzed mutagenicity of benzidine (BZ) and 3,3'-dichlorobenzidine (DCB) to Salmonella TA 98. When catalyzed by Sg from untreated rats, the mutagenicity of DCB and BZ was 601 +/- 101 and 79 +/- 25 (His/sup +/ revertants/plate) respectively, but was 345 +/- 55 and 226 +/- 30 respectively, when catalyzed by microsomes (P) from untreated rats. PB-pretreatment enhanced the Sg-catalyzed mutagenicity of DCB and BZ (2.3-fold and 1.7-fold, respectively) and the P-catalyzed mutagenicity of DCB (1.7-fold), but totally inhibited the P-catalyzed mutagenicity of BZ. In TCPO-supplemented activating systems from PB-pretreated rats, the mutagenicity of DCB was enhanced in both Sg and P (1.9-fold and 1.6-fold, respectively), whereas that of BZ was unchanged. Added EH enhanced the P-catalyzed mutagenicity of DCB (1.4-fold) but had no effect on that of BZ, suggesting that the activity of the enzyme on DCB metabolites may not be entirely detoxifying. The data suggest that epoxidation may contribute to the activation of DCB but not BZ.

  5. Improving the thermostability of a methyl parathion hydrolase by adding the ionic bond on protein surface.

    PubMed

    Su, Yidan; Tian, Jian; Wang, Ping; Chu, Xiaoyu; Liu, Guoan; Wu, Ningfeng; Fan, Yunliu

    2011-10-01

    The thermostability of the methyl parathion hydrolase (MPH_OCH) from Ochrobactrum sp. M231 was improved using site-directed mutagenesis. Two prolines (Pro76 and Pro78) located on the protein surface were selected for mutations after inspection of the sequence alignment of MPH_OCH and OPHC2, a thermostable organophosphorus hydrolase from Pseudomonas pseudoalcaligenes C2-1. The temperature of the double-point mutant (P76D/P78K) at which the mutant lost 50% of its activity (T50) was approximately 68 °C, which is higher than that of WT enzyme (64 °C), P76D (67 °C), and P78K (59 °C). Structural analysis of P76D/P78K indicated that the substituted residues (Asp76 and Lys78) could generate an ionic bond and increase the structural electrostatic energy, which could then increase the stability of the protein. These results also suggest that the thermal stability of proteins could be improved by adding the ionic bond on protein surface.

  6. 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. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  7. Expanded insecticide catabolic activity gained by a single nucleotide substitution in a bacterial carbamate hydrolase gene.

    PubMed

    Öztürk, Başak; Ghequire, Maarten; Nguyen, Thi Phi Oanh; De Mot, René; Wattiez, Ruddy; Springael, Dirk

    2016-12-01

    Carbofuran-mineralizing strain Novosphingobium sp. KN65.2 produces the CfdJ enzyme that converts the N-methylcarbamate insecticide to carbofuran phenol. Purified CfdJ shows a remarkably low KM towards carbofuran. Together with the carbaryl hydrolase CehA of Rhizobium sp. strain AC100, CfdJ represents a new protein family with several uncharacterized bacterial members outside the proteobacteria. Although both enzymes differ by only four amino acids, CehA does not recognize carbofuran as a substrate whereas CfdJ also hydrolyzes carbaryl. None of the CfdJ amino acids that differ from CehA were shown to be silent regarding carbofuran hydrolytic activity but one particular amino acid substitution, i.e., L152 to F152, proved crucial. CfdJ is more efficient in degrading methylcarbamate pesticides with an aromatic side chain whereas CehA is more efficient in degrading the oxime carbamate nematicide oxamyl. The presence of common flanking sequences suggest that the cfdJ gene is located on a remnant of the mobile genetic element Tnceh carrying cehA. Our results suggest that these enzymes can be acquired through horizontal gene transfer and can evolve to degrade new carbamate substrates by limited amino acid substitutions. We demonstrate that a carbaryl hydrolase can gain the additional capacity to degrade carbofuran by a single nucleotide transversion. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

  8. Catalytic Mechanism of a Novel Glycoside Hydrolase Family 16 "Elongating" β-Transglycosylase.

    PubMed

    Qin, Zhen; Yang, Shaoqing; Zhao, Liming; You, Xin; Yan, Qiaojuan; Jiang, Zhengqiang

    2017-02-03

    Carbohydrates are complex macromolecules in biological metabolism. Enzymatic synthesis of carbohydrates is recognized as a powerful tool to overcome the problems associated with large scale synthesis of carbohydrates. Novel enzymes with significant transglycosylation ability are still in great demand in glycobiology studies. Here we report a novel glycoside hydrolase family 16 "elongating" β-transglycosylase from Paecilomyces thermophila (PtBgt16A), which efficiently catalyzes the synthesis of higher polymeric oligosaccharides using β-1,3/1,4-oligosaccharides as donor/acceptor substrates. Further structural information reveals that PtBgt16A has a binding pocket around the -1 subsite. The catalytic mechanism of PtBgt16A is partly similar to an exo-glycoside hydrolase, which cleaves the substrate from the non-reducing end one by one. However, PtBgt16A releases the reducing end product and uses the remainder glucosyl as a transglycosylation donor. This catalytic mechanism has similarity with the catalytic mode of amylosucrase, which catalyzes the transglycosylation products gradually extend by one glucose unit. PtBgt16A thus has the potential to be a tool enzyme for the enzymatic synthesis of new β-oligosaccharides and glycoconjugates. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. 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.

  10. Heavy Chain Single Domain Antibodies to Detect Native Human Soluble Epoxide Hydrolase

    PubMed Central

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

    2015-01-01

    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 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

  11. 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.

  12. Cold-active hydrolases producing bacteria from two different sub-glacial Himalayan lakes.

    PubMed

    Sahay, Harmesh; Babu, Bandamaravuri Kishore; Singh, Surendra; Kaushik, Rajeev; Saxena, Anil K; Arora, Dilip K

    2013-08-01

    Microorganisms, native to the cold environments have successfully acclimatized their physiological, metabolic, and biological features, exhibiting uniqueness in their enzymes, proteins, and membrane structures. These cold-active enzymes have immense biotechnological potential. The diversity of culturable bacteria in two different water lakes (the sub-glacial freshwater and the brackish) of Himalayas was analyzed using SYBR green staining and cultural methods. A total of 140 bacteria were isolated and were grouped as psychrophiles, psychrotrophs, and psychrotolerant organisms, based on their optimal temperature for growth. The amplified ribosomal DNA restriction analysis using three restriction enzymes facilitated the grouping of these isolates into 96 genotypes at ≥85% polymorphism. Phylogenetic analysis using 16S rRNA gene sequences revealed that the bacterial strains from both lakes belonged to Firmicutes, Proteobacteria (α, β, and γ) or Actinobacteria. Screening of the germplasm for the activity of different cold-active hydrolases such as protease, amylase, xylanase, and cellulase, revealed that about 16 isolates were positive, and exhibiting a wide range of stability at various temperature and pH. Our results suggest that the distinctly different ecosystems of sub-glacial freshwater and brackish water lakes have diverse groups of bacteria, which can be an excellent source of extracellular hydrolases with a wide range of thermal stability. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. 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

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

    PubMed

    Schinke, Claudia; Germani, José C

    2012-03-01

    Macrophomina phaseolina, phylum Ascomycota, is a phytopathogenic fungus distributed worldwide in hot dry areas. There are few studies on its secreted lipases and none on its colony radial growth rate, an indicator of fungal ability to use nutrients for growth, on media other than potato-dextrose agar. In this study, 13 M. phaseolina isolates collected in different Brazilian regions were screened for fast-growth and the production of hydrolases of industrial interest, especially alkaline lipases. Hydrolase detection and growth rate determination were done on citric pectin, gelatin, casein, soluble starch, and olive oil as substrates. Ten isolates were found to be active on all substrates tested. The most commonly detected enzymes were pectinases, amylases, and lipases. The growth rate on pectin was significantly higher (P < 0.05), while the growth rates on the different media identified CMM 2105, CMM 1091, and PEL as the fastest-growing isolates. The lipase activity of four isolates grown on olive oil was followed for 4 days by measuring the activity in the cultivation broth. The specific lipolytic activity of isolate PEL was significantly higher at 96 h (130 mU mg protein(-1)). The broth was active at 37 °C, pH 8, indicating the potential utility of the lipases of this isolate in mild alkaline detergents. There was a strong and positive correlation (0.86) between radial growth rate and specific lipolytic activity.

  15. Crystallization and preliminary X-ray diffraction studies of cyanuric acid hydrolase from Azorhizobium caulinodans.

    PubMed

    Cho, Seunghee; Shi, Ke; Wackett, Lawrence P; Aihara, Hideki

    2013-08-01

    Cyanuric acid is synthesized industrially and forms during the microbial metabolism of s-triazine herbicides. Cyanuric acid is metabolized by some microorganisms via cyanuric acid hydrolase (CAH), which opens the s-triazine ring as a prelude to further metabolism. CAH is a member of the rare cyanuric acid hydrolase/barbiturase family. Here, the crystallization and preliminary X-ray diffraction analysis of CAH from Azorhizobium caulinodans are reported. CAH was cocrystallized with barbituric acid, a close analog of cyanuric acid that is a tight-binding competitive inhibitor. Crystals suitable for X-ray diffraction experiments were grown in conditions containing PEG 8K or magnesium sulfate as precipitants. An X-ray diffraction data set was collected from CAH-barbituric acid crystals to 2.7 Å resolution. The crystals were found to belong to space group I4₁22, with unit-cell parameters a = b = 237.9, c = 105.3 Å, α = β = γ = 90°.

  16. The role of human glutathione transferases and epoxide hydrolases in the metabolism of xenobiotics.

    PubMed Central

    Seidegård, J; Ekström, G

    1997-01-01

    Human glutathione transferases (GSTs) are a multigene family of enzymes that are involved in the metabolism of a wide range of electrophilic compounds of both exogenous and endogenous origin. GSTs are generally recognized as detoxifying enzymes by catalyzing the conjugation of these compounds with glutathione, but they may also be involved in activation of some carcinogens. The memmalian GSTs can be differentiated into four classes of cytosolic enzymes and two membrane bound enzymes. Human epoxide hydrolases (EHs) catalyze the addition of water to epoxides to form the corresponding dihydrodiol. The enzymatic hydration is essentially irreversible and produces mainly metabolites of lower reactivity that can be conjugated and excreted. The reaction of EHs is therefore generally regarded as detoxifying. The mammalian EHs can be distinguished by their physical and enzymatic properties. Microsomal EH (mEH) exhibits a broad substrate specificity, while the soluble EH (sEH) is an enzyme with a "complementary" substrate specificity to mEH. Cholesterol EH and leukotriene A4 hydrolase are two EHs with very limited substrate specificity. The activities of either GSTs or EHs expressed in vivo exhibit a relatively large interindividual variation, which might be explained by induction, inhibition, or genetic factors. These variations in levels or activities of individual isoenzymes are of importance with respect to an individual's susceptibility to genotoxic effects. This article gives a general overview of GSTs and EHs, discussing the modulation of activities, determination of these enzymes ex vivo, and the polymorphic expression of some isoenzymes. PMID:9255563

  17. Studies on the structure-bound sedimentability of some rat liver lysosome hydrolases

    PubMed Central

    Baccino, F. M.; Rita, G. A.; Zuretti, Maria Franca

    1971-01-01

    1. Lysosome-rich fractions from rat liver were subjected to several disruptive procedures: osmotic lysis or freezing and thawing in different media, shearing forces in a high-speed blender, treatment with Triton X-100. 2. The soluble and particulate phases were then separated by high-speed centrifugation and assayed for their content of acid phosphatase, β-galactosidase, β-N-acetylglucosaminidase, acid proteinase, acid ribonuclease, acid deoxyribonuclease and protein. 3. The degree of elution of these hydrolases appeared to depend on both the enzyme species and the treatment. The resulting patterns of solubilization were rather complex, so that a clear-cut discrimination between soluble and structure-bound enzymes could not always be traced. 4. Although only β-galactosidase was readily solubilizable after all treatments, acid proteinase could also be extensively eluted from the sedimentable material in the presence of EDTA and acid phosphatase was fully extracted by Triton X-100. On the other hand, considerable proportions of the other activities could not be solubilized by any of the procedures used. 5. In other experiments, the adsorbability of hydrolases on subcellular structures was investigated by measuring the partition between sedimentable particles and soluble fraction of solubilized enzymes added to `intact' liver homogenates. 6. Large proportions of acid proteinase, ribonuclease and deoxyribonuclease, and almost all of β-N-acetylglucosaminidase, were found to be adsorbed on the particulate material. PMID:5118107

  18. 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.

  19. Mutational and nucleotide sequence analysis of S-adenosyl-L-homocysteine hydrolase from Rhodobacter capsulatus.

    PubMed Central

    Sganga, M W; Aksamit, R R; Cantoni, G L; Bauer, C E

    1992-01-01

    The genetic locus ahcY, encoding the enzyme S-adenosyl-L-homocysteine hydrolase (EC 3.3.1.1) from the bacterium Rhodobacter capsulatus, has been mapped by mutational analysis to within a cluster of genes involved in regulating the induction and maintenance of the bacterial photosynthetic apparatus. Sequence analysis demonstrates that ahcY encodes a 51-kDa polypeptide that displays 64% sequence identity to its human homolog. Insertion mutants in ahcY lack detectable S-adenosyl-L-homocysteine hydrolase activity and, as a consequence, S-adenosyl-L-homocysteine accumulates in the cells, resulting in a 16-fold decrease in the intracellular ratio of S-adenosyl-L-methionine to S-adenosyl-L-homocysteine as compared to wild-type cells. The ahcY disrupted strain fails to grow in minimal medium; however, growth is restored in minimal medium supplemented with methionine or homocysteine or in a complex medium, thereby indicating that the hydrolysis of S-adenosyl-L-homocysteine plays a key role in the metabolism of sulfur-containing amino acids. The ahcY mutant, when grown in supplemented medium, synthesizes significantly reduced levels of bacteriochlorophyll, indicating that modulation of the intracellular ratio of S-adenosyl-L-methionine to S-adenosyl-L-homocysteine may be an important factor in regulating bacteriochlorophyll biosynthesis. PMID:1631127

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

    PubMed Central

    Matsuzawa, Tomohiko; Mitsuishi, Yasushi; Kameyama, Akihiko

    2016-01-01

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

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

    PubMed

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

    2016-03-04

    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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Bacillus licheniformis trehalose-6-phosphate hydrolase structures suggest keys to substrate specificity.

    PubMed

    Lin, Min Guan; Chi, Meng Chun; Naveen, Vankadari; Li, Yi Ching; Lin, Long Liu; Hsiao, Chwan Deng

    2016-01-01

    Trehalose-6-phosphate hydrolase (TreA) belongs to glycoside hydrolase family 13 (GH13) and catalyzes the hydrolysis of trehalose 6-phosphate (T6P) to yield glucose and glucose 6-phosphate. The products of this reaction can be further metabolized by the energy-generating glycolytic pathway. Here, crystal structures of Bacillus licheniformis TreA (BlTreA) and its R201Q mutant complexed with p-nitrophenyl-α-D-glucopyranoside (R201Q-pPNG) are presented at 2.0 and 2.05 Å resolution, respectively. The overall structure of BlTreA is similar to those of other GH13 family enzymes. However, detailed structural comparisons revealed that the catalytic site of BlTreA contains a long loop that adopts a different conformation from those of other GH13 family members. Unlike the homologous regions of Bacillus cereus oligo-1,6-glucosidase (BcOgl) and Erwinia rhapontici isomaltulose synthase (NX-5), the surface potential of the BlTreA active site exhibits a largely positive charge contributed by the four basic residues His281, His282, Lys284 and Lys292. Mutation of these residues resulted in significant decreases in the enzymatic activity of BlTreA. Strikingly, the (281)HHLK(284) motif and Lys292 play critical roles in substrate discrimination by BlTreA.

  3. 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.

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

    SciTech Connect

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

    2016-02-01

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

  5. 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

  6. N-Arylmethyl substituted iminoribitol derivatives as inhibitors of a purine specific nucleoside hydrolase.

    PubMed

    Goeminne, Annelies; Berg, Maya; McNaughton, Michael; Bal, Gunther; Surpateanu, Georgiana; Van der Veken, Pieter; De Prol, Stijn; Versées, Wim; Steyaert, Jan; Haemers, Achiel; Augustyns, Koen

    2008-07-15

    A key enzyme within the purine salvage pathway of parasites, nucleoside hydrolase, is proposed as a good target for new antiparasitic drugs. We have developed N-arylmethyl-iminoribitol derivatives as a novel class of inhibitors against a purine specific nucleoside hydrolase from Trypanosoma vivax. Several of our inhibitors exhibited low nanomolar activity, with 1,4-dideoxy-1,4-imino-N-(8-quinolinyl)methyl-d-ribitol (UAMC-00115, K(i) 10.8nM), N-(9-deaza-adenin-9-yl)methyl-1,4-dideoxy-1,4-imino-d-ribitol (K(i) 4.1nM), and N-(9-deazahypoxanthin-9-yl)methyl-1,4-dideoxy-1,4-imino-d-ribitol (K(i) 4.4nM) being the three most active compounds. Docking studies of the most active inhibitors revealed several important interactions with the enzyme. Among these interactions are aromatic stacking of the nucleobase mimic with two Trp-residues, and hydrogen bonds between the hydroxyl groups of the inhibitors and amino acid residues in the active site. During the course of these docking studies we also identified a strong interaction between the Asp40 residue from the enzyme and the inhibitor. This is an interaction which has not previously been considered as being important.

  7. Signature motifs identify an Acinetobacter Cif virulence factor with epoxide hydrolase activity.

    PubMed

    Bahl, Christopher D; Hvorecny, Kelli L; Bridges, Andrew A; Ballok, Alicia E; Bomberger, Jennifer M; Cady, Kyle C; O'Toole, George A; Madden, Dean R

    2014-03-14

    Endocytic recycling of the cystic fibrosis transmembrane conductance regulator (CFTR) is blocked by the CFTR inhibitory factor (Cif). Originally discovered in Pseudomonas aeruginosa, Cif is a secreted epoxide hydrolase that is transcriptionally regulated by CifR, an epoxide-sensitive repressor. In this report, we investigate a homologous protein found in strains of the emerging nosocomial pathogens Acinetobacter nosocomialis and Acinetobacter baumannii ("aCif"). Like Cif, aCif is an epoxide hydrolase that carries an N-terminal secretion signal and can be purified from culture supernatants. When applied directly to polarized airway epithelial cells, mature aCif triggers a reduction in CFTR abundance at the apical membrane. Biochemical and crystallographic studies reveal a dimeric assembly with a stereochemically conserved active site, confirming our motif-based identification of candidate Cif-like pathogenic EH sequences. Furthermore, cif expression is transcriptionally repressed by a CifR homolog ("aCifR") and is induced in the presence of epoxides. Overall, this Acinetobacter protein recapitulates the essential attributes of the Pseudomonas Cif system and thus may facilitate airway colonization in nosocomial lung infections.

  8. Signature Motifs Identify an Acinetobacter Cif Virulence Factor with Epoxide Hydrolase Activity*

    PubMed Central

    Bahl, Christopher D.; Hvorecny, Kelli L.; Bridges, Andrew A.; Ballok, Alicia E.; Bomberger, Jennifer M.; Cady, Kyle C.; O'Toole, George A.; Madden, Dean R.

    2014-01-01

    Endocytic recycling of the cystic fibrosis transmembrane conductance regulator (CFTR) is blocked by the CFTR inhibitory factor (Cif). Originally discovered in Pseudomonas aeruginosa, Cif is a secreted epoxide hydrolase that is transcriptionally regulated by CifR, an epoxide-sensitive repressor. In this report, we investigate a homologous protein found in strains of the emerging nosocomial pathogens Acinetobacter nosocomialis and Acinetobacter baumannii (“aCif”). Like Cif, aCif is an epoxide hydrolase that carries an N-terminal secretion signal and can be purified from culture supernatants. When applied directly to polarized airway epithelial cells, mature aCif triggers a reduction in CFTR abundance at the apical membrane. Biochemical and crystallographic studies reveal a dimeric assembly with a stereochemically conserved active site, confirming our motif-based identification of candidate Cif-like pathogenic EH sequences. Furthermore, cif expression is transcriptionally repressed by a CifR homolog (“aCifR”) and is induced in the presence of epoxides. Overall, this Acinetobacter protein recapitulates the essential attributes of the Pseudomonas Cif system and thus may facilitate airway colonization in nosocomial lung infections. PMID:24474692

  9. Lethal Effect of a Heterologous Murein Hydrolase on Penicillin-Treated Streptococcus sanguis

    PubMed Central

    Horne, Diane; Tomasz, Alexander

    1980-01-01

    Nine strains of Streptococcus sanguis exhibited tolerance to benzylpenicillin: the growth of each strain was susceptible to penicillin with minimal inhibitory concentrations of 0.1 μg/ml or lower, but the bacteriolytic and bactericidal effects were limited in each case. The tolerance of these bacteria was also reflected in the large discrepancies between the minimal inhibitory and minimal bactericidal concentrations for benzylpenicillin. The hypothesis that a natural deficiency of endogenous murein hydrolase (autolysin) in this species accounts for the penicillin tolerance was tested by using a heterologous murein hydrolase, the C-phage-associated lysin. In seven of the strains, addition of the lysin to the culture together with penicillin or other cell wall inhibitors resulted in lysis and rapid loss of viability. The enzyme alone did not appreciably affect normally growing cultures. The irreversible effects of penicillin plus lysin were drastically reduced in the presence of the bacteriostatic agents chloramphenicol and cerulenin. Speculations based on experiments are presented for the mechanisms by which penicillin treatment sensitizes these bacteria to an exogenous lytic enzyme. Similar phenomena requiring cooperation of host factors and penicillin may occur during infection, since somewhat similar although less pronounced results were obtained by addition of human lysozyme to penicillin-treated S. sanguis. PMID:6104471

  10. Organophosphate and Pyrethroid Hydrolase Activities of Mutant Esterases from the Cotton Bollworm Helicoverpa armigera

    PubMed Central

    Li, Yongqiang; Farnsworth, Claire A.; Coppin, Chris W.; Teese, Mark G.; Liu, Jian-Wei; Scott, Colin; Zhang, Xing; Russell, Robyn J.; Oakeshott, John G.

    2013-01-01

    Two mutations have been found in five closely related insect esterases (from four higher Diptera and a hymenopteran) which each confer organophosphate (OP) hydrolase activity on the enzyme and OP resistance on the insect. One mutation converts a Glycine to an Aspartate, and the other converts a Tryptophan to a Leucine in the enzymes’ active site. One of the dipteran enzymes with the Leucine mutation also shows enhanced activity against pyrethroids. Introduction of the two mutations in vitro into eight esterases from six other widely separated insect groups has also been reported to increase substantially the OP hydrolase activity of most of them. These data suggest that the two mutations could contribute to OP, and possibly pyrethroid, resistance in a variety of insects. We therefore introduced them in vitro into eight Helicoverpa armigera esterases from a clade that has already been implicated in OP and pyrethroid resistance. We found that they do not generally enhance either OP or pyrethroid hydrolysis in these esterases but the Aspartate mutation did increase OP hydrolysis in one enzyme by about 14 fold and the Leucine mutation caused a 4–6 fold increase in activity (more in one case) of another three against some of the most insecticidal isomers of fenvalerate and cypermethrin. The Aspartate enzyme and one of the Leucine enzymes occur in regions of the H. armigera esterase isozyme profile that have been previously implicated in OP and pyrethroid resistance, respectively. PMID:24204917

  11. The role of a purine-specific nucleoside hydrolase in spore germination of Bacillus thuringiensis.

    PubMed

    Liang, Liang; He, Xihong; Liu, Gang; Tan, Huarong

    2008-05-01

    A homologous gene (iunH) of a putative nucleoside hydrolase (NH), which had been identified from the exosporia of Bacillus cereus and Bacillus anthracis spores, was cloned from Bacillus thuringiensis subsp. kurstaki. Disruption of iunH did not affect the vegetative growth and sporulation of Bacillus thuringiensis, but promoted both inosine- and adenosine-induced spore germination. The inosine- or adenosine-induced germination rate decreased when the wild-type iunH gene was overexpressed in Bacillus thuringiensis. The iunH gene product was characterized as a purine-specific NH. The kinetic parameters of IunH with inosine as substrate were K(m)=399+/-115 microM, k(cat)=48.9+/-8.5 s(-1) and k(cat)/K(m)=1.23 x 10(5) M(-1) s(-1). The optimal pH and temperature for IunH were found to be pH 6 and 80 degrees C. Meanwhile, the specific activity of inosine hydrolase in intact spores of the wild-type strain with inosine as substrate was 2.89+/-0.23x10(-2) micromol min(-1) (mg dry wt)(-1). These results indicate that IunH is important in moderating inosine- or adenosine-induced germination of Bacillus thuringiensis spores.

  12. Mycobacterium tuberculosis FtsX extracellular domain activates the peptidoglycan hydrolase, RipC

    PubMed Central

    Mavrici, Daniela; Marakalala, Mohlopheni J.; Holton, James M.; Prigozhin, Daniil M.; Gee, Christine L.; Zhang, Yanjia J.; Rubin, Eric J.; Alber, Tom

    2014-01-01

    Bacterial growth and cell division are coordinated with hydrolysis of the peptidoglycan (PG) layer of the cell wall, but the mechanisms of regulation of extracellular PG hydrolases are not well understood. Here we report the biochemical, structural, and genetic analysis of the Mycobacterium tuberculosis homolog of the transmembrane PG-hydrolase regulator, FtsX. The purified FtsX extracellular domain binds the PG peptidase Rv2190c/RipC N-terminal segment, causing a conformational change that activates the enzyme. Deletion of ftsEX and ripC caused similar phenotypes in Mycobacterium smegmatis, as expected for genes in a single pathway. The crystal structure of the FtsX extracellular domain reveals an unprecedented fold containing two lobes connected by a flexible hinge. Mutations in the hydrophobic cleft between the lobes reduce RipC binding in vitro and inhibit FtsX function in M. smegmatis. These studies suggest how FtsX recognizes RipC and support a model in which a conformational change in FtsX links the cell division apparatus with PG hydrolysis. PMID:24843173

  13. Determinants of murein hydrolase targeting to cross-wall of Staphylococcus aureus peptidoglycan.

    PubMed

    Frankel, Matthew B; Schneewind, Olaf

    2012-03-23

    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.

  14. 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

  15. 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