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Sample records for amidohydrolases

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

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

    2017-12-26

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

  2. Toward Photopharmacological Antimicrobial Chemotherapy Using Photoswitchable Amidohydrolase Inhibitors.

    Science.gov (United States)

    Weston, Claire E; Krämer, Andreas; Colin, Felix; Yildiz, Özkan; Baud, Matthias G J; Meyer-Almes, Franz-Josef; Fuchter, Matthew J

    2017-02-10

    Photopharmacological agents exhibit light-dependent biological activity and may have potential in the development of new antimicrobial agents/modalities. Amidohydrolase enzymes homologous to the well-known human histone deacetylases (HDACs) are present in bacteria, including resistant organisms responsible for a significant number of hospital-acquired infections and deaths. We report photopharmacological inhibitors of these enzymes, using two classes of photoswitches embedded in the inhibitor pharmacophore: azobenzenes and arylazopyrazoles. Although both classes of inhibitor show excellent inhibitory activity (nM IC 50 values) of the target enzymes and promising differential activity of the switchable E- and Z-isomeric forms, the arylazopyrazoles exhibit better intrinsic photoswitch performance (more complete switching, longer thermal lifetime of the Z-isomer). We also report protein-ligand crystal structures of the E-isomers of both an azobenzene and an arylazopyrazole inhibitor, bound to bacterial histone deacetylase-like amidohydrolases (HDAHs). These structures not only uncover interactions important for inhibitor binding but also reveal conformational differences between the two photoswitch inhibitor classes. As such, our data may pave the way for the design of improved photopharmacological agents targeting the HDAC superfamily.

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

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    Cheng-Yang Huang

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

  4. Enzymatic production of 5-aminovalerate from l-lysine using l-lysine monooxygenase and 5-aminovaleramide amidohydrolase

    OpenAIRE

    Liu, Pan; Zhang, Haiwei; Lv, Min; Hu, Mandong; Li, Zhong; Gao, Chao; Xu, Ping; Ma, Cuiqing

    2014-01-01

    5-Aminovalerate is a potential C5 platform chemical for synthesis of valerolactam, 5-hydroxyvalerate, glutarate, and 1,5-pentanediol. It is a metabolite of l-lysine catabolism through the aminovalerate pathway in Pseudomonas putida. l-Lysine monooxygenase (DavB) and 5-aminovaleramide amidohydrolase (DavA) play key roles in the biotransformation of l-lysine into 5-aminovalerate. Here, DavB and DavA of P. putida KT2440 were expressed, purified, and coupled for the production of 5-aminovalerate ...

  5. Polyamine Deacetylase Structure and Catalysis: Prokaryotic Acetylpolyamine Amidohydrolase and Eukaryotic HDAC10.

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    Shinsky, Stephen; Christianson, David W

    2018-03-13

    Polyamines such as putrescine, spermidine, and spermine are small aliphatic cations that serve myriad biological functions in all forms of life. While polyamine biosynthesis and cellular trafficking pathways are generally well defined, it is only recently that the molecular basis of reversible polyamine acetylation has been established. In particular, enzymes that catalyze polyamine deacetylation reactions have been identified and structurally characterized: histone deacetylase 10 (HDAC10) from Homo sapiens and Danio rerio (zebrafish) is a highly specific N8-acetylspermidine deacetylase, and its prokaryotic counterpart, acetylpolyamine amidohydrolase (APAH) from Mycoplana ramosa, is a broad-specificity polyamine deacetylase. Similar to the greater family of HDACs, which mainly serve as lysine deacetylases, both enzymes adopt the characteristic arginase-deacetylase fold and employ a Zn2+-activated water molecule for catalysis. In contrast with HDACs, however, the active sites of HDAC10 and APAH are sterically constricted to enforce specificity for long, slender polyamine substrates and exclude bulky peptides and proteins containing acetyl-L-lysine. Crystal structures of APAH and D. rerio HDAC10 reveal that quaternary structure, i.e., dimer assembly, provides the steric constriction that directs the polyamine substrate specificity of APAH, whereas tertiary structure - a unique 310 helix defined by the P(E,A)CE motif - provides the steric constriction that directs the polyamine substrate specificity of HDAC10. Given the recent identification of HDAC10 and spermidine as mediators of autophagy, HDAC10 is rapidly emerging as a biomarker and target for the design of isozyme-selective inhibitors that will suppress autophagic responses to cancer chemotherapy, thereby rendering cancer cells more susceptible to cytotoxic drugs.

  6. The Cell Lysis Activity of the Streptococcus agalactiae Bacteriophage B30 Endolysin Relies on the Cysteine, Histidine-Dependent Amidohydrolase/Peptidase Domain

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    Donovan, David M.; Foster-Frey, Juli; Dong, Shengli; Rousseau, Geneviève M.; Moineau, Sylvain; Pritchard, David G.

    2006-01-01

    The Streptococcus agalactiae bacteriophage B30 endolysin contains three domains: cysteine, histidine-dependent amidohydrolase/peptidase (CHAP), Acm glycosidase, and the SH3b cell wall binding domain. Truncations and point mutations indicated that the Acm domain requires the SH3b domain for activity, while the CHAP domain is responsible for nearly all the cell lysis activity. PMID:16820517

  7. Functional Identification and Structure Determination of Two Novel Prolidases from cog1228 in the Amidohydrolase Superfamily

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    Xiang, Dao Feng; Patskovsky, Yury; Xu, Chengfu; Fedorov, Alexander A.; Fedorov, Elena V.; Sisco, Abby A.; Sauder, J. Michael; Burley, Stephen K.; Almo, Steven C.; Raushel, Frank M. (Einstein); (TAM); (Lilly)

    2010-12-07

    Two uncharacterized enzymes from the amidohydrolase superfamily belonging to cog1228 were cloned, expressed, and purified to homogeneity. The two proteins, Sgx9260c (gi|44242006) and Sgx9260b (gi|44479596), were derived from environmental DNA samples originating from the Sargasso Sea. The catalytic function and substrate profiles for Sgx9260c and Sgx9260b were determined using a comprehensive library of dipeptides and N-acyl derivative of L-amino acids. Sgx9260c catalyzes the hydrolysis of Gly-L-Pro, L-Ala-L-Pro, and N-acyl derivatives of L-Pro. The best substrate identified to date is N-acetyl-L-Pro with a value of k{sub cat}/K{sub m} of 3 x 10{sup 5} M{sup -1} s{sup -1}. Sgx9260b catalyzes the hydrolysis of L-hydrophobic L-Pro dipeptides and N-acyl derivatives of L-Pro. The best substrate identified to date is N-propionyl-L-Pro with a value of k{sub cat}/K{sub m} of 1 x 10{sup 5} M{sup -1} s{sup -1}. Three-dimensional structures of both proteins were determined by X-ray diffraction methods (PDB codes 3MKV and 3FEQ). These proteins fold as distorted ({beta}/{alpha})8-barrels with two divalent cations in the active site. The structure of Sgx9260c was also determined as a complex with the N-methylphosphonate derivative of L-Pro (PDB code 3N2C). In this structure the phosphonate moiety bridges the binuclear metal center, and one oxygen atom interacts with His-140. The {alpha}-carboxylate of the inhibitor interacts with Tyr-231. The proline side chain occupies a small substrate binding cavity formed by residues contributed from the loop that follows {beta}-strand 7 within the ({beta}/{alpha})8-barrel. A total of 38 other proteins from cog1228 are predicted to have the same substrate profile based on conservation of the substrate binding residues. The structure of an evolutionarily related protein, Cc2672 from Caulobacter crecentus, was determined as a complex with the N-methylphosphonate derivative of L-arginine (PDB code 3MTW).

  8. Enzymatic production of 5-aminovalerate from L-lysine using L-lysine monooxygenase and 5-aminovaleramide amidohydrolase.

    Science.gov (United States)

    Liu, Pan; Zhang, Haiwei; Lv, Min; Hu, Mandong; Li, Zhong; Gao, Chao; Xu, Ping; Ma, Cuiqing

    2014-07-11

    5-Aminovalerate is a potential C5 platform chemical for synthesis of valerolactam, 5-hydroxyvalerate, glutarate, and 1,5-pentanediol. It is a metabolite of l-lysine catabolism through the aminovalerate pathway in Pseudomonas putida. L-Lysine monooxygenase (DavB) and 5-aminovaleramide amidohydrolase (DavA) play key roles in the biotransformation of L-lysine into 5-aminovalerate. Here, DavB and DavA of P. putida KT2440 were expressed, purified, and coupled for the production of 5-aminovalerate from L-lysine. Under optimal conditions, 20.8 g/L 5-aminovalerate was produced from 30 g/L L-lysine in 12 h. Because L-lysine is an industrial fermentation product, the two-enzyme coupled system presents a promising alternative for the production of 5-aminovalerate.

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

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    Hicks, Katherine A.; Ealick, Steven E.

    2016-05-25

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

  10. Characterization of the metallo-dependent amidohydrolases responsible for “auxiliary” leucinyl removal in the biosynthesis of 2,2′-bipyridine antibiotics

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    Ming Chen

    2017-06-01

    Full Text Available 2,2′-Bipyridine (2,2′-BiPy is an attractive core structure present in a number of biologically active natural products, including the structurally related antibiotics caerulomycins (CAEs and collismycins (COLs. Their biosynthetic pathways share a similar key 2,2′-BiPy-l-leucine intermediate, which is desulfurated or sulfurated at C5, arises from a polyketide synthase/nonribosomal peptide synthetase hybrid assembly line. Focusing on the common off-line modification steps, we here report that the removal of the “auxiliary” l-leucine residue relies on the metallo-dependent amidohydrolase activity of CaeD or ColD. This activity leads to the production of similar 2,2′-BiPy carboxylate products that then receive an oxime functionality that is characteristic for both CAEs and COLs. Unlike many metallo-dependent amidohydrolase superfamily proteins that have been previously reported, these proteins (particularly CaeD exhibited a strong zinc ion-binding capacity that was proven by site-specific mutagenesis studies to be essential to proteolytic activity. The kinetics of the conversions that respectively involve CaeD and ColD were analyzed, showing the differences in the efficiency and substrate specificity of these two proteins. These findings would generate interest in the metallo-dependent amidohydrolase superfamily proteins that are involved in the biosynthesis of bioactive natural products.

  11. Evolutionary Expansion of the Amidohydrolase Superfamily in Bacteria in Response to the Synthetic Compounds Molinate and Diuron

    Science.gov (United States)

    Sugrue, Elena; Fraser, Nicholas J.; Hopkins, Davis H.; Carr, Paul D.; Khurana, Jeevan L.; Oakeshott, John G.; Scott, Colin

    2015-01-01

    The amidohydrolase superfamily has remarkable functional diversity, with considerable structural and functional annotation of known sequences. In microbes, the recent evolution of several members of this family to catalyze the breakdown of environmental xenobiotics is not well understood. An evolutionary transition from binuclear to mononuclear metal ion coordination at the active sites of these enzymes could produce large functional changes such as those observed in nature, but there are few clear examples available to support this hypothesis. To investigate the role of binuclear-mononuclear active-site transitions in the evolution of new function in this superfamily, we have characterized two recently evolved enzymes that catalyze the hydrolysis of the synthetic herbicides molinate (MolA) and phenylurea (PuhB). In this work, the crystal structures, mutagenesis, metal ion analysis, and enzyme kinetics of both MolA and PuhB establish that these enzymes utilize a mononuclear active site. However, bioinformatics and structural comparisons reveal that the closest putative ancestor of these enzymes had a binuclear active site, indicating that a binuclear-mononuclear transition has occurred. These proteins may represent examples of evolution modifying the characteristics of existing catalysts to satisfy new requirements, specifically, metal ion rearrangement leading to large leaps in activity that would not otherwise be possible. PMID:25636851

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

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

    2011-11-18

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

  13. Structural investigations of N-carbamoylputrescine amidohydrolase from Medicago truncatula: insights into the ultimate step of putrescine biosynthesis in plants

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    Bartosz eSekula

    2016-03-01

    Full Text Available Putrescine, 1,4-diaminobutane, is an intermediate in the biosynthesis of more complexed polyamines, spermidine and spermine. Unlike other eukaryotes, plants have evolved a multistep pathway for putrescine biosynthesis that utilizes arginine. In the final reaction, N-carbamoylputrescine is hydrolyzed to putrescine by N-carbamoylputrescine amidohydrolase (CPA, EC 3.5.1.53. During the hydrolysis, consecutive nucleophilic attacks on the substrate by Cys158 and water lead to formation of putrescine and two by-products, ammonia and carbon dioxide. CPA from the model legume plant, Medicago truncatula (MtCPA, was investigated in this work. Four crystal structures were determined: the wild-type MtCPA in complex with the reaction intermediate, N-(dihydroxymethylputrescine as well as with cadaverine, which is a longer analogue of putrescine; and also structures of MtCPA-C158S mutant unliganded and with putrescine. MtCPA assembles into octamers, which resemble an incomplete left-handed helical twist. The active site of MtCPA is funnel-like shaped, and its entrance is walled with a contribution of the neighboring protein subunits. Deep inside the catalytic cavity, Glu48, Lys121 and Cys158 form the catalytic triad. In this studies, we have highlighted the key residues, highly conserved among the plant kingdom, responsible for the activity and selectivity of MtCPA towards N-carbamoylputrescine. Moreover, since, according to previous reports, a close MtCPA relative from Arabidopsis thaliana, along with several other nitrilase-like proteins, are subjected to allosteric regulation by substrates, we have used the structural information to indicate a putative secondary binding site. Based on the docking experiment, we postulate that this site is adjacent to the entrance to the catalytic pocket.

  14. The thermodynamic signature of ligand binding to histone deacetylase-like amidohydrolases is most sensitive to the flexibility in the L2-loop lining the active site pocket.

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    Meyners, Christian; Krämer, Andreas; Yildiz, Özkan; Meyer-Almes, Franz-Josef

    2017-07-01

    The analysis of the thermodynamic driving forces of ligand-protein binding has been suggested to be a key component for the selection and optimization of active compounds into drug candidates. The binding enthalpy as deduced from isothermal titration calorimetry (ITC) is usually interpreted assuming single-step binding of a ligand to one conformation of the target protein. Although successful in many cases, these assumptions are oversimplified approximations of the reality with flexible proteins and complicated binding mechanism in many if not most cases. The relationship between protein flexibility and thermodynamic signature of ligand binding is largely understudied. Directed mutagenesis, X-ray crystallography, enzyme kinetics and ITC methods were combined to dissect the influence of loop flexibility on the thermodynamics and mechanism of ligand binding to histone deacetylase (HDAC)-like amidohydrolases. The general ligand-protein binding mechanism comprises an energetically demanding gate opening step followed by physical binding. Increased flexibility of the L2-loop in HDAC-like amidohydrolases facilitates access of ligands to the binding pocket resulting in predominantly enthalpy-driven complex formation. The study provides evidence for the great importance of flexibility adjacent to the active site channel for the mechanism and observed thermodynamic driving forces of molecular recognition in HDAC like enzymes. The flexibility or malleability in regions adjacent to binding pockets should be given more attention when designing better drug candidates. The presented case study also suggests that the observed binding enthalpy of protein-ligand systems should be interpreted with caution, since more complicated binding mechanisms may obscure the significance regarding potential drug likeness. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Expression of Recombinant pET22b-LysK-Cysteine/Histidine-Dependent Amidohydrolase/Peptidase Bacteriophage Therapeutic Protein in Escherichia coli BL21 (DE3).

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    Kashani, Hamed Haddad; Moniri, Rezvan

    2015-08-01

    Bacteriophage-encoded endolysins are a group of enzymes that act by digesting the peptidoglycan of bacterial cell walls. LysK has been reported to lyse live staphylococcal cultures. LysK proteins containing only the cysteine/histidine-dependent amidohydrolase/peptidase (CHAP) domain has the capability to show lytic activity against live clinical staphylococcal isolates, including methicillin-resistant Staphylococcus aureus (MRSA). The aim of this study was to clone and express LysK-CHAP domain in Escherichia coli BL21 (DE3) using pET22b as a secretion vector. The pET22b plasmid was used, which encoded a pelB secretion signal under the control of the strong bacteriophage T7 promoter. The E. coli cloning strains DH5α and BL21 (DE3) were grown at 37°C with aeration in the Luria-Bertani medium. A plasmid encoding LysK-CHAP in a pET22b backbone was constructed. The pET22b vector containing LysK-CHAP sequences were digested with NcoI and HindIII restriction enzymes. Cloning accuracy was confirmed by electrophoresis. The pET22b-LysK plasmid was used to transform the E. coli strain BL21. Isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to a final concentration of 1mM to induce T7 RNA polymerase-based expression. Finally, western blot confirmed the expression of target protein. In this study, after double digestion of pEX and pET22b vectors with HindIII and NcoI, LysK gene was cloned into two HindIII and NcoI sites in pET22b vector, and then transformed to E. coli DH5α. Cloning was confirmed with double digestion and analyzed with agarose gel. The recombinant pET22b-LysK plasmid was transformed to E. coli BL21 and the expression was induced by IPTG. The expression was confirmed by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting method. Observation of a 28.5 kDa band confirmed LysK protein expression. In the present study, LysK-CHAP domain was successfully cloned and expressed at the pET22b vector and E. coli BL21 (DE3).

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

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    Juan eLi

    2015-11-01

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

  17. Knocking down expression of the auxin-amidohydrolase IAR3 alters defense responses in Solanaceae family plants

    Czech Academy of Sciences Publication Activity Database

    D'Ippolito, S.; Vaňková, Radomíra; Joosten, M.H.A.J.; Casalongue, C.A.; Fiol, D.F.

    2016-01-01

    Roč. 253, DEC (2016), s. 31-39 ISSN 0168-9452 Institutional support: RVO:61389030 Keywords : Auxin * Biotic stress * Cladosporium fulvum Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.437, year: 2016

  18. Structure and mechanism of Escherichia coli glutathionylspermidine amidase belonging to the family of cysteine; histidine-dependent amidohydrolases/peptidases

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    Pai, Chien-Hua; Wu, Hsing-Ju; Lin, Chun-Hung; Wang, Andrew H-J

    2011-01-01

    The bifunctional Escherichia coli glutathionylspermidine synthetase/amidase (GspSA) catalyzes both the synthesis and hydrolysis of Gsp. Its amidase domain (GspA), which catalyzes the hydrolysis of Gsp into glutathione and spermidine, plays an important role in redox sensing and protein S-thiolation. To gain insight of the regulation and catalytic mechanism of and further understand the recycling of the Gsp dimer and Gsp-S-protein adducts, we solved two crystal structures of GspA and GspSA bot...

  19. Knocking down expression of the auxin-amidohydrolase IAR3 alters defense responses in Solanaceae family plants.

    Science.gov (United States)

    D'Ippolito, Sebastian; Vankova, Radomira; Joosten, Matthieu H A J; Casalongué, Claudia A; Fiol, Diego F

    2016-12-01

    In plants, indole-3-acetic acid (IAA) amido hydrolases (AHs) participate in auxin homeostasis by releasing free IAA from IAA-amino acid conjugates. We investigated the role of IAR3, a member of the IAA amido hydrolase family, in the response of Solanaceous plants challenged by biotrophic and hemi-biotrophic pathogens. By means of genome inspection and phylogenic analysis we firstly identified IAA-AH sequences and putative IAR3 orthologs in Nicotiana benthamiana, tomato and potato. We evaluated the involvement of IAR3 genes in defense responses by using virus-induced gene silencing. We observed that N. benthamiana and tomato plants with knocked-down expression of IAR3 genes contained lower levels of free IAA and presented altered responses to pathogen attack, including enhanced basal defenses and higher tolerance to infection in susceptible plants. We showed that IAR3 genes are consistently up-regulated in N. benthamiana and tomato upon inoculation with Phytophthora infestans and Cladosporium fulvum respectively. However, IAR3 expression decreased significantly when hypersensitive response was triggered in transgenic tomato plants coexpressing the Cf-4 resistance gene and the avirulence factor Avr4. Altogether, our results indicate that changes in IAR3 expression lead to alteration in auxin homeostasis that ultimately affects plant defense responses. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  20. Mining of the pyrrolamide antibiotics analogs in Streptomyces netropsis reveals the amidohydrolase-dependent "iterative strategy" underlying the pyrrole polymerization.

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    Chunlin Hao

    Full Text Available In biosynthesis of natural products, potential intermediates or analogs of a particular compound in the crude extracts are commonly overlooked in routine assays due to their low concentration, limited structural information, or because of their insignificant bio-activities. This may lead into an incomplete and even an incorrect biosynthetic pathway for the target molecule. Here we applied multiple compound mining approaches, including genome scanning and precursor ion scan-directed mass spectrometry, to identify potential pyrrolamide compounds in the fermentation culture of Streptomyces netropsis. Several novel congocidine and distamycin analogs were thus detected and characterized. A more reasonable route for the biosynthesis of pyrrolamides was proposed based on the structures of these newly discovered compounds, as well as the functional characterization of several key biosynthetic genes of pyrrolamides. Collectively, our results implied an unusual "iterative strategy" underlying the pyrrole polymerization in the biosynthesis of pyrrolamide antibiotics.

  1. Mining of the pyrrolamide antibiotics analogs in Streptomyces netropsis reveals the amidohydrolase-dependent "iterative strategy" underlying the pyrrole polymerization.

    Science.gov (United States)

    Hao, Chunlin; Huang, Sheng; Deng, Zixin; Zhao, Changming; Yu, Yi

    2014-01-01

    In biosynthesis of natural products, potential intermediates or analogs of a particular compound in the crude extracts are commonly overlooked in routine assays due to their low concentration, limited structural information, or because of their insignificant bio-activities. This may lead into an incomplete and even an incorrect biosynthetic pathway for the target molecule. Here we applied multiple compound mining approaches, including genome scanning and precursor ion scan-directed mass spectrometry, to identify potential pyrrolamide compounds in the fermentation culture of Streptomyces netropsis. Several novel congocidine and distamycin analogs were thus detected and characterized. A more reasonable route for the biosynthesis of pyrrolamides was proposed based on the structures of these newly discovered compounds, as well as the functional characterization of several key biosynthetic genes of pyrrolamides. Collectively, our results implied an unusual "iterative strategy" underlying the pyrrole polymerization in the biosynthesis of pyrrolamide antibiotics.

  2. Staphylococcus haemolyticus prophage ΦSH2 endolysin relies on Cysteine, Histidine-dependent Amidohydrolases/Peptidases activity for lysis ‘from without’

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    Schmelcher, Mathias; Korobova, Olga; Schischkova, Nina; Kiseleva, Natalia; Kopylov, Paul; Pryamchuk, Sergey; Donovan, David M.; Abaev, Igor

    2014-01-01

    Staphylococcus aureus is an important pathogen, with methicillin-resistant (MRSA) and multi-drug resistant strains becoming increasingly prevalent in both human and veterinary clinics. S. aureus causing bovine mastitis yields high annual losses to the dairy industry. Conventional treatment of mastitis by broad range antibiotics is often not successful and may contribute to development of antibiotic resistance. Bacteriophage endolysins present a promising new source of antimicrobials. The endolysin of prophage ΦSH2 of Staphylococcus haemolyticus strain JCSC1435 (ΦSH2 lysin) is a peptidoglycan hydrolase consisting of two catalytic domains (CHAP and amidase) and an SH3b cell wall binding domain. In this work, we demonstrated its lytic activity against live staphylococcal cells and investigated the contribution of each functional module to bacterial lysis by testing a series of deletion constructs in zymograms and turbidity reduction assays. The CHAP domain exhibited three-fold higher activity than the full length protein and optimum activity in physiological saline. This activity was further enhanced by the presence of bivalent calcium ions. The SH3b domain was shown to be required for full activity of the complete ΦSH2 lysin. The full length enzyme and the CHAP domain showed activity against multiple staphylococcal strains, including MRSA strains, mastitis isolates, and CoNS. PMID:23026556

  3. Mining of the Pyrrolamide Antibiotics Analogs in Streptomyces netropsis Reveals the Amidohydrolase-Dependent “Iterative Strategy” Underlying the Pyrrole Polymerization

    Science.gov (United States)

    Deng, Zixin; Zhao, Changming; Yu, Yi

    2014-01-01

    In biosynthesis of natural products, potential intermediates or analogs of a particular compound in the crude extracts are commonly overlooked in routine assays due to their low concentration, limited structural information, or because of their insignificant bio-activities. This may lead into an incomplete and even an incorrect biosynthetic pathway for the target molecule. Here we applied multiple compound mining approaches, including genome scanning and precursor ion scan-directed mass spectrometry, to identify potential pyrrolamide compounds in the fermentation culture of Streptomyces netropsis. Several novel congocidine and distamycin analogs were thus detected and characterized. A more reasonable route for the biosynthesis of pyrrolamides was proposed based on the structures of these newly discovered compounds, as well as the functional characterization of several key biosynthetic genes of pyrrolamides. Collectively, our results implied an unusual “iterative strategy” underlying the pyrrole polymerization in the biosynthesis of pyrrolamide antibiotics. PMID:24901640

  4. Arabidopsis CDS blastp result: AK066796 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK066796 J013079D18 At4g37560.1 formamidase, putative / formamide amidohydrolase, p...utative similar to SP|Q50228 Formamidase (EC 3.5.1.49) (Formamide amidohydrolase) {Methylophilus methylotrophus}; contains Pfam profile PF03069: Acetamidase/Formamidase family 0.0 ...

  5. Arabidopsis CDS blastp result: AK066328 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK066328 J013059B20 At4g37550.1 formamidase, putative / formamide amidohydrolase, p...utative similar to SP|Q50228 Formamidase (EC 3.5.1.49) (Formamide amidohydrolase) {Methylophilus methylotrophus}; contains Pfam profile PF03069: Acetamidase/Formamidase family 2e-86 ...

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

    Lifescience Database Archive (English)

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

  7. NCBI nr-aa BLAST: CBRC-PABE-03-0001 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PABE-03-0001 ref|NP_001017603.1| N-acylsphingosine amidohydrolase 3 [Danio rer...io] sp|Q568I2|ASAH3_BRARE Alkaline ceramidase 1 (Alkaline CDase-1) (AlkCDase 1) (Acylsphingosine deacylase 3) (N-acylsphingos

  8. LysK CHAP endopeptidase domain is required for lysis of live staphylococcal cells.

    Science.gov (United States)

    LysK is a staphylococcal bacteriophage endolysin composed of three domains, an N-terminal cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) endopeptidase domain (cleaves between D-alanine of the stem peptide and glycine of the cross-bridge peptide) a mid-protein amidase 2 domain (N-ace...

  9. Optimised production of L-glutaminase: A tumour inhibitor from ...

    African Journals Online (AJOL)

    L-Glutaminase, an amidohydrolase enzyme has been a choice of interest in the treatment of lymphoblastic leukemia. This study investigates the production and optimization of extracellular glutaminase enzyme using several agro-industrial residues by Aspergillus flavus KUGF009 using SSF (solid state fermentation).

  10. Deciphering Physiological Functions of AHL Quorum Quenching Acylases

    NARCIS (Netherlands)

    Utari, Putri D.; Vogel, Jan; Quax, Wim J.

    2017-01-01

    N-Acylhomoserine lactone (AHL)-acylase (also known as amidase or amidohydrolase) is a class of enzyme that belongs to the Ntn-hydrolase superfamily. As the name implies, AHL-acylases are capable of hydrolysing AHLs, the most studied signaling molecules for quorum sensing in Gram-negative bacteria.

  11. A Common Catalytic Mechanism for Proteins of the HutI Family

    Energy Technology Data Exchange (ETDEWEB)

    Tyagi,R.; Eswaramoorthy, S.; Burley, S.; Raushel, F.; Swaminathan, S.

    2008-01-01

    Imidazolonepropionase (HutI) (imidazolone-5-propanote hydrolase, EC 3.5.2.7) is a member of the amidohydrolase superfamily and catalyzes the conversion of imidazolone-5-propanoate to N-formimino-l-glutamate in the histidine degradation pathway. We have determined the three-dimensional crystal structures of HutI from Agrobacterium tumefaciens (At-HutI) and an environmental sample from the Sargasso Sea Ocean Going Survey (Es-HutI) bound to the product [N-formimino-l-glutamate (NIG)] and an inhibitor [3-(2, 5-dioxoimidazolidin-4-yl)propionic acid (DIP)], respectively. In both structures, the active site is contained within each monomer, and its organization displays the landmark feature of the amidohydrolase superfamily, showing a metal ligand (iron), four histidines, and one aspartic acid. A catalytic mechanism involving His265 is proposed on the basis of the inhibitor-bound structure. This mechanism is applicable to all HutI forms.

  12. Biochemical characterization of a new nicotinamidase from an unclassified bacterium thriving in a geothermal water stream microbial mat community

    OpenAIRE

    Zapata-P?rez, Rub?n; Mart?nez-Mo?ino, Ana-Bel?n; Garc?a-Saura, Antonio-Gin?s; Cabanes, Juana; Takami, Hideto; S?nchez-Ferrer, ?lvaro

    2017-01-01

    Nicotinamidases are amidohydrolases that convert nicotinamide into nicotinic acid, contributing to NAD+ homeostasis in most organisms. In order to increase the number of nicotinamidases described to date, this manuscript characterizes a nicotinamidase obtained from a metagenomic library fosmid clone (JFF054_F02) obtained from a geothermal water stream microbial mat community in a Japanese epithermal mine. The enzyme showed an optimum temperature of 90?C, making it the first hyperthermophilic ...

  13. Enhancing the Promiscuous Phosphotriesterase Activity of a Thermostable Lactonase (GkaP) for the Efficient Degradation of Organophosphate Pesticides

    OpenAIRE

    Zhang, Yu; An, Jiao; Ye, Wei; Yang, Guangyu; Qian, Zhi-Gang; Chen, Hai-Feng; Cui, Li; Feng, Yan

    2012-01-01

    The phosphotriesterase-like lactonase (PLL) enzymes in the amidohydrolase superfamily hydrolyze various lactones and exhibit latent phosphotriesterase activities. These enzymes serve as attractive templates for in vitro evolution of neurotoxic organophosphates (OPs) with hydrolytic capabilities that can be used as bioremediation tools. Here, a thermostable PLL from Geobacillus kaustophilus HTA426 (GkaP) was targeted for joint laboratory evolution with the aim of enhancing its catalytic effici...

  14. Gene Sequence and Properties of an s-Triazine Ring-Cleavage Enzyme from Pseudomonas sp. Strain NRRLB-12227

    Science.gov (United States)

    Karns, Jeffrey S.

    1999-01-01

    Pesticides based on the s-triazine ring structure are widely used in cultivation of food crops. Cleavage of the s-triazine ring is an important step in the mineralization of s-triazine compounds and hence in their complete removal from the environment. Cyanuric acid amidohydrolase cleaves cyanuric acid (2,4,6-trihydroxy-s-triazine), which yields carbon dioxide and biuret; the biuret is subject to further metabolism, which yields CO2 and ammonia. The trzD gene encoding cyanuric acid amidohydrolase was cloned into pMMB277 from Pseudomonas sp. strain NRRLB-12227, a strain that is capable of utilizing s-triazines as nitrogen sources. Hydrolysis of cyanuric acid was detected in crude extracts of Escherichia coli containing the cloned gene by monitoring the disappearance of cyanuric acid and the appearance of biuret by high-performance liquid chromatography (HPLC). DEAE and hydrophobic interaction HPLC were used to purify cyanuric acid amidohydrolase to homogeneity, and a spectrophotometric assay for the purified enzyme was developed. The purified enzyme had an apparent Km of 0.05 mM for cyanuric acid at pH 8.0. The enzyme did not cleave any other s-triazine or hydroxypyrimidine compound, although barbituric acid (2,4,6-trihydroxypyrimidine) was found to be a strong competitive inhibitor. Neither the nucleotide sequence of trzD nor the amino acid sequence of the gene product exhibited a significant level of similarity to any known gene or protein. PMID:10427042

  15. Potentials for Soil Enzyme as Indicators of Ecological Management

    Science.gov (United States)

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

    1997-01-01

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

  16. Inhibitory effect of different product analogues on β-alanine synthase: A thermodynamic and fluorescence analysis

    International Nuclear Information System (INIS)

    Andujar-Sanchez, Montserrat; Martinez-Gomez, Ana Isabel; Martinez-Rodriguez, Sergio; Clemente-Jimenez, Josefa Maria; Heras-Vazquez, Francisco Javier Las; Rodriguez-Vico, Felipe; Jara-Perez, Vicente

    2009-01-01

    The enzyme N-carbamoyl-β-alanine amidohydrolase catalyse the hydrolysis of N-carbamoyl-β-alanine or N-carbamoyl-β-aminoisobutyric acid to β-alanine or 3-aminoisobutyric acid, under the release of carbon-dioxide and ammonia. This work studies the inhibition of N-carbamoyl-β-alanine amidohydrolase from Agrobacterium tumefaciens C58 (Atβcar) by different carboxylic acid compounds that differ in number of carbons, and position and size of ramification, while the binding thermodynamics of the inhibitors are studied by isothermal titration calorimetry (ITC) and fluorescence. From the binding constants and inhibition studies, we conclude that propionate is the most efficient inhibitor among those tested. Substitution of the linear alkyl acids in positions 2 and 3 resulted in a drastic decrease of the affinity. The thermodynamic parameters show that a conformational change is triggered upon ligand binding. Binding enthalpy ΔH b is negative in all cases for all ligands, and thus, Van der Waals interactions and hydrogen bonding are most probably the major sources for this term. The process is entropically favoured at all temperatures and pH studied, most probably due to the liberation of water molecules accompanying the conformational change of the enzyme

  17. Inhibitory effect of different product analogues on {beta}-alanine synthase: A thermodynamic and fluorescence analysis

    Energy Technology Data Exchange (ETDEWEB)

    Andujar-Sanchez, Montserrat; Martinez-Gomez, Ana Isabel; Martinez-Rodriguez, Sergio; Clemente-Jimenez, Josefa Maria; Heras-Vazquez, Francisco Javier Las; Rodriguez-Vico, Felipe [Departamento de Quimica Fisica, Bioquimica y Quimica Inorganica, Facultad de Ciencias Experimentales, Universidad de Almeria, Carretera de Sacramento s/n, La Canada de San Urbano, Almeria 04120 (Spain); Jara-Perez, Vicente [Departamento de Quimica Fisica, Bioquimica y Quimica Inorganica, Facultad de Ciencias Experimentales, Universidad de Almeria, Carretera de Sacramento s/n, La Canada de San Urbano, Almeria 04120 (Spain)], E-mail: vjara@ual.es

    2009-02-15

    The enzyme N-carbamoyl-{beta}-alanine amidohydrolase catalyse the hydrolysis of N-carbamoyl-{beta}-alanine or N-carbamoyl-{beta}-aminoisobutyric acid to {beta}-alanine or 3-aminoisobutyric acid, under the release of carbon-dioxide and ammonia. This work studies the inhibition of N-carbamoyl-{beta}-alanine amidohydrolase from Agrobacterium tumefaciens C58 (At{beta}car) by different carboxylic acid compounds that differ in number of carbons, and position and size of ramification, while the binding thermodynamics of the inhibitors are studied by isothermal titration calorimetry (ITC) and fluorescence. From the binding constants and inhibition studies, we conclude that propionate is the most efficient inhibitor among those tested. Substitution of the linear alkyl acids in positions 2 and 3 resulted in a drastic decrease of the affinity. The thermodynamic parameters show that a conformational change is triggered upon ligand binding. Binding enthalpy {delta}H{sub b} is negative in all cases for all ligands, and thus, Van der Waals interactions and hydrogen bonding are most probably the major sources for this term. The process is entropically favoured at all temperatures and pH studied, most probably due to the liberation of water molecules accompanying the conformational change of the enzyme.

  18. Crystallization, diffraction data collection and preliminary crystallographic analysis of hexagonal crystals of Pseudomonas aeruginosa amidase

    International Nuclear Information System (INIS)

    Andrade, Jorge; Karmali, Amin; Carrondo, Maria A.; Frazão, Carlos

    2007-01-01

    Crystals of aliphatic amidase (acylamide amidohydrolase; EC 3.5.1.4) from P. aeruginosa were obtained in space group P6 3 22 and diffracted to 1.25 Å resolution. The aliphatic amidase (acylamide amidohydrolase; EC 3.5.1.4) from Pseudomonas aeruginosa is a hexameric enzyme composed of six identical subunits with a molecular weight of ∼38 kDa. Since microbial amidases are very important enzymes in industrial biocatalysis, the structural characterization of this enzyme will help in the design of novel catalytic activities of commercial interest. The present study reports the successful crystallization of the wild-type amidase from P. aeruginosa. Native crystals were obtained and a complete data set was collected at 1.4 Å resolution, although the crystals showed diffraction to 1.25 Å resolution. The crystals were found to belong to space group P6 3 22, with unit-cell parameters a = b = 102.60, c = 151.71 Å, and contain one molecule in the asymmetric unit

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-06-28

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

  1. Functional analysis of 14 genes that constitute the purine catabolic pathway in Bacillus subtilis and evidence for a novel regulon controlled by the PucR transcription activator

    DEFF Research Database (Denmark)

    Schultz, Anna Charlotte; Nygaard, P.; Saxild, Hans Henrik

    2001-01-01

    expression of five genes (pucA, pucB, pucC, pucD, and pucE). Uricase activity is encoded by the pucL and pucM genes, and a uric acid transport system is encoded by pucJ and pucK. Allantoinase is encoded by the pucH gene, and allantoin permease is encoded by the pucI gene. Allantoate amidohydrolase is encoded...... acid, allantoin, and uric acid were all found to function as effector molecules for PucR-dependent regulation of puc gene expression. When cells were grown in the presence of glutamate plus allantoin, a 3- to 10-fold increase in expression was seen for most of the genes. However, expression of the puc...

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

    DEFF Research Database (Denmark)

    Gojkovic, Zoran; Sandrini, Michael; Piskur, Jure

    2001-01-01

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

  3. Activity screening of environmental metagenomic libraries reveals novel carboxylesterase families

    Science.gov (United States)

    Popovic, Ana; Hai, Tran; Tchigvintsev, Anatoly; Hajighasemi, Mahbod; Nocek, Boguslaw; Khusnutdinova, Anna N.; Brown, Greg; Glinos, Julia; Flick, Robert; Skarina, Tatiana; Chernikova, Tatyana N.; Yim, Veronica; Brüls, Thomas; Paslier, Denis Le; Yakimov, Michail M.; Joachimiak, Andrzej; Ferrer, Manuel; Golyshina, Olga V.; Savchenko, Alexei; Golyshin, Peter N.; Yakunin, Alexander F.

    2017-01-01

    Metagenomics has made accessible an enormous reserve of global biochemical diversity. To tap into this vast resource of novel enzymes, we have screened over one million clones from metagenome DNA libraries derived from sixteen different environments for carboxylesterase activity and identified 714 positive hits. We have validated the esterase activity of 80 selected genes, which belong to 17 different protein families including unknown and cyclase-like proteins. Three metagenomic enzymes exhibited lipase activity, and seven proteins showed polyester depolymerization activity against polylactic acid and polycaprolactone. Detailed biochemical characterization of four new enzymes revealed their substrate preference, whereas their catalytic residues were identified using site-directed mutagenesis. The crystal structure of the metal-ion dependent esterase MGS0169 from the amidohydrolase superfamily revealed a novel active site with a bound unknown ligand. Thus, activity-centered metagenomics has revealed diverse enzymes and novel families of microbial carboxylesterases, whose activity could not have been predicted using bioinformatics tools. PMID:28272521

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

    Directory of Open Access Journals (Sweden)

    Ronald Guillermo Peláez Sanchez

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

  5. Function Discovery and Structural Characterization of a Methylphosphonate Esterase

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-05-12

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

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

    Science.gov (United States)

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

    2010-12-01

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

  7. Enhancing the promiscuous phosphotriesterase activity of a thermostable lactonase (GkaP) for the efficient degradation of organophosphate pesticides.

    Science.gov (United States)

    Zhang, Yu; An, Jiao; Ye, Wei; Yang, Guangyu; Qian, Zhi-Gang; Chen, Hai-Feng; Cui, Li; Feng, Yan

    2012-09-01

    The phosphotriesterase-like lactonase (PLL) enzymes in the amidohydrolase superfamily hydrolyze various lactones and exhibit latent phosphotriesterase activities. These enzymes serve as attractive templates for in vitro evolution of neurotoxic organophosphates (OPs) with hydrolytic capabilities that can be used as bioremediation tools. Here, a thermostable PLL from Geobacillus kaustophilus HTA426 (GkaP) was targeted for joint laboratory evolution with the aim of enhancing its catalytic efficiency against OP pesticides. By a combination of site saturation mutagenesis and whole-gene error-prone PCR approaches, several improved variants were isolated. The most active variant, 26A8C, accumulated eight amino acid substitutions and demonstrated a 232-fold improvement over the wild-type enzyme in reactivity (k(cat)/K(m)) for the OP pesticide ethyl-paraoxon. Concomitantly, this variant showed a 767-fold decrease in lactonase activity with δ-decanolactone, imparting a specificity switch of 1.8 × 10(5)-fold. 26A8C also exhibited high hydrolytic activities (19- to 497-fold) for several OP pesticides, including parathion, diazinon, and chlorpyrifos. Analysis of the mutagenesis sites on the GkaP structure revealed that most mutations are located in loop 8, which determines substrate specificity in the amidohydrolase superfamily. Molecular dynamics simulation shed light on why 26A8C lost its native lactonase activity and improved the promiscuous phosphotriesterase activity. These results permit us to obtain further insights into the divergent evolution of promiscuous enzymes and suggest that laboratory evolution of GkaP may lead to potential biological solutions for the efficient decontamination of neurotoxic OP compounds.

  8. Catalytic Mechanism and Three-Dimensional Structure of Adenine Deaminase

    Energy Technology Data Exchange (ETDEWEB)

    Kamat, S.S.; Swaminathan, S.; Bagaria, A.; Kumaran, D.; Holmes-Hampton, G. P.; Fan, H.; Sali, A.; Sauder, J. M.; Burley, S. K.; Lindahl, P. A.; Raushel, F. M.

    2011-03-22

    Adenine deaminase (ADE) catalyzes the conversion of adenine to hypoxanthine and ammonia. The enzyme isolated from Escherichia coli using standard expression conditions was low for the deamination of adenine (k{sub cat} = 2.0 s{sup -1}; k{sub cat}/K{sub m} = 2.5 x 10{sup 3} M{sup -1} s{sup -1}). However, when iron was sequestered with a metal chelator and the growth medium was supplemented with Mn{sup 2+} prior to induction, the purified enzyme was substantially more active for the deamination of adenine with kcat and kcat/Km values of 200 s{sup -1} and 5 x 10{sup 5} M{sup -1} s{sup -1}, respectively. The apoenzyme was prepared and reconstituted with Fe{sup 2+}, Zn{sup 2+}, or Mn{sup 2+}. In each case, two enzyme equivalents of metal were necessary for reconstitution of the deaminase activity. This work provides the first example of any member of the deaminase subfamily of the amidohydrolase superfamily to utilize a binuclear metal center for the catalysis of a deamination reaction. [Fe{sup II}/Fe{sup II}]-ADE was oxidized to [Fe{sup III}/Fe{sup III}]-ADE with ferricyanide with inactivation of the deaminase activity. Reducing [Fe{sup III}/Fe{sup III}]-ADE with dithionite restored the deaminase activity, and thus, the diferrous form of the enzyme is essential for catalytic activity. No evidence of spin coupling between metal ions was evident by electron paramagnetic resonance or Moessbauer spectroscopy. The three-dimensional structure of adenine deaminase from Agrobacterium tumefaciens (Atu4426) was determined by X-ray crystallography at 2.2 {angstrom} resolution, and adenine was modeled into the active site on the basis of homology to other members of the amidohydrolase superfamily. On the basis of the model of the adenine-ADE complex and subsequent mutagenesis experiments, the roles for each of the highly conserved residues were proposed. Solvent isotope effects, pH-rate profiles, and solvent viscosity were utilized to propose a chemical reaction mechanism and the

  9. Catalytic Mechanism and Three-Dimensional Structure of Adenine Deaminase

    Energy Technology Data Exchange (ETDEWEB)

    S Kamat; A Bagaria; D Kumaran; G Holmes-Hampton; H Fan; A Sali; J Sauder; S Burley; P Lindahl; et. al.

    2011-12-31

    Adenine deaminase (ADE) catalyzes the conversion of adenine to hypoxanthine and ammonia. The enzyme isolated from Escherichia coli using standard expression conditions was low for the deamination of adenine (k{sub cat} = 2.0 s{sup -1}; k{sub cat}/K{sub m} = 2.5 x 10{sup 3} M{sup -1} s{sup -1}). However, when iron was sequestered with a metal chelator and the growth medium was supplemented with Mn{sup 2+} prior to induction, the purified enzyme was substantially more active for the deamination of adenine with k{sub cat} and k{sub cat}/K{sub m} values of 200 s{sup -1} and 5 x 10{sup 5} M{sup -1} s{sup -1}, respectively. The apoenzyme was prepared and reconstituted with Fe{sup 2+}, Zn{sup 2+}, or Mn{sup 2+}. In each case, two enzyme equivalents of metal were necessary for reconstitution of the deaminase activity. This work provides the first example of any member of the deaminase subfamily of the amidohydrolase superfamily to utilize a binuclear metal center for the catalysis of a deamination reaction. [Fe{sup II}/Fe{sup II}]-ADE was oxidized to [Fe{sup III}/Fe{sup III}]-ADE with ferricyanide with inactivation of the deaminase activity. Reducing [Fe{sup III}/Fe{sup III}]-ADE with dithionite restored the deaminase activity, and thus, the diferrous form of the enzyme is essential for catalytic activity. No evidence of spin coupling between metal ions was evident by electron paramagnetic resonance or Moessbauer spectroscopy. The three-dimensional structure of adenine deaminase from Agrobacterium tumefaciens (Atu4426) was determined by X-ray crystallography at 2.2 {angstrom} resolution, and adenine was modeled into the active site on the basis of homology to other members of the amidohydrolase superfamily. On the basis of the model of the adenine-ADE complex and subsequent mutagenesis experiments, the roles for each of the highly conserved residues were proposed. Solvent isotope effects, pH-rate profiles, and solvent viscosity were utilized to propose a chemical reaction

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-12-31

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

  11. Sequence analysis of the aminoacylase-1 family. A new proposed signature for metalloexopeptidases.

    Science.gov (United States)

    Biagini, A; Puigserver, A

    2001-03-01

    The amino acid sequence analysis of the human and porcine aminoacylases-1, the carboxypeptidase S precursor from Saccharomyces cerevisiae, the succinyl-diaminopimelate desuccinylase from Escherichia coli, Haemophilus influenzae and Corynebacterium glutamicum, the acetylornithine deacetylase from Escherichia coli and Dictyostelium discoideum and the carboxypeptidase G(2) precursor from Pseudomonas strain, using the Basic Local Alignment Search Tool (BLAST) and the Position-Specific Iterated BLAST (PSI-BLAST), allowed us to suggest that all these enzymes, which share common functional and biochemical features, belong to the same structural family. The three amino acid blocks which were found to be highly conserved, using the CLUSTAL W program, could be assigned to the catalytic active site, based on the general three-dimensional structure of the carboxypeptidase G(2) from the Pseudomonas strain precursor. Six additional proteins with the same signature have been retrieved after performing two successive PSI-BLAST iterations using the sequence of the conserved motif, namely Lactobacillus delbrueckii aminoacyl-histidine dipeptidase, Streptomyces griseus aminopeptidase, Saccharomyces cerevisiae aminopeptidase Y precursor, two Bacillus stearothermophilus N-carbamyl-L-amino acid amidohydrolases and Pseudomonas sp. hydantoin utilization protein C. The three conserved amino acid motifs corresponded to the following blocks: (i) [S, G, A]-H-x-D-x-V; (ii) G-x-x-D; and (iii) x-E-E. This new sequence signature is clearly different from that commonly reported in the literature for proteins belonging to the ArgE/DapE/CPG2/YscS family.

  12. X-ray structure of imidazolonepropionase from Agrobacterium tumefaciens at 1.87 Å resolution

    Energy Technology Data Exchange (ETDEWEB)

    Tyagi, Rajiv; Kumaran, Desigan; Burley, Stephen K.; Swaminathan, Subramanyam (SGX); (BNL)

    2010-01-12

    Histidine degradation in Agrobacterium tumefaciens involves four enzymes, including histidase (EC 4.3.1.3), urocanase (EC 4.2.1.49), imidazolonepropionase (EC 3.5.2.7), and N-formylglutamate amidohydrolase (EC 3.5.3.8). The third enzyme of the pathway, imidazolone-propionase, a 45.6 kDa protein, catalyzes conversion of imidazolone-5-propanoate to N-forminio-L-glutamate. Initial studies of the role of imidazolonepropionase in histidine degradation were published in 1953. Subsequent publications have been limited to enzyme kinetics, crystallization, and a recently reported structure determination. The imidazolonepropionases are members of metallodepenent-hydrolases (or amidohydroase) superfamily, which includs ureases, adenosine deaminases, phosphotriesterases, dihydroorotases, allantoinases, hydantoinases, adenine and cytosine deaminases, imidazolonepropionases, aryldial-kylphosphatases, chlorohydrolases, and formylmethanofuran dehydroases. Proteins belonging to this large group share a common three-dimensional structural motif (an eightfold {alpha}/{beta} or TIM barrel) with similar active sites. Most superfamily members also share a conserved metal binding site, involving four histidine residues and one aspartic acid. Imidazolonepropionase is one of the targets selected for X-ray crystallpgrahpic structure determination by the New York Structural GenomiX Research Consortium (NYSGXRC) Target ID: 9252b to correlate the structure function relationship of poorly studied by important enzyme. Here they report the crystal structure of imidazolonepropionase from Agrobacterium tumefaciens determined at 1.87 {angstrom} resolution.

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

    Science.gov (United States)

    El Shora, Hamed M; Ali, Awatif S

    2016-03-01

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

  14. Chlorella viruses contain genes encoding a complete polyamine biosynthetic pathway

    Science.gov (United States)

    Baumann, Sascha; Sander, Adrianne; Gurnon, James R.; Yanai-Balser, Giane; VanEtten, James L.; Piotrowski, Markus

    2007-01-01

    Two genes encoding the putative polyamine biosynthetic enzymes agmatine iminohydrolase (AIH) and N-carbamoylputrescine amidohydrolase (CPA) were cloned from the chloroviruses PBCV-1, NY-2A and MT325. They were expressed in Escherichia coli to form C-terminal (His)6-tagged proteins and the recombinant proteins were purified by Ni2+- binding affinity chromatography. The biochemical properties of the two enzymes are similar to AIH and CPA enzymes from Arabidopsis thaliana and Pseudomonas aeruginosa. Together with the previously known virus genes encoding ornithine/arginine decarboxlyase (ODC/ADC) and homospermidine synthase, the chloroviruses have genes that encode a complete set of functional enzymes that synthesize the rare polyamine homospermidine from arginine via agmatine, N-carbamoylputrescine and putrescine. The PBCV-1 aih and cpa genes are expressed early during virus infection together with the odc/adc gene, suggesting that biosynthesis of putrescine is important in early stages of viral replication. The aih and cpa genes are widespread in the chlorella viruses. PMID:17101165

  15. Amine-synthesizing enzyme N-substituted formamide deformylase: screening, purification, characterization, and gene cloning.

    Science.gov (United States)

    Fukatsu, Hiroshi; Hashimoto, Yoshiteru; Goda, Masahiko; Higashibata, Hiroki; Kobayashi, Michihiko

    2004-09-21

    N-substituted formamide was produced through the hydration of an isonitrile by isonitrile hydratase in the isonitrile metabolism. The former compound was further degraded by a microorganism, strain F164, which was isolated from soil through an acclimatization culture. The N-substituted formamide-degrading microorganism was identified as Arthrobacter pascens. The microbial degradation was found to proceed through an enzymatic reaction, the N-substituted formamide being hydrolyzed to yield the corresponding amine and formate. The enzyme, designated as N-substituted formamide deformylase (NfdA), was purified and characterized. The native enzyme had a molecular mass of approximately 61 kDa and consisted of two identical subunits. It stoichiometrically catalyzed the hydrolysis of N-benzylformamide (an N-substituted formamide) to benzylamine and formate. Of all of the N-substituted formamides tested, N-benzylformamide was the most suitable substrate for the enzyme. However, no amides were accepted as substrates. The gene (nfdA) encoding this enzyme was also cloned. The deduced amino acid sequence of nfdA exhibited the highest overall sequence identity (28%) with those of regulatory proteins among known proteins. Only the N-terminal region (residues 58-72) of NfdA also showed significant sequence identity (27-73%) to that of each member of the amidohydrolase superfamily, although there was no similarity in the overall sequence except in the above limited region.

  16. Purification, characterization and in-silico analysis of nitrilase from Gordonia terrae.

    Science.gov (United States)

    Kumar, Vijay; Seth, Amit; Kumari, Vijaya; Kumar, Virender; Bhalla, Tek C

    2015-01-01

    An inducible and aromatic nitrilase from Gordonia terrae was purified with a yield of 19%. The enzyme had turnover number of 63 s⁻¹ x 10⁻¹, Km 1.4 mM and Vmax 95 Umg⁻¹ protein for benzonitrile. The nitrilase of G. terrae was active at basic pH (7-10), moderate temperature (20-45 °C) and has a half-life of 4 h at 35 °C. MALDI analysis and amino acid sequence deduced from cloned nucleotide fragment showed 97% homology with putative amidohydrolase of Gordonia sputi NBRC 100414 and G. namibiensis. The enzyme showed regioselectivity towards hydroxybenzonitriles, as different position of hydroxyl group i.e. meta-, para- and orthosubstitutions on benzonitrile effect enzyme activity. The in-silico interactions of these substrates with the predicted 3D model of this enzyme also showed differential interaction between hydroxyl group of substrates and the polar amino acids surrounding enzyme's active site. This leads to different proximity and orientation of substrates vis-a-vis their interaction with catalytic residues.

  17. The gene for indole-3-acetyl-L-aspartic acid hydrolase from Enterobacter agglomerans: molecular cloning, nucleotide sequence, and expression in Escherichia coli.

    Science.gov (United States)

    Chou, J C; Mulbry, W W; Cohen, J D

    1998-08-01

    A 5.5-kb DNA fragment containing the indole-3-acetyl-aspartic acid (IAA-asp) hydrolase gene (iaaspH) was isolated from Enterobacter agglomerans strain GK12 using a hybridization probe based on the N-terminal amino acid sequence of the protein. The DNA sequence of a 2.4-kb region of this fragment was determined and revealed a 1311-nucleotide ORF large enough to encode the 45-kDa IAA-asp hydrolase. A 1.5-kb DNA fragment containing iaaspH was subcloned into the Escherichia coli expression plasmid pTTQ8 to yield plasmid pJCC2. Extracts of IPTG-induced E. coli cultures containing the pJCC2 recombinant plasmid showed IAA-asp hydrolase levels 5 to 10-fold higher than those in E. agglomerans extracts. Homology searches revealed that the IAA-asp hydrolase was similar to a variety of amidohydrolases. In addition, IAA-asp hydrolase showed 70% sequence identity to a putative thermostable carboxypeptidase of E. coli.

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

    Science.gov (United States)

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

    2015-12-01

    Streptococcus suis serotype 2 (S. suis 2) is a zoonotic pathogen that exhibits high-level resistance and multi-drug resistance to classic antibiotics and causes serious human casualties and heavy economic losses in the swine industry worldwide. Therefore, alternative therapies or novel antibacterial agents need to be developed to combat this pathogen. A novel endolysin derived from the S. suis temperate phage phi7917, termed Ly7917, was identified, which had broad lytic activity against S. suis type 1, 2, 7 and 9. Ly7917 consisted of an N-terminal cysteine, histidine-dependent amidohydrolases/peptidase catalytic domain and C-terminal SH3b cell wall binding domain. The endolysin maintained activity at high pH and its catalytic activity could be improved by addition of 10 μM 1.5 mM Ca(2+). In animal studies, 90% of BALB/c mice challenged with typical virulent strain HA9801 of S. suis 2 were protected by Ly7917 treatment. The bacterial load in the blood of HA9801-challenged mice was efficiently reduced almost 50% by Ly7917 while that of penicillin-G-treated mice kept almost unchanged. Our data suggest that Ly7917 may be an alternative therapeutic agent for infections caused by virulent S. suis strains. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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

    Science.gov (United States)

    Chang, Yoonjee; Ryu, Sangryeol

    2017-01-01

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

  20. Characterization of the Lytic Capability of a LysK-Like Endolysin, Lys-phiSA012, Derived from a Polyvalent Staphylococcus aureus Bacteriophage.

    Science.gov (United States)

    Fujiki, Jumpei; Nakamura, Tomohiro; Furusawa, Takaaki; Ohno, Hazuki; Takahashi, Hiromichi; Kitana, Junya; Usui, Masaru; Higuchi, Hidetoshi; Tanji, Yasunori; Tamura, Yutaka; Iwano, Hidetomo

    2018-02-24

    Antibiotic-resistant bacteria (ARB) have spread widely and rapidly, with their increased occurrence corresponding with the increased use of antibiotics. Infections caused by Staphylococcus aureus have a considerable negative impact on human and livestock health. Bacteriophages and their peptidoglycan hydrolytic enzymes (endolysins) have received significant attention as novel approaches against ARB, including S. aureus . In the present study, we purified an endolysin, Lys-phiSA012, which harbors a cysteine/histidine-dependent amidohydrolase/peptidase (CHAP) domain, an amidase domain, and a SH3b cell wall binding domain, derived from a polyvalent S. aureus bacteriophage which we reported previously. We demonstrate that Lys-phiSA012 exhibits high lytic activity towards staphylococcal strains, including methicillin-resistant S. aureus (MRSA). Analysis of deletion mutants showed that only mutants possessing the CHAP and SH3b domains could lyse S. aureus , indicating that lytic activity of the CHAP domain depended on the SH3b domain. The presence of at least 1 mM Ca 2+ and 100 µM Zn 2+ enhanced the lytic activity of Lys-phiSA012 in a turbidity reduction assay. Furthermore, a minimum inhibitory concentration (MIC) assay showed that the addition of Lys-phiSA012 decreased the MIC of oxacillin. Our results suggest that endolysins are a promising approach for replacing current antimicrobial agents and may contribute to the proper use of antibiotics, leading to the reduction of ARB.

  1. Characterization of the Lytic Capability of a LysK-Like Endolysin, Lys-phiSA012, Derived from a Polyvalent Staphylococcus aureus Bacteriophage

    Directory of Open Access Journals (Sweden)

    Jumpei Fujiki

    2018-02-01

    Full Text Available Antibiotic-resistant bacteria (ARB have spread widely and rapidly, with their increased occurrence corresponding with the increased use of antibiotics. Infections caused by Staphylococcus aureus have a considerable negative impact on human and livestock health. Bacteriophages and their peptidoglycan hydrolytic enzymes (endolysins have received significant attention as novel approaches against ARB, including S. aureus. In the present study, we purified an endolysin, Lys-phiSA012, which harbors a cysteine/histidine-dependent amidohydrolase/peptidase (CHAP domain, an amidase domain, and a SH3b cell wall binding domain, derived from a polyvalent S. aureus bacteriophage which we reported previously. We demonstrate that Lys-phiSA012 exhibits high lytic activity towards staphylococcal strains, including methicillin-resistant S. aureus (MRSA. Analysis of deletion mutants showed that only mutants possessing the CHAP and SH3b domains could lyse S. aureus, indicating that lytic activity of the CHAP domain depended on the SH3b domain. The presence of at least 1 mM Ca2+ and 100 µM Zn2+ enhanced the lytic activity of Lys-phiSA012 in a turbidity reduction assay. Furthermore, a minimum inhibitory concentration (MIC assay showed that the addition of Lys-phiSA012 decreased the MIC of oxacillin. Our results suggest that endolysins are a promising approach for replacing current antimicrobial agents and may contribute to the proper use of antibiotics, leading to the reduction of ARB.

  2. Lytic activity of the staphylolytic Twort phage endolysin CHAP domain is enhanced by the SH3b cell wall binding domain.

    Science.gov (United States)

    Becker, Stephen C; Swift, Steven; Korobova, Olga; Schischkova, Nina; Kopylov, Pavel; Donovan, David M; Abaev, Igor

    2015-01-01

    Increases in the prevalence of antibiotic-resistant strains of Staphylococcus aureus have elicited efforts to develop novel antimicrobials to treat these drug-resistant pathogens. One potential treatment repurposes the lytic enzymes produced by bacteriophages as antimicrobials. The phage Twort endolysin (PlyTW) harbors three domains, a cysteine, histidine-dependent amidohydrolases/peptidase domain (CHAP), an amidase-2 domain and a SH3b-5 cell wall binding domain (CBD). Our results indicate that the CHAP domain alone is necessary and sufficient for lysis of live S. aureus, while the amidase-2 domain is insufficient for cell lysis when provided alone. Loss of the CBD results in ∼10X reduction of enzymatic activity in both turbidity reduction and plate lysis assays compared to the full length protein. Deletion of the amidase-2 domain resulted in a protein (PlyTW Δ172-373) with lytic activity that exceeded the activity of the full length construct in both the turbidity reduction and plate lysis assays. Addition of Ca(2+) enhanced the turbidity reduction activity of both the full length protein and truncation constructs harboring the CHAP domain. Chelation by addition of EDTA or the addition of zinc inhibited the activity of all PlyTW constructs. Published by Oxford University Press on behalf of FEMS 2014. This work is written by US Government employees and is in the public domain in the US.

  3. An unexpected vestigial protein complex reveals the evolutionary origins of an s-triazine catabolic enzyme.

    Science.gov (United States)

    Esquirol, Lygie; Peat, Thomas S; Wilding, Matthew; Liu, Jian-Wei; French, Nigel G; Hartley, Carol J; Onagi, Hideki; Nebl, Thomas; Easton, Christopher J; Newman, Janet; Scott, Colin

    2018-03-09

    Cyanuric acid is a metabolic intermediate of s-triazines, such as atrazine (a common herbicide) and melamine (used in resins and plastics). Cyanuric acid is mineralized to ammonia and carbon dioxide by the soil bacterium Pseudomonas sp. strain ADP via three hydrolytic enzymes (AtzD, AtzE, and AtzF). Here, we report the purification and biochemical and structural characterization of AtzE. Contrary to previous reports, we found that AtzE is not a biuret amidohydrolase, but instead catalyzes the hydrolytic deamination of 1-carboxybiuret. X-ray crystal structures of apo AtzE and AtzE bound with the suicide inhibitor phenyl phosphorodiamidate revealed that the AtzE enzyme complex consists of two independent molecules in the asymmetric unit. We also show that AtzE forms an α2β2 heterotetramer with a hitherto unidentified 68-amino-acid-long protein (AtzG) encoded in the cyanuric acid mineralization operon from Pseudomonas sp. strain ADP. Moreover, we observed that AtzG is essential for the production of soluble, active AtzE and that this obligate interaction is a vestige of their shared evolutionary origin. We propose that AtzEG was likely recruited into the cyanuric acid-mineralizing pathway from an ancestral glutamine transamidosome that required protein-protein interactions to enforce the exclusion of solvent from the transamidation reaction. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Marine microbial L-asparaginase: Biochemistry, molecular approaches and applications in tumor therapy and in food industry.

    Science.gov (United States)

    Izadpanah Qeshmi, Fatemeh; Homaei, Ahmad; Fernandes, Pedro; Javadpour, Sedigheh

    2018-03-01

    The marine environment is a rich source of biological and chemical diversity. It covers more than 70% of the Earth's surface and features a wide diversity of habitats, often displaying extreme conditions, where marine organisms thrive, offering a vast pool for microorganisms and enzymes. Given the dissimilarity between marine and terrestrial habitats, enzymes and microorganisms, either novel or with different and appealing features as compared to terrestrial counterparts, may be identified and isolated. L-asparaginase (E.C. 3.5.1.1), is among the relevant enzymes that can be obtained from marine sources. This amidohydrolase acts on L-asparagine and produce L-aspartate and ammonia, accordingly it has an acknowledged chemotherapeutic application, namely in acute lymphoblastic leukemia. Moreover, L-asparaginase is also of interest in the food industry as it prevents acrylamide formation. Terrestrial organisms have been largely tapped for L-asparaginases, but most failed to comply with criteria for practical applications, whereas marine sources have only been marginally screened. This work provides an overview on the relevant features of this enzyme and the framework for its application, with a clear emphasis on the use of L-asparaginase from marine sources. The review envisages to highlight the unique properties of marine L-asparaginases that could make them good candidates for medical applications and industries, especially in food safety. Copyright © 2018 Elsevier GmbH. All rights reserved.

  5. Inactivation of chloramphenicol and florfenicol by a novel chloramphenicol hydrolase.

    Science.gov (United States)

    Tao, Weixin; Lee, Myung Hwan; Wu, Jing; Kim, Nam Hee; Kim, Jin-Cheol; Chung, Eunsook; Hwang, Eul Chul; Lee, Seon-Woo

    2012-09-01

    Chloramphenicol and florfenicol are broad-spectrum antibiotics. Although the bacterial resistance mechanisms to these antibiotics have been well documented, hydrolysis of these antibiotics has not been reported in detail. This study reports the hydrolysis of these two antibiotics by a specific hydrolase that is encoded by a gene identified from a soil metagenome. Hydrolysis of chloramphenicol has been recognized in cell extracts of Escherichia coli expressing a chloramphenicol acetate esterase gene, estDL136. A hydrolysate of chloramphenicol was identified as p-nitrophenylserinol by liquid chromatography-mass spectroscopy and proton nuclear magnetic resonance spectroscopy. The hydrolysis of these antibiotics suggested a promiscuous amidase activity of EstDL136. When estDL136 was expressed in E. coli, EstDL136 conferred resistance to both chloramphenicol and florfenicol on E. coli, due to their inactivation. In addition, E. coli carrying estDL136 deactivated florfenicol faster than it deactivated chloramphenicol, suggesting that EstDL136 hydrolyzes florfenicol more efficiently than it hydrolyzes chloramphenicol. The nucleotide sequences flanking estDL136 encode proteins such as amidohydrolase, dehydrogenase/reductase, major facilitator transporter, esterase, and oxidase. The most closely related genes are found in the bacterial family Sphingomonadaceae, which contains many bioremediation-related strains. Whether the gene cluster with estDL136 in E. coli is involved in further chloramphenicol degradation was not clear in this study. While acetyltransferases for chloramphenicol resistance and drug exporters for chloramphenicol or florfenicol resistance are often detected in numerous microbes, this is the first report of enzymatic hydrolysis of florfenicol resulting in inactivation of the antibiotic.

  6. Biochemical characterization of a new nicotinamidase from an unclassified bacterium thriving in a geothermal water stream microbial mat community.

    Science.gov (United States)

    Zapata-Pérez, Rubén; Martínez-Moñino, Ana-Belén; García-Saura, Antonio-Ginés; Cabanes, Juana; Takami, Hideto; Sánchez-Ferrer, Álvaro

    2017-01-01

    Nicotinamidases are amidohydrolases that convert nicotinamide into nicotinic acid, contributing to NAD+ homeostasis in most organisms. In order to increase the number of nicotinamidases described to date, this manuscript characterizes a nicotinamidase obtained from a metagenomic library fosmid clone (JFF054_F02) obtained from a geothermal water stream microbial mat community in a Japanese epithermal mine. The enzyme showed an optimum temperature of 90°C, making it the first hyperthermophilic bacterial nicotinamidase to be characterized, since the phylogenetic analysis of this fosmid clone placed it in a clade of uncultured geothermal bacteria. The enzyme, named as UbNic, not only showed an alkaline optimum pH, but also a biphasic pH dependence of its kcat, with a maximum at pH 9.5-10.0. The two pKa values obtained were 4.2 and 8.6 for pKes1 and pKes2, respectively. These results suggest a possible flexible catalytic mechanism for nicotinamidases, which reconciles the two previously proposed mechanisms. In addition, the enzyme showed a high catalytic efficiency, not only toward nicotinamide, but also toward other nicotinamide analogs. Its mutational analysis showed that a tryptophan (W83) is needed in one of the faces of the active site to maintain low Km values toward all the substrates tested. Furthermore, UbNic proved to contain a Fe2+ ion in its metal binding site, and was revealed to belong to a new nicotinamidase subgroup. All these characteristics, together with its high pH- and thermal stability, distinguish UbNic from previously described nicotinamidases, and suggest that a wide diversity of enzymes remains to be discovered in extreme environments.

  7. A QM/MM study of the catalytic mechanism of nicotinamidase.

    Science.gov (United States)

    Sheng, Xiang; Liu, Yongjun

    2014-02-28

    Nicotinamidase (Pnc1) is a member of Zn-dependent amidohydrolases that hydrolyzes nicotinamide (NAM) to nicotinic acid (NA), which is a key step in the salvage pathway of NAD(+) biosynthesis. In this paper, the catalytic mechanism of Pnc1 has been investigated by using a combined quantum-mechanical/molecular-mechanical (QM/MM) approach based on the recently obtained crystal structure of Pnc1. The reaction pathway, the detail of each elementary step, the energetics of the whole catalytic cycle, and the roles of key residues and Zn-binding site are illuminated. Our calculation results indicate that the catalytic water molecule comes from the bulk solvent, which is then deprotonated by residue D8. D8 functions as a proton transfer station between C167 and NAM, while the activated C167 serves as the nucleophile. The residue K122 only plays a role in stabilizing intermediates and transition states. The oxyanion hole formed by the amide backbone nitrogen atoms of A163 and C167 has the function to stabilize the hydroxyl anion of nicotinamide. The Zn-binding site rather than a single Zn(2+) ion acts as a Lewis acid to influence the reaction. Two elementary steps, the activation of C167 in the deamination process and the decomposition of catalytic water in the hydrolysis process, correspond to the large energy barriers of 25.7 and 28.1 kcal mol(-1), respectively, meaning that both of them contribute a lot to the overall reaction barrier. Our results may provide useful information for the design of novel and efficient Pnc1 inhibitors and related biocatalytic applications.

  8. Biochemical characterization of a new nicotinamidase from an unclassified bacterium thriving in a geothermal water stream microbial mat community.

    Directory of Open Access Journals (Sweden)

    Rubén Zapata-Pérez

    Full Text Available Nicotinamidases are amidohydrolases that convert nicotinamide into nicotinic acid, contributing to NAD+ homeostasis in most organisms. In order to increase the number of nicotinamidases described to date, this manuscript characterizes a nicotinamidase obtained from a metagenomic library fosmid clone (JFF054_F02 obtained from a geothermal water stream microbial mat community in a Japanese epithermal mine. The enzyme showed an optimum temperature of 90°C, making it the first hyperthermophilic bacterial nicotinamidase to be characterized, since the phylogenetic analysis of this fosmid clone placed it in a clade of uncultured geothermal bacteria. The enzyme, named as UbNic, not only showed an alkaline optimum pH, but also a biphasic pH dependence of its kcat, with a maximum at pH 9.5-10.0. The two pKa values obtained were 4.2 and 8.6 for pKes1 and pKes2, respectively. These results suggest a possible flexible catalytic mechanism for nicotinamidases, which reconciles the two previously proposed mechanisms. In addition, the enzyme showed a high catalytic efficiency, not only toward nicotinamide, but also toward other nicotinamide analogs. Its mutational analysis showed that a tryptophan (W83 is needed in one of the faces of the active site to maintain low Km values toward all the substrates tested. Furthermore, UbNic proved to contain a Fe2+ ion in its metal binding site, and was revealed to belong to a new nicotinamidase subgroup. All these characteristics, together with its high pH- and thermal stability, distinguish UbNic from previously described nicotinamidases, and suggest that a wide diversity of enzymes remains to be discovered in extreme environments.

  9. The members of M20D peptidase subfamily from Burkholderia cepacia, Deinococcus radiodurans and Staphylococcus aureus (HmrA) are carboxydipeptidases, primarily specific for Met-X dipeptides.

    Science.gov (United States)

    Jamdar, Sahayog N; Are, Venkata N; Navamani, Mallikarjunan; Kumar, Saurabh; Nagar, Vandan; Makde, Ravindra D

    2015-12-01

    Three members of peptidase family M20D from Burkholderia cepacia (BcepM20D; Uniprot accession no. A0A0F7GQ23), Deinococcus radiodurans R1 (DradM20D; Uniprot accession no. Q9RTP6) and Staphylococcus aureus (HmrA; Uniprot accession no. Q99Q45) were characterized in terms of their preference for various substrates. The results thus reveal that all the enzymes including HmrA lack endopeptidase as well as aminopeptidase activities and possess strong carboxypeptidase activity. Further, the amidohydrolase activity exerted on other substrates like N-Acetyl-Amino acids, N-Carbobenzoxyl-Amino acids and Indole acetic acid (IAA)-Amino acids is due to the ability of these enzymes to accommodate different types of chemical groups other than the amino acid at the S1 pocket. Further, data on peptide hydrolysis strongly suggests that all the three enzymes are primarily carboxydipeptidases exhibiting highest catalytic efficiency (kcat/Km 5-36 × 10(5) M(-1) s(-1)) for Met-X substrates, where -X could be Ala/Gly/Ser/Tyr/Phe/Leu depending on the source organism. The supportive evidence for the substrate specificities was also provided with the molecular docking studies carried out using structure of SACOL0085 and homology modelled structure of BcepM20D. The preference for different substrates, their binding at active site of the enzyme and possible role of these enzymes in recycling of methionine are discussed in this study. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Structure and evolutionary conservation of the plant N-end rule pathway.

    Science.gov (United States)

    Graciet, Emmanuelle; Mesiti, Francesca; Wellmer, Frank

    2010-03-01

    The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal amino acid residue. While some N-terminal residues result in metabolically stable proteins, other, so-called destabilizing residues, lead to rapid protein turnover. The N-end rule pathway, which mediates the recognition and degradation of proteins with N-terminal destabilizing residues, is present in all organisms examined, including prokaryotes. This protein degradation pathway has a hierarchical organization in which some N-terminal residues, called primary destabilizing residues, are directly recognized by specific ubiquitin ligases. Other destabilizing residues, termed secondary and tertiary destabilizing residues, require modifications before the corresponding proteins can be targeted for degradation by ubiquitin ligases. In eukaryotes, the N-end rule pathway is a part of the ubiquitin/proteasome system and is known to play essential roles in a broad range of biological processes in fungi, animals and plants. While the structure of the N-end rule pathway has been extensively studied in yeast and mammals, knowledge of its organization in plants is limited. Using both tobacco and Arabidopsis, we identified the complete sets destabilizing and stabilizing N-terminal residues. We also characterized the hierarchical organization of the plant N-end rule by identifying and determining the specificity of two distinct N-terminal amidohydrolases (Nt-amidases) of Arabidopsis that are essential for the destabilizing activity of the tertiary destabilizing residues Asn and Gln. Our results indicate that both the N-end rule itself and mechanistic aspects of the N-end rule pathway in angiosperms are very similar to those of mammals.

  11. The cis-state of an azobenzene photoswitch is stabilized through specific interactions with a protein surface.

    Science.gov (United States)

    Korbus, Michael; Backé, Sarah; Meyer-Almes, Franz-Josef

    2015-03-01

    The photocontrol of protein function like enzyme activity has been the subject of many investigations to enable reversible and spatiotemporally defined cascading biochemical reactions without the need for separation in miniaturized and parallelized assay setups for academic and industrial applications. A photoswitchable amidohydrolase variant from Bordetella/Alcaligenes with the longest reported half-life (approximately 30 h) for the cis-state of the attached azobenzene group was chosen as a model system to dissect the underlying mechanism and molecular interactions that caused the enormous deceleration of the thermal cis-to-trans relaxation of the azobenzene photoswitch. A systematic site-directed mutagenesis study on the basis of molecular dynamics simulation data was employed to investigate enzyme and thermal cis-to-trans relaxation kinetics in dependence on selected amino acid substitution, which revealed a prominent histidine and a hydrophobic cluster as molecular determinants for the stabilization of the cis-isomer of the attached azobenzene moiety on the protein surface. The nature of the involved interactions consists of polar, hydrophobic, and possibly aromatic Π-Π contributions. The elucidated principles behind the stabilization of the cis-state of azobenzene derivatives on a protein surface can be exploited to design improved biologically inspired photoswitches. Moreover, the findings open the door to highly long-lived cis-states of azobenzene groups yielding improved bistable photoswitches that can be controlled by single light-pulses rather than continuous irradiation with UV light that causes potential photodamage to the employed biomolecules. Copyright © 2015 John Wiley & Sons, Ltd.

  12. Transcriptome Analysis of Agmatine and Putrescine Catabolism in Pseudomonas aeruginosa PAO1▿ †

    Science.gov (United States)

    Chou, Han Ting; Kwon, Dong-Hyeon; Hegazy, Mohamed; Lu, Chung-Dar

    2008-01-01

    Polyamines (putrescine, spermidine, and spermine) are major organic polycations essential for a wide spectrum of cellular processes. The cells require mechanisms to maintain homeostasis of intracellular polyamines to prevent otherwise severe adverse effects. We performed a detailed transcriptome profile analysis of Pseudomonas aeruginosa in response to agmatine and putrescine with an emphasis in polyamine catabolism. Agmatine serves as the precursor compound for putrescine (and hence spermidine and spermine), which was proposed to convert into 4-aminobutyrate (GABA) and succinate before entering the tricarboxylic acid cycle in support of cell growth, as the sole source of carbon and nitrogen. Two acetylpolyamine amidohydrolases, AphA and AphB, were found to be involved in the conversion of agmatine into putrescine. Enzymatic products of AphA were confirmed by mass spectrometry analysis. Interestingly, the alanine-pyruvate cycle was shown to be indispensable for polyamine utilization. The newly identified dadRAX locus encoding the regulator alanine transaminase and racemase coupled with SpuC, the major putrescine-pyruvate transaminase, were key components to maintaining alanine homeostasis. Corresponding mutant strains were severely hampered in polyamine utilization. On the other hand, an alternative γ-glutamylation pathway for the conversion of putrescine into GABA is present in some organisms. Subsequently, GabD, GabT, and PA5313 were identified for GABA utilization. The growth defect of the PA5313 gabT double mutant in GABA suggested the importance of these two transaminases. The succinic-semialdehyde dehydrogenase activity of GabD and its induction by GABA were also demonstrated in vitro. Polyamine utilization in general was proven to be independent of the PhoPQ two-component system, even though a modest induction of this operon was induced by polyamines. Multiple potent catabolic pathways, as depicted in this study, could serve pivotal roles in the control of

  13. Characterization of a novel endolysin LysSA11 and its utility as a potent biocontrol agent against Staphylococcus aureus on food and utensils.

    Science.gov (United States)

    Chang, Yoonjee; Kim, Minsik; Ryu, Sangryeol

    2017-12-01

    Here we show that the LysSA11 endolysin, derived from the virulent Staphylococcus aureus phage SA11, has lytic activity against staphylococcal strains. Bioinformatics analysis revealed an enzymatically active CHAP (cysteine, histidine-dependent amidohydrolases/peptidases) domain at the N-terminus of LysSA11 that showed amidase activity. A novel cell wall binding domain (CBD) in the C-terminus could bind to a broad spectrum of staphylococcal cells. The bactericidal activity of LysSA11 was determined in food and utensils artificially contaminated with methicillin-resistant S. aureus (MRSA). The amounts of MRSA bacteria in milk and on ham were significantly reduced by 1.44-log CFU/mL and 3.12-log CFU/cm 3 , respectively, within 15 min at refrigeration temperature (4 °C) and by 2.02-log CFU/mL and 3.37-log CFU/cm 2 , respectively, within 15 min at room temperature (25 °C). Moreover, a polypropylene plastic cutting board and a stainless steel knife artificially contaminated with approximately 4-log CFU/cm 2 of MRSA also showed complete bacterial elimination after a 30-min treatment with 1.35 μM of LysSA11. The data presented here strongly suggest that the novel CBD-containing staphylococcal endolysin LysSA11 can be used both as a food antimicrobial and as a practical sanitizer for utensils. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Increasing the stability of the bacteriophage endolysin PlyC using rationale-based FoldX computational modeling.

    Science.gov (United States)

    Heselpoth, Ryan D; Yin, Yizhou; Moult, John; Nelson, Daniel C

    2015-04-01

    Endolysins are bacteriophage-derived peptidoglycan hydrolases that represent an emerging class of proteinaceous therapeutics. While the streptococcal endolysin PlyC has been validated in vitro and in vivo for its therapeutic efficacy, the inherent thermosusceptible structure of the enzyme correlates to transient long-term stability, thereby hindering the feasibility of developing the enzyme as an antimicrobial. Here, we thermostabilized the cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain of the PlyCA catalytic subunit of PlyC using a FoldX-driven computational protein engineering approach. Using a combination of FoldX and Rosetta algorithms, as well as visual inspection, a final list of PlyC point mutant candidates with predicted stabilizing ΔΔG values was assembled and thermally characterized. Five of the eight point mutations were found experimentally to be destabilizing, a result most likely attributable to computationally modeling a complex and dynamic nine-subunit holoenzyme with a corresponding 3.3-Å X-ray crystal structure. However, one of the mutants, PlyC (PlyCA) T406R, was shown experimentally to increase the thermal denaturation temperature by ∼2.2°C and kinetic stability 16-fold over wild type. This mutation is expected to introduce a thermally advantageous hydrogen bond between the Q106 side chain of the N-terminal glycosyl hydrolase domain and the R406 side chain of the C-terminal CHAP domain. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  15. Biochemical and biophysical characterization of PlyGRCS, a bacteriophage endolysin active against methicillin-resistant Staphylococcus aureus.

    Science.gov (United States)

    Linden, Sara B; Zhang, Helena; Heselpoth, Ryan D; Shen, Yang; Schmelcher, Mathias; Eichenseher, Fritz; Nelson, Daniel C

    2015-01-01

    The increasing rate of resistance of pathogenic bacteria, such as Staphylococcus aureus, to classical antibiotics has driven research toward identification of other means to fight infectious disease. One particularly viable option is the use of bacteriophage-encoded peptidoglycan hydrolases, called endolysins or enzybiotics. These enzymes lyse the bacterial cell wall upon direct contact, are not inhibited by traditional antibiotic resistance mechanisms, and have already shown great promise in the areas of food safety, human health, and veterinary science. We have identified and characterized an endolysin, PlyGRCS, which displays dose-dependent antimicrobial activity against both planktonic and biofilm S. aureus, including methicillin-resistant S. aureus (MRSA). The spectrum of lytic activity for this enzyme includes all S. aureus and Staphylococcus epidermidis strains tested, but not other Gram-positive pathogens. The contributions of the PlyGRCS putative catalytic and cell wall binding domains were investigated through deletion analysis. The cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) catalytic domain displayed activity by itself, though reduced, indicating the necessity of the binding domain for full activity. In contrast, the SH3_5 binding domain lacked activity but was shown to interact directly with the staphylococcal cell wall via fluorescent microscopy. Site-directed mutagenesis studies determined that the active site residues in the CHAP catalytic domain were C29 and H92, and its catalytic functionality required calcium as a co-factor. Finally, biochemical assays coupled with mass spectrometry analysis determined that PlyGRCS displays both N-acetylmuramoyl-L-alanine amidase and D-alanyl-glycyl endopeptidase hydrolytic activities despite possessing only a single catalytic domain. These results indicate that PlyGRCS has the potential to become a revolutionary therapeutic option to combat bacterial infections.

  16. A Novel Chimeric Endolysin with Antibacterial Activity against Methicillin-Resistant Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Hamed Haddad Kashani

    2017-06-01

    Full Text Available Cysteine/histidine-dependent amidohydrolase/peptidase (CHAP and amidase are known as catalytic domains of the bacteriophage-derived endolysin LysK and were previously reported to show lytic activity against methicillin-resistant Staphylococcus aureus (MRSA. In the current study, the in silico design and analysis of chimeric CHAP-amidase model was applied to enhance the stability and solubility of protein, which was achieved through improving the properties of primary, secondary and tertiary structures. The coding gene sequence of the chimeric CHAP-amidase was synthesized and subcloned into the pET-22(+ expression vector, and the recombinant protein was expressed in E. coli BL21 (DE3 strain. Subsequent affinity-based purification yielded ~12 mg soluble protein per liter of E. coli culture. Statistical analysis indicated that concentrations of ≥1 μg/mL of the purified protein have significant antibacterial activity against S. aureus MRSA252 cells. The engineered chimeric CHAP-amidase exhibited 3.2 log reduction of MRSA252 cell counts at the concentration of 10 μg/mL. A synergistic interaction between CHAP-amidase and vancomycin was detected by using checkerboard assay and calculating the fractional inhibitory concentration (FIC index. This synergistic effect was shown by 8-fold reduction in the minimum inhibitory concentration of vancomycin. The chimeric CHAP-amidase displayed strong antibacterial activity against S. aureus, S. epidermidis, and enterococcus. However, it did not indicate any significant antibacterial activity against E. coli and Lactococcus lactis. Taken together, these findings suggest that our chimeric CHAP-amidase might represent potential to be used for the development of efficient antibacterial therapies targeting MRSA and certain Gram-positive bacteria.

  17. From callus to embryo: a proteomic view on the development and maturation of somatic embryos in Cyclamen persicum.

    Science.gov (United States)

    Rode, Christina; Lindhorst, Kathrin; Braun, Hans-Peter; Winkelmann, Traud

    2012-05-01

    In this study, the proteome structures following the pathway in somatic embryogenesis of Cyclamen persicum were analysed via high-resolution 2D-SDS-PAGE with two objectives: (1) to identify the significant physiological processes during somatic embryogenesis in Cyclamen and (2) to improve the maturation of somatic embryos. Therefore, the effects of maturation-promoting plant growth regulator abscisic acid (ABA) and high sucrose levels on torpedo-shaped embryos were investigated. In total, 108 proteins of differential abundance were identified using a combination of tandem mass spectrometry and a digital proteome reference map. In callus, enzymes related to energy supply were especially distinct, most likely due to energy demand caused by fast growth and cell division. The switch from callus to globular embryo as well as from globular to torpedo-shaped embryo was associated with controlled proteolysis via the ubiquitin-26S proteasome pathway. Storage compound accumulation was first detected 21 days after transfer to plant growth regulator (PGR)-free medium in early torpedo-shaped embryos. Increase in abundance of auxin-amidohydrolase during embryogenesis suggests a possible increase in auxin release in the late embryo stages of Cyclamen. A development-specific isoelectric point switch of catalases has been reported for the first time for somatic embryogenesis. Several proteins were identified to represent markers for the different developmental stages analysed. High sucrose levels and ABA treatment promoted the accumulation of storage compounds in torpedo-shaped embryos. Additionally, proteins of the primary metabolic pathways were decreased in the proteomes of ABA-treated embryos. Thus, ABA and high sucrose concentration in the culture medium improved maturation and consequently the quality of somatic embryos in C. persicum.

  18. Accessing carboxylesterase diversity from termite hindgut symbionts through metagenomics.

    Science.gov (United States)

    Rashamuse, Konanani; Mabizela-Mokoena, Nobalanda; Sanyika, Tendai Walter; Mabvakure, Batsirai; Brady, Dean

    2012-01-01

    A shotgun metagenomic library was constructed from termite hindgut symbionts and subsequently screened for esterase activities. A total of 68 recombinant clones conferring esterolytic phenotypes were identified, of which the 14 most active were subcloned and sequenced. The nucleotide lengths of the esterase-encoding open reading frames (ORFs) ranged from 783 to 2,592 bp and encoded proteins with predicted molecular masses of between 28.8 and 97.5 kDa. The highest identity scores in the GenBank database, from a global amino acid alignment ranged from 39 to 83%. The identified ORFs revealed the presence of the G-X-S-X-D, G-D-S-X, and S-X-X-K sequence motifs that have been reported to harbour a catalytic serine residue in other previously reported esterase primary structures. Five of the ORFs (EstT5, EstT7, EstT9, EstT10, and EstT12) could not be classified into any of the original eight esterase families. One of the ORFs (EstT9) showed a unique primary structure consisting of an amidohydrolase-esterase fusion. Six of the 14 esterase-encoding genes were recombinantly expressed in Escherichia coli and the purified enzymes exhibited temperature optima of between 40-50°C. Substrate-profiling studies revealed that the characterised enzymes were 'true' carboxylesterases based on their preferences for short to medium chain length p-nitrophenyl ester substrates. This study has demonstrated a successful application of a metagenomic approach in accessing novel esterase-encoding genes from the gut of termites that could otherwise have been missed by classical culture enrichment approaches. Copyright © 2012 S. Karger AG, Basel.

  19. A Novel Chimeric Endolysin with Antibacterial Activity against Methicillin-Resistant Staphylococcus aureus.

    Science.gov (United States)

    Haddad Kashani, Hamed; Fahimi, Hossein; Dasteh Goli, Yasaman; Moniri, Rezvan

    2017-01-01

    Cysteine/histidine-dependent amidohydrolase/peptidase (CHAP) and amidase are known as catalytic domains of the bacteriophage-derived endolysin LysK and were previously reported to show lytic activity against methicillin-resistant Staphylococcus aureus (MRSA). In the current study, the in silico design and analysis of chimeric CHAP-amidase model was applied to enhance the stability and solubility of protein, which was achieved through improving the properties of primary, secondary and tertiary structures. The coding gene sequence of the chimeric CHAP-amidase was synthesized and subcloned into the pET-22(+) expression vector, and the recombinant protein was expressed in E. coli BL21 (DE3) strain. Subsequent affinity-based purification yielded ~12 mg soluble protein per liter of E. coli culture. Statistical analysis indicated that concentrations of ≥1 μg/mL of the purified protein have significant antibacterial activity against S. aureus MRSA 252 cells. The engineered chimeric CHAP-amidase exhibited 3.2 log reduction of MRSA 252 cell counts at the concentration of 10 μg/mL. A synergistic interaction between CHAP-amidase and vancomycin was detected by using checkerboard assay and calculating the fractional inhibitory concentration (FIC) index. This synergistic effect was shown by 8-fold reduction in the minimum inhibitory concentration of vancomycin. The chimeric CHAP-amidase displayed strong antibacterial activity against S. aureus, S. epidermidis , and enterococcus . However, it did not indicate any significant antibacterial activity against E. coli and Lactococcus lactis . Taken together, these findings suggest that our chimeric CHAP-amidase might represent potential to be used for the development of efficient antibacterial therapies targeting MRSA and certain Gram-positive bacteria.

  20. Quantum Mechanics/Molecular Mechanics Simulations Identify the Ring-Opening Mechanism of Creatininase.

    Science.gov (United States)

    Jitonnom, Jitrayut; Mujika, Jon I; van der Kamp, Marc W; Mulholland, Adrian J

    2017-12-05

    Creatininase catalyzes the conversion of creatinine (a biosensor for kidney function) to creatine via a two-step mechanism: water addition followed by ring opening. Water addition is common to other known cyclic amidohydrolases, but the precise mechanism for ring opening is still under debate. The proton donor in this step is either His178 or a water molecule bound to one of the metal ions, and the roles of His178 and Glu122 are unclear. Here, the two possible reaction pathways have been fully examined by means of combined quantum mechanics/molecular mechanics simulations at the SCC-DFTB/CHARMM22 level of theory. The results indicate that His178 is the main catalytic residue for the whole reaction and explain its role as proton shuttle during the ring-opening step. In the first step, His178 provides electrostatic stabilization to the gem-diolate tetrahedral intermediate. In the second step, His178 abstracts the hydroxyl proton of the intermediate and delivers it to the cyclic amide nitrogen, leading to ring opening. The latter is the rate-limiting step with a free energy barrier of 18.5 kcal/mol, in agreement with the experiment. We find that Glu122 must be protonated during the enzyme reaction, so that it can form a stable hydrogen bond with its neighboring water molecule. Simulations of the E122Q mutant showed that this replacement disrupts the H-bond network formed by three conserved residues (Glu34, Ser78, and Glu122) and water, increasing the energy barrier. Our computational studies provide a comprehensive explanation for previous structural and kinetic observations, including why the H178A mutation causes a complete loss of activity but the E122Q mutation does not.

  1. Structural and Kinetic Characterization of the 4-Carboxy-2-hydroxymuconate Hydratase from the Gallate and Protocatechuate 4,5-Cleavage Pathways of Pseudomonas putida KT2440*

    Science.gov (United States)

    Mazurkewich, Scott; Brott, Ashley S.; Kimber, Matthew S.; Seah, Stephen Y. K.

    2016-01-01

    The bacterial catabolism of lignin and its breakdown products is of interest for applications in industrial processing of ligno-biomass. The gallate degradation pathway of Pseudomonas putida KT2440 requires a 4-carboxy-2-hydroxymuconate (CHM) hydratase (GalB), which has a 12% sequence identity to a previously identified CHM hydratase (LigJ) from Sphingomonas sp. SYK-6. The structure of GalB was determined and found to be a member of the PIG-L N-acetylglucosamine deacetylase family; GalB is structurally distinct from the amidohydrolase fold of LigJ. LigJ has the same stereospecificity as GalB, providing an example of convergent evolution for catalytic conversion of a common metabolite in bacterial aromatic degradation pathways. Purified GalB contains a bound Zn2+ cofactor; however the enzyme is capable of using Fe2+ and Co2+ with similar efficiency. The general base aspartate in the PIG-L deacetylases is an alanine in GalB; replacement of the alanine with aspartate decreased the GalB catalytic efficiency for CHM by 9.5 × 104-fold, and the variant enzyme did not have any detectable hydrolase activity. Kinetic analyses and pH dependence studies of the wild type and variant enzymes suggested roles for Glu-48 and His-164 in the catalytic mechanism. A comparison with the PIG-L deacetylases led to a proposed mechanism for GalB wherein Glu-48 positions and activates the metal-ligated water for the hydration reaction and His-164 acts as a catalytic acid. PMID:26867578

  2. Structural and Kinetic Characterization of the 4-Carboxy-2-hydroxymuconate Hydratase from the Gallate and Protocatechuate 4,5-Cleavage Pathways of Pseudomonas putida KT2440.

    Science.gov (United States)

    Mazurkewich, Scott; Brott, Ashley S; Kimber, Matthew S; Seah, Stephen Y K

    2016-04-01

    The bacterial catabolism of lignin and its breakdown products is of interest for applications in industrial processing of ligno-biomass. The gallate degradation pathway ofPseudomonas putidaKT2440 requires a 4-carboxy-2-hydroxymuconate (CHM) hydratase (GalB), which has a 12% sequence identity to a previously identified CHM hydratase (LigJ) fromSphingomonassp. SYK-6. The structure of GalB was determined and found to be a member of the PIG-LN-acetylglucosamine deacetylase family; GalB is structurally distinct from the amidohydrolase fold of LigJ. LigJ has the same stereospecificity as GalB, providing an example of convergent evolution for catalytic conversion of a common metabolite in bacterial aromatic degradation pathways. Purified GalB contains a bound Zn(2+)cofactor; however the enzyme is capable of using Fe(2+)and Co(2+)with similar efficiency. The general base aspartate in the PIG-L deacetylases is an alanine in GalB; replacement of the alanine with aspartate decreased the GalB catalytic efficiency for CHM by 9.5 × 10(4)-fold, and the variant enzyme did not have any detectable hydrolase activity. Kinetic analyses and pH dependence studies of the wild type and variant enzymes suggested roles for Glu-48 and His-164 in the catalytic mechanism. A comparison with the PIG-L deacetylases led to a proposed mechanism for GalB wherein Glu-48 positions and activates the metal-ligated water for the hydration reaction and His-164 acts as a catalytic acid. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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

    Science.gov (United States)

    Bae, Narkhyun; Pollak, Arnold; Lubec, Gert

    2011-07-01

    L-Asparaginase from Erwinia chrysanthemi (ASPG_ERWCH; UniProtKB accession number P06608 (Erwinase(®))) and L-asparaginase 2 from Escherichia coli (ASPG2_ECOLI; UniProtKB accession number P00805 (Medac(®))), both L-asparagine amidohydrolases, are widely used for the treatment of acute lymphoblastic leukemia. A series of serious side effects have been reported and this warrants studies into the protein chemistry of the medical products sold. Mass spectrometry (MS) data on ASPG_ERWCH and ASPG2_ECOLI have not been published so far and herein a gel-based proteomics study was performed to provide information about sequence and modifications of the commercially available medical products. ASPG_ERWCH and ASPG2_ECOLI were applied onto two-dimensional gel electrophoresis, spots were in-gel digested with several proteases and resulting peptides and protein modifications were analysed by nano-ESI-LC-MS/MS. Four spots were observed for ASPG_ERWCH, six spots were observed for ASPG2_ECOLI and the identified proteins showed high sequence coverage without sequence conflicts. Several protein modifications including technical and posttranslational modifications were demonstrated. Protein modifications are known to change physicochemical, immunochemical, biological and pharmacological properties and results from this work may challenge re-designing of the product including possible removal of the modifications by the manufacturer because it is not known whether they are contributing to the serious adverse effects of the protein drug. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Structure-Based and Random Mutagenesis Approaches Increase the Organophosphate-Degrading Activity of a Phosphotriesterase Homologue from Deinococcus radiodurans

    Energy Technology Data Exchange (ETDEWEB)

    Hawwa, Renda; Larsen, Sonia D.; Ratia, Kiira; Mesecar, Andrew D.; (UIC)

    2010-11-09

    An enzyme from the amidohydrolase family from Deinococcus radiodurans (Dr-OPH) with homology to phosphotriesterase has been shown to exhibit activity against both organophosphate (OP) and lactone compounds. We have characterized the physical properties of Dr-OPH and have found it to be a highly thermostable enzyme, remaining active after 3 h of incubation at 60 C and withstanding incubation at temperatures up to 70 C. In addition, it can withstand concentrations of at least 200 mg/mL. These properties make Dr-OPH a promising candidate for development in commercial applications. However, compared to the most widely studied OP-degrading enzyme, that from Pseudomonas diminuta, Dr-OPH has low hydrolytic activity against certain OP substrates. Therefore, we sought to improve the OP-degrading activity of Dr-OPH, specifically toward the pesticides ethyl and methyl paraoxon, using structure-based and random approaches. Site-directed mutagenesis, random mutagenesis, and site-saturation mutagenesis were utilized to increase the OP-degrading activity of Dr-OPH. Out of a screen of more than 30,000 potential mutants, a total of 26 mutant enzymes were purified and characterized kinetically. Crystal structures of w.t. Dr-OPH, of Dr-OPH in complex with a product analog, and of 7 mutant enzymes were determined to resolutions between 1.7 and 2.4 {angstrom}. Information from these structures directed the design and production of 4 additional mutants for analysis. In total, our mutagenesis efforts improved the catalytic activity of Dr-OPH toward ethyl and methyl paraoxon by 126- and 322-fold and raised the specificity for these two substrates by 557- and 183-fold, respectively. Our work highlights the importance of an iterative approach to mutagenesis, proving that large rate enhancements are achieved when mutations are made in already active mutants. In addition, the relationship between the kinetic parameters and the introduced mutations has allowed us to hypothesize on those

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

    Science.gov (United States)

    Lagares, Antonio; Roux, Indra; Valverde, Claudio

    2016-06-01

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

  6. Complete Nucleotide Sequence and Organization of the Atrazine Catabolic Plasmid pADP-1 from Pseudomonas sp. Strain ADP

    Science.gov (United States)

    Martinez, Betsy; Tomkins, Jeffrey; Wackett, Lawrence P.; Wing, Rod; Sadowsky, Michael J.

    2001-01-01

    The complete 108,845-nucleotide sequence of catabolic plasmid pADP-1 from Pseudomonas sp. strain ADP was determined. Plasmid pADP-1 was previously shown to encode AtzA, AtzB, and AtzC, which catalyze the sequential hydrolytic removal of s-triazine ring substituents from the herbicide atrazine to yield cyanuric acid. Computational analyses indicated that pADP-1 encodes 104 putative open reading frames (ORFs), which are predicted to function in catabolism, transposition, and plasmid maintenance, transfer, and replication. Regions encoding transfer and replication functions of pADP-1 had 80 to 100% amino acid sequence identity to pR751, an IncPβ plasmid previously isolated from Enterobacter aerogenes. pADP-1 was shown to contain a functional mercury resistance operon with 99% identity to Tn5053. Complete copies of transposases with 99% amino acid sequence identity to TnpA from IS1071 and TnpA from Pseudomonas pseudoalcaligenes were identified and flank each of the atzA, atzB, and atzC genes, forming structures resembling nested catabolic transposons. Functional analyses identified three new catabolic genes, atzD, atzE, and atzF, which participate in atrazine catabolism. Crude extracts from Escherichia coli expressing AtzD hydrolyzed cyanuric acid to biuret. AtzD showed 58% amino acid sequence identity to TrzD, a cyanuric acid amidohydrolase, from Pseudomonas sp. strain NRRLB-12227. Two other genes encoding the further catabolism of cyanuric acid, atzE and atzF, reside in a contiguous cluster adjacent to a potential LysR-type transcriptional regulator. E. coli strains bearing atzE and atzF were shown to encode a biuret hydrolase and allophanate hydrolase, respectively. atzDEF are cotranscribed. AtzE and AtzF are members of a common amidase protein family. These data reveal the complete structure of a catabolic plasmid and show that the atrazine catabolic genes are dispersed on three disparate regions of the plasmid. These results begin to provide insight into how

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

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    Donovan David M

    2011-06-01

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

  8. Functional Characterization of CYP94-Genes and Identification of a Novel Jasmonate Catabolite in Flowers.

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    Viktoria Bruckhoff

    Full Text Available Over the past decades much research focused on the biosynthesis of the plant hormone jasmonyl-isoleucine (JA-Ile. While many details about its biosynthetic pathway as well about its physiological function are established nowadays, knowledge about its catabolic fate is still scarce. Only recently, the hormonal inactivation mechanisms became a stronger research focus. Two major pathways have been proposed to inactivate JA-Ile: i The cleavage of the jasmonyl-residue from the isoleucine moiety, a reaction that is catalyzed by specific amido-hydrolases, or ii, the sequential oxidation of the ω-end of the pentenyl side-chain. This reaction is catalyzed by specific members of the cytochrome P450 (CYP subfamily CYP94: CYP94B1, CYP94B3 and CYP94C1. In the present study, we further investigated the oxidative fate of JA-Ile by expanding the analysis on Arabidopsis thaliana mutants, lacking only one (cyp94b1, cyp94b2, cyp94b3, cyp94c1, two (cyp94b1xcyp94b2, cyp94b1xcyp94b3, cyp94b2xcyp94b3, three (cyp94b1xcyp94b2xcyp94b3 or even four (cyp94b1xcyp94b2xcyp94b3xcyp94c1 CYP94 functionalities. The results obtained in the present study show that CYP94B1, CYP94B2, CYP94B3 and CYP94C1 are responsible for catalyzing the sequential ω-oxidation of JA-Ile in a semi-redundant manner. While CYP94B-enzymes preferentially hydroxylate JA-Ile to 12-hydroxy-JA-Ile, CYP94C1 catalyzes primarily the subsequent oxidation, yielding 12-carboxy-JA-Ile. In addition, data obtained from investigating the triple and quadruple mutants let us hypothesize that a direct oxidation of unconjugated JA to 12-hydroxy-JA is possible in planta. Using a non-targeted metabolite fingerprinting analysis, we identified unconjugated 12-carboxy-JA as novel jasmonate derivative in floral tissues. Using the same approach, we could show that deletion of CYP94-genes might not only affect JA-homeostasis but also other signaling pathways. Deletion of CYP94B1, for example, led to accumulation of metabolites