WorldWideScience

Sample records for amidohydrolases

  1. Characterization of unexplored amidohydrolase enzyme-pterin deaminase.

    Science.gov (United States)

    Jayaraman, Angayarkanni; Thandeeswaran, Murugesan; Priyadarsini, Ulaganathan; Sabarathinam, Shanmugam; Nawaz, K A Ayub; Palaniswamy, Muthusamy

    2016-06-01

    Pterin deaminase is an amidohydrolase enzyme hydrolyzing pteridines to form lumazine derivatives and ammonia. The enzyme captured the attention of scientists as early as 1959 and had been patented for its application as an anticancer agent. It is ubiquitously present in prokaryotes and has been reported in some eukaryotes such as honey bee, silkworm and rats. The enzyme has been observed to have a spectrum of substrates with the formation of respective lumazines. The role of the substrates of the enzyme in various metabolic pathways warrants a significant role in the biological activity of both prokaryotes and eukaryotes. Even though the functions of the enzyme have been explored in prokaryotes, their niche in the eukaryotic system is not clear. There is very few information on the structural and functional properties of the enzyme. This review has been congregated to emphasize the significance of pterin deaminase and analyzes the lacunae in understanding the biological characters of the enzyme. PMID:27094187

  2. A stable three-enzyme creatinine biosensor. 3. Immobilization of creatinine amidohydrolase and sensor development.

    Science.gov (United States)

    Berberich, Jason A; Chan, Andy; Boden, Mark; Russell, Alan J

    2005-03-01

    We describe the development of an extended use amperometric three-enzyme creatinine biosensor and the successful chemical modification and immobilization of the enzyme creatinine amidohydrolase using polyurethane prepolymers. Creatinine amidohydrolase is significantly stabilized by immobilization in polyurethane polymers. The half-life increases from six to more than 80 days in buffer at 37 degrees C. The effect of silver ions leached from amperometric reference electrodes on enzyme and sensor performance is discussed. The use of cellulose acetate cover membranes to prevent silver from reaching the enzyme is investigated. Sensors prepared with cover membranes have half-lives almost an order of magnitude greater than those prepared with no cover membrane over the silver electrode. The complete biosensor has been constructed on a clinical blood analyzer platform and is stable for many days. PMID:16701796

  3. Directed Evolution of a Thermostable Quorum-quenching Lactonase from the Amidohydrolase Superfamily*

    OpenAIRE

    Chow, Jeng Yeong; Xue, Bo; Lee, Kang Hao; Tung, Alvin; Wu, Long; Robinson, Robert C.; Yew, Wen Shan

    2010-01-01

    A thermostable quorum-quenching lactonase from Geobacillus kaustophilus HTA426 (GI: 56420041) was used as an initial template for in vitro directed evolution experiments. This enzyme belongs to the phosphotriesterase-like lactonase (PLL) group of enzymes within the amidohydrolase superfamily that hydrolyze N-acylhomoserine lactones (AHLs) that are involved in virulence pathways of quorum-sensing pathogenic bacteria. Here we have determined the N-butyryl-l-homoserine lactone-liganded structure...

  4. The crystal structure of the amidohydrolase VinJ shows a unique hydrophobic tunnel for its interaction with polyketide substrates.

    Science.gov (United States)

    Shinohara, Yuji; Miyanaga, Akimasa; Kudo, Fumitaka; Eguchi, Tadashi

    2014-03-18

    VinJ is an amidohydrolase belonging to the serine peptidase family that catalyzes the hydrolysis of the terminal aminoacyl moiety of a polyketide intermediate during the biosynthesis of vicenistatin. Herein, we report the crystal structure of VinJ. VinJ possesses a unique hydrophobic tunnel for the recognition of the polyketide chain moiety of its substrate in the cap domain. Taken together with the results of phylogenetic analysis, our results suggest that VinJ represents a new amidohydrolase family that is different from the known α/β hydrolase type serine peptidases. PMID:24530530

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

    DEFF Research Database (Denmark)

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

    2003-01-01

    Slime mold, plant and insect dihydropyrimidine amidohydrolases (DHPases, EC 3.5.2.2), which catalyze the second step of pyrimidine and several anti-cancer drug degradations, were cloned and shown to functionally replace a defective DHPase enzyme in the yeast Saccharomyces kluyveri. The yeast...... and slime mold DHPases were over-expressed, shown to contain two zinc ions, characterized for their properties and compared to those of the calf liver enzyme. In general, the kinetic parameters varied widely among the enzymes, the mammalian DHPase having the highest catalytic efficiency. The ring opening...

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

    DEFF Research Database (Denmark)

    Gojkovic, Zoran; Rislund, L.; Andersen, B.; Sandrini, Michael; Cook, P.F.; Schnackerz, K.D.; Piskur, Jure

    2003-01-01

    Slime mold, plant and insect dihydropyrimidine amidohydrolases (DHPases, EC 3.5.2.2), which catalyze the second step of pyrimidine and several anti-cancer drug degradations, were cloned and shown to functionally replace a defective DHPase enzyme in the yeast Saccharomyces kluyveri. The yeast and...... slime mold DHPases were over-expressed, shown to contain two zinc ions, characterized for their properties and compared to those of the calf liver enzyme. In general, the kinetic parameters varied widely among the enzymes, the mammalian DHPase having the highest catalytic efficiency. The ring opening...

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

    OpenAIRE

    Cummings, Jennifer; Fedorov, Alexander A.; Xu, Chengfu; Brown, Shoshana; Fedorov, Elena; Patricia C Babbitt; Almo, Steven C.; Raushel, Frank M.

    2009-01-01

    The catalytic activities of three members of the amidohydrolase superfamily were discovered using amino acid substrate libraries. Bb3285 from Bordetella bronchiseptica, Gox1177 from Gluconobacter oxydans, and Sco4986 from Streptomyces coelicolor are currently annotated as d-aminoacylases or N-acetyl-d-glutamate deacetylases. These three enzymes are 22−34% identical to one another in amino acid sequence. Substrate libraries containing nearly all combinations of N-formyl-d-Xaa, N-acetyl-d-Xaa, ...

  8. CbiZ, an amidohydrolase enzyme required for salvaging the coenzyme B12 precursor cobinamide in archaea

    OpenAIRE

    Woodson, Jesse D.; Escalante-Semerena, Jorge C.

    2004-01-01

    The existence of a pathway for salvaging the coenzyme B12 precursor dicyanocobinamide (Cbi) from the environment was established by genetic and biochemical means. The pathway requires the function of a previously unidentified amidohydrolase enzyme that converts adenosylcobinamide to adenosylcobyric acid, a bona fide intermediate of the de novo coenzyme B12 biosynthetic route. The cbiZ gene of the methanogenic archaeon Methanosarcina mazei strain Göl was cloned, was overproduced in Escherichia...

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

    Directory of Open Access Journals (Sweden)

    Cheng-Yang Huang

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

  10. Hormone crosstalk in wound stress response: wound-inducible amidohydrolases can simultaneously regulate jasmonate and auxin homeostasis in Arabidopsis thaliana

    Science.gov (United States)

    Zhang, Tong; Poudel, Arati N.; Jewell, Jeremy B.; Kitaoka, Naoki; Staswick, Paul; Matsuura, Hideyuki; Koo, Abraham J.

    2016-01-01

    Jasmonate (JA) and auxin are essential hormones in plant development and stress responses. While the two govern distinct physiological processes, their signaling pathways interact at various levels. Recently, members of the Arabidopsis indole-3-acetic acid (IAA) amidohydrolase (IAH) family were reported to metabolize jasmonoyl-isoleucine (JA-Ile), a bioactive form of JA. Here, we characterized three IAH members, ILR1, ILL6, and IAR3, for their function in JA and IAA metabolism and signaling. Expression of all three genes in leaves was up-regulated by wounding or JA, but not by IAA. Purified recombinant proteins showed overlapping but distinct substrate specificities for diverse amino acid conjugates of JA and IAA. Perturbed patterns of the endogenous JA profile in plants overexpressing or knocked-out for the three genes were consistent with ILL6 and IAR3, but not ILR1, being the JA amidohydrolases. Increased turnover of JA-Ile in the ILL6- and IAR3-overexpressing plants created symptoms of JA deficiency whereas increased free IAA by overexpression of ILR1 and IAR3 made plants hypersensitive to exogenous IAA conjugates. Surprisingly, ILL6 overexpression rendered plants highly resistant to exogenous IAA conjugates, indicating its interference with IAA conjugate hydrolysis. Fluorescent protein-tagged IAR3 and ILL6 co-localized with the endoplasmic reticulum-localized JA-Ile 12-hydroxylase, CYP94B3. Together, these results demonstrate that in wounded leaves JA-inducible amidohydrolases contribute to regulate active IAA and JA-Ile levels, promoting auxin signaling while attenuating JA signaling. This mechanism represents an example of a metabolic-level crosstalk between the auxin and JA signaling pathways. PMID:26672615

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-01-01

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

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

    OpenAIRE

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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).

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Hicks, Katherine A.; Ealick, Steven E.

    2016-05-25

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

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

    OpenAIRE

    Kashani, Hamed Haddad; Moniri, Rezvan

    2015-01-01

    Objectives 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...

  17. 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; Jackson, Colin J

    2015-04-01

    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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-09-27

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

  19. Structural Investigations of N-carbamoylputrescine Amidohydrolase from Medicago truncatula: Insights into the Ultimate Step of Putrescine Biosynthesis in Plants.

    Science.gov (United States)

    Sekula, Bartosz; Ruszkowski, Milosz; Malinska, Maura; Dauter, Zbigniew

    2016-01-01

    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-(dihydroxymethyl)putrescine as well as with cadaverine, which is a longer analog 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 toward 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. PMID:27066023

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-01-01

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

  1. Annotating enzymes of uncertain function: the deacylation of D-amino acids by members of the amidohydrolase superfamily.

    Science.gov (United States)

    Cummings, Jennifer A; Fedorov, Alexander A; Xu, Chengfu; Brown, Shoshana; Fedorov, Elena; Babbitt, Patricia C; Almo, Steven C; Raushel, Frank M

    2009-07-14

    The catalytic activities of three members of the amidohydrolase superfamily were discovered using amino acid substrate libraries. Bb3285 from Bordetella bronchiseptica, Gox1177 from Gluconobacter oxidans, and Sco4986 from Streptomyces coelicolor are currently annotated as d-aminoacylases or N-acetyl-d-glutamate deacetylases. These three enzymes are 22-34% identical to one another in amino acid sequence. Substrate libraries containing nearly all combinations of N-formyl-d-Xaa, N-acetyl-d-Xaa, N-succinyl-d-Xaa, and l-Xaa-d-Xaa were used to establish the substrate profiles for these enzymes. It was demonstrated that Bb3285 is restricted to the hydrolysis of N-acyl-substituted derivatives of d-glutamate. The best substrates for this enzyme are N-formyl-d-glutamate (k(cat)/K(m) = 5.8 x 10(6) M(-1) s(-1)), N-acetyl-d-glutamate (k(cat)/K(m) = 5.2 x 10(6) M(-1) s(-1)), and l-methionine-d-glutamate (k(cat)/K(m) = 3.4 x 10(5) M(-1) s(-1)). Gox1177 and Sco4986 preferentially hydrolyze N-acyl-substituted derivatives of hydrophobic d-amino acids. The best substrates for Gox1177 are N-acetyl-d-leucine (k(cat)/K(m) = 3.2 x 10(4) M(-1) s(-1)), N-acetyl-d-tryptophan (k(cat)/K(m) = 4.1 x 10(4) M(-1) s(-1)), and l-tyrosine-d-leucine (k(cat)/K(m) = 1.5 x 10(4) M(-1) s(-1)). A fourth protein, Bb2785 from B. bronchiseptica, did not have d-aminoacylase activity. The best substrates for Sco4986 are N-acetyl-d-phenylalanine and N-acetyl-d-tryptophan. The three-dimensional structures of Bb3285 in the presence of the product acetate or a potent mimic of the tetrahedral intermediate were determined by X-ray diffraction methods. The side chain of the d-glutamate moiety of the inhibitor is ion-paired to Arg-295, while the alpha-carboxylate is ion-paired with Lys-250 and Arg-376. These results have revealed the chemical and structural determinants for substrate specificity in this protein. Bioinformatic analyses of an additional approximately 250 sequences identified as members of this group

  2. Identification and characterization of a new gene from Variovorax paradoxus Iso1 encoding N-acyl-D-amino acid amidohydrolase responsible for D-amino acid production.

    Science.gov (United States)

    Lin, Pei-Hsun; Su, Shiun-Cheng; Tsai, Ying-Chieh; Lee, Chia-Yin

    2002-10-01

    An N-acyl-d-amino acid amidohydrolase (N-D-AAase) was identified in cell extracts of a strain, Iso1, isolated from an environment containing N-acetyl-d-methionine. The bacterium was classified as Variovorax paradoxus by phylogenetic analysis. The gene was cloned and sequenced. The gene consisted of a 1467-bp ORF encoding a polypeptide of 488 amino acids. The V. paradoxusN-D-AAase showed significant amino acid similarity to the N-acyl-d-amino acid amidohydrolases of the two eubacteria Alcaligenes xylosoxydans A-6 (44-56% identity), Alcaligenes facelis DA1 (54% identity) and the hyperthermophilic archaeon Pyrococcus abyssi (42% identity). After over-expression of the N-D-AAase protein in Escherichia coli, the enzyme was purified by multistep chromatography. The native molecular mass was 52.8 kDa, which agreed with the predicted molecular mass of 52 798 Da and the enzyme appeared to be a monomer protein by gel-filtration chromatography. A homogenous protein with a specific activity of 516 U.mg-1 was finally obtained. After peptide sequencing by LC/MS/MS, the results were in agreement with the deduced amino acid sequence of the N-D-AAase. The pI of the enzyme was 5.12 and it had an optimal pH and temperature of 7.5 and 50 degrees C, respectively. After 30 min heat treatment at 45 degrees C, between pH 6 and pH 8, 80% activity remained. The N-D-AAase had higher hydrolysing activity against N-acetyl-d-amino acid derivates containing d-methionine, d-leucine and d-alanine and against N-chloroacetyl-d-phenylalanine. Importantly, the enzyme does not act on the N-acetyl-l-amino acid derivatives. The enzyme was inhibited by chelating agents and certain metal ions, but was activated by 1 mm of Co2+ and Mg2+. Thus, the N-D-AAase from V. paradoxus can be considered a chiral specific and metal-dependent enzyme. PMID:12354118

  3. Directed evolution and structural analysis of N-carbamoyl-D-amino acid amidohydrolase provide insights into recombinant protein solubility in Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Shimin; Li, Chunhong; Zhang, Weiwen; Cai, Yuanheng; Yang, Yunlin; Yang, Sheng; Jiang, Weihong

    2007-03-15

    One of the greatest bottlenecks in producing recombinant proteins in Escherichia coli is that over-expressed target proteins are mostly present in an insoluble form without any biological activity. N-carbamoyl-D-amino-acid amidohydrolase (DCase) is an important enzyme involved in semi-synthesis of β-lactam antibiotics in industry. In this study, in order to determine the amino acid sites responsible for solubility in DCase, error-prone PCR and DNA shuffling techniques are applied to randomly mutate its encoding sequence, followed by an efficient screening based on structural complementation. Several mutants of DCase with reduced aggregation are isolated. Solubility tests of these mutants and several other mutants generated by site-directed mutagenesis indicate that three amino acid residues of DCase (A18, Y30 and K34) are related to the protein solubility in DCase. In silico structural modeling analyses further suggest that hydrophilicity and/or negative charge at these three residues may be responsible for the increased solubility of DCase proteins in E. coli. Based on the information, multiple engineering designated mutants were constructed by site-directed mutagenesis; among them, a triple mutant A18T/Y30N/K34E (named as DCase-M3) can be over-expressed in E. coli with up to 80% of DCase-M3 protein as soluble. DCase-M3 is purified to homogeneity and a comparative analysis with WT DCase demonstrates that DCase-M3 enzyme is similar to the native DCase in terms of its kinetic and thermodynamic properties. The study provides new insights on recombinant protein solubility in E. coli.

  4. Murein lytic enzyme TgaA of Bifidobacterium bifidum MIMBb75 modulates dendritic cell maturation through its cysteine- and histidine-dependent amidohydrolase/peptidase (CHAP) amidase domain.

    Science.gov (United States)

    Guglielmetti, Simone; Zanoni, Ivan; Balzaretti, Silvia; Miriani, Matteo; Taverniti, Valentina; De Noni, Ivano; Presti, Ilaria; Stuknyte, Milda; Scarafoni, Alessio; Arioli, Stefania; Iametti, Stefania; Bonomi, Francesco; Mora, Diego; Karp, Matti; Granucci, Francesca

    2014-09-01

    Bifidobacteria are Gram-positive inhabitants of the human gastrointestinal tract that have evolved close interaction with their host and especially with the host's immune system. The molecular mechanisms underlying such interactions, however, are largely unidentified. In this study, we investigated the immunomodulatory potential of Bifidobacterium bifidum MIMBb75, a bacterium of human intestinal origin commercially used as a probiotic. Particularly, we focused our attention on TgaA, a protein expressed on the outer surface of MIMBb75's cells and homologous to other known bacterial immunoactive proteins. TgaA is a peptidoglycan lytic enzyme containing two active domains: lytic murein transglycosylase (LT) and cysteine- and histidine-dependent amidohydrolase/peptidase (CHAP). We ran immunological experiments stimulating dendritic cells (DCs) with the B. bifidum MIMBb75 and TgaA, with the result that both the bacterium and the protein activated DCs and triggered interleukin-2 (IL-2) production. In addition, we observed that the heterologous expression of TgaA in Bifidobacterium longum transferred to the bacterium the ability to induce IL-2. Subsequently, immunological experiments performed using two purified recombinant proteins corresponding to the single domains LT and CHAP demonstrated that the CHAP domain is the immune-reactive region of TgaA. Finally, we also showed that TgaA-dependent activation of DCs requires the protein CD14, marginally involves TRIF, and is independent of Toll-like receptor 4 (TLR4) and MyD88. In conclusion, our study suggests that the bacterial CHAP domain is a novel microbe-associated molecular pattern actively participating in the cross talk mechanisms between bifidobacteria and the host's immune system. PMID:24814791

  5. 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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    O. M. Artyushenko

    2006-02-01

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

  7. Immobilization of Escherichia coli cells with penicillin-amidohydrolase activity on solid polymeric carriers.

    Science.gov (United States)

    Zurková, E; Drobník, J; Kálal, J; Svec, F; Tyrácková, V; Vojtísek, V; Zeman, R

    1983-09-01

    Whole cells of Escherichia coli containing the enzyme penicillinamidohydrolase EC 3.5.1.11 were immobilized on the surface of modified macroporous copolymers of glycidylmethacrylate with ethylenedimethacrylate and of copolymers of methacrylaldehyde (MA) with divinylbenzene (DVB) by means of glutaraldehyde. These polymeric carriers were modified before cell binding by using ammonia or polyamines, especially ethylenediamine and hexamethylenediamine (HMDA). The highest specific activity and the largest yield in cell immobilization were achieved with the macroporous copolymer of MA and DVB modified with HMDA. The material thus obtained was used in repeated conversions of benzylpenicillin to 6-aminopenicillanic acid in a stirred batch reactor. PMID:18574818

  8. The substrate specificity and the catalytic mechanism of N-carbamyl- D-amino acid amidohydrolase: A theoretical investigation

    Science.gov (United States)

    Han, Wei-Wei; Zhan, Dong Ling; Luo, Quan; Zhou, Yi-Han; Yao, Yuan; Li, Ze-Sheng; Feng, Yan

    2009-04-01

    N-carbamyl- D-amino acid amidohydrolasecatalyzes the hydrolysis of N-carbamyl- D-amino acids to D-amino acids, ammonia and the carbon dioxide. The docking studies validate that D-NCAase possesses of preference for D-enantiomers, predict that Gly194 and Arg174 may take part in the catalytic mechanism, and Glu136 is essential to maintain the stable conformation for catalysis. The initial step of the acylation reaction catalyzed by D-NCAase has been studied by density functional calculations. It was furthermore demonstrated that Lys126, His143, and Asn196 decrease the reaction barrier, while Asn172 raise the barrier. The structural and mechanistic insights obtained from computational study should be valuable for the mechanisms of cysteine proteases.

  9. Murein Lytic Enzyme TgaA of Bifidobacterium bifidum MIMBb75 Modulates Dendritic Cell Maturation through Its Cysteine- and Histidine-Dependent Amidohydrolase/Peptidase (CHAP) Amidase Domain

    OpenAIRE

    S. Guglielmetti; Zanoni, I.; S. Balzaretti; M. Miriani; V. Taverniti; I. De Noni; I. Presti; M. Stuknyte; A. Scarafoni; S. Arioli; Iametti, S; F. Bonomi; Mora, D.; Karp, M; F. Granucci

    2014-01-01

    Bifidobacteria are Gram-positive inhabitants of the human gastrointestinal tract that have evolved close interaction with their host and especially with the host's immune system. The molecular mechanisms underlying such interactions, however, are largely unidentified. In this study, we investigated the immunomodulatory potential of Bifidobacterium bifidum MIMBb75, a bacterium of human intestinal origin commercially used as a probiotic. Particularly, we focused our attention on TgaA, a protein...

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

    Lifescience Database Archive (English)

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

  11. 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% ...

  12. 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.

  13. AcEST: BP915558 [AcEST

    Lifescience Database Archive (English)

    Full Text Available 3.4 tr|Q8CRD2|Q8CRD2_STAES Amino acid amidohydrolase OS=Staphylococc... 34 4.4 tr|Q5HLR5|Q5HLR5_STAEQ Amidohydrolase fami...V70K_TYMV 69 kDa protein OS=Turnip yellow mosaic virus... 30 4.7 sp|Q2KA45|PUR5_RHIEC Phosphoribosylformylglycinamid...1 sp|Q49WJ5|PUR5_STAS1 Phosphoribosylformylglycinamidine cyclo-lig... 30 6.1 sp|Q...4L580|PUR5_STAHJ Phosphoribosylformylglycinamidine cyclo-lig... 30 6.1 sp|Q9ERZ0|HEMGN_MOUSE Hemogen OS=Mus ...-tRNA--protein transferase O... 31 2.8 sp|P32870|CYA1_DROME Ca(2+)/calmodulin-responsive adenyla

  14. Molecular biology of microbial ureases.

    OpenAIRE

    Mobley, H L; Island, M D; Hausinger, R P

    1995-01-01

    Urease (urea amidohydrolase; EC 3.5.1.5) catalyzes the hydrolysis of urea to yield ammonia and carbamate. The latter compound spontaneously decomposes to yield another molecule of ammonia and carbonic acid. The urease phenotype is widely distributed across the bacterial kingdom, and the gene clusters encoding this enzyme have been cloned from numerous bacterial species. The complete nucleotide sequence, ranging from 5.15 to 6.45 kb, has been determined for five species including Bacillus sp. ...

  15. Effects of homologues and analogues of palmitoylethanolamide upon the inactivation of the endocannabinoid anandamide

    OpenAIRE

    Jonsson, Kent-Olov; Vandevoorde, Séverine; Lambert, Didier M; Tiger, Gunnar; Fowler, Christopher J.

    2001-01-01

    The ability of a series of homologues and analogues of palmitoylethanolamide to inhibit the uptake and fatty acid amidohydrolase (FAAH)-catalysed hydrolysis of [3H]-anandamide ([3H]-AEA) has been investigated.Palmitoylethanolamide and homologues with chain lengths from 12–18 carbon atoms inhibited rat brain [3H]-AEA metabolism with pI50 values of ∼5. Homologues with chain lengths ⩽eight carbon atoms gave

  16. 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...

  17. Molecular cloning and characterization of an amidase from Arabidopsis thaliana capable of converting indole-3-acetamide into the plant growth hormone, indole-3-acetic acid

    OpenAIRE

    Pollmann, Stephan; Neu, Daniel; Weiler, Elmar W.

    2003-01-01

    Acylamidohydrolases from higher plants have not been characterized or cloned so far. AtAMI1 is the first member of this enzyme family from a higher plant and was identified in the genome of Arabidopsis thaliana based on sequence homology with the catalytic-domain sequence of bacterial acylamidohydrolases, particularly those that exhibit indole-3-acetamide amidohydrolase activity. AtAMI1 polypeptide and mRNA are present in leaf tissues, as shown by immunoblotting and RT-PCR, respectively. AtAM...

  18. Genetic and bibliographic information: ASAH1 [GenLibi

    Lifescience Database Archive (English)

    Full Text Available ASAH1 N-acylsphingosine amidohydrolase (acid ceramidase) 1 human Farber lipogranulomatosis (MeSH ... C10) > Central Nervous System Diseases (C10.228) > Brain ... Diseases (C10.228.140) > Brain ... Diseases, Metabolic ... (C10.228.140.163) > Brain ... Diseases, Metabolic, Inborn (C10.228.140.163.100) ... 6.320) > Metabolism, Inborn Errors (C16.320.565) > Brain ... Diseases, Metabolic, Inborn (C16.320.565.189) > Ly ... ic Diseases (C18) > Metabolic Diseases (C18.452) > Brain ... Diseases, Metabolic (C18.452.132) > Brain ... Diseases ...

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

    International Nuclear Information System (INIS)

    Crystals of aliphatic amidase (acylamide amidohydrolase; EC 3.5.1.4) from P. aeruginosa were obtained in space group P6322 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 P6322, with unit-cell parameters a = b = 102.60, c = 151.71 Å, and contain one molecule in the asymmetric unit

  20. 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.

  1. 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.

  2. Identification of the Fluvirucin B2 (Sch 38518) Biosynthetic Gene Cluster from Actinomadura fulva subsp. indica ATCC 53714: substrate Specificity of the β-Amino Acid Selective Adenylating Enzyme FlvN.

    Science.gov (United States)

    Miyanaga, Akimasa; Hayakawa, Yuki; Numakura, Mario; Hashimoto, Junko; Teruya, Kuniko; Hirano, Takashi; Shin-Ya, Kazuo; Kudo, Fumitaka; Eguchi, Tadashi

    2016-05-01

    Fluvirucins are 14-membered macrolactam polyketides that show antifungal and antivirus activities. Fluvirucins have the β-alanine starter unit at their polyketide skeletons. To understand the construction mechanism of the β-alanine moiety in fluvirucin biosyntheses, we have identified the biosynthetic cluster of fluvirucin B2 produced from Actinomadura fulva subsp. indica ATCC 53714. The identified gene cluster contains three polyketide synthases, four characteristic β-amino acid-carrying enzymes, one decarboxylase, and one amidohydrolase. We next investigated the activity of the adenylation enzyme FlvN, which is a key enzyme for the selective incorporation of a β-amino acid substrate. FlvN showed strong preference for l-aspartate over other amino acids such as β-alanine. Based on these results, we propose a biosynthetic pathway for fluvirucin B2. PMID:26818633

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

    Directory of Open Access Journals (Sweden)

    José P Leite

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

  4. 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.

  5. Cloning, purification, crystallization and preliminary structural studies of penicillin V acylase from Bacillus subtilis

    Energy Technology Data Exchange (ETDEWEB)

    Rathinaswamy, Priya; Pundle, Archana V.; Prabhune, Asmita A.; SivaRaman, Hepzibah [Division of Biochemical Sciences, National Chemical Laboratory, Pune 411 008 (India); Brannigan, James A., E-mail: jab@ysbl.york.ac.uk; Dodson, Guy G. [Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5YW (United Kingdom); Suresh, C. G., E-mail: jab@ysbl.york.ac.uk [Division of Biochemical Sciences, National Chemical Laboratory, Pune 411 008 (India)

    2005-07-01

    An unannotated protein reported from B. subtilis has been expressed in E. coli and identified as possessing penicillin V acylase activity. The crystallization and preliminary crystallographic analysis of this penicillin V acylase is presented. Penicillin acylase proteins are amidohydrolase enzymes that cleave penicillins at the amide bond connecting the side chain to their β-lactam nucleus. An unannotated protein from Bacillus subtilis has been expressed in Escherichia coli, purified and confirmed to possess penicillin V acylase activity. The protein was crystallized using the hanging-drop vapour-diffusion method from a solution containing 4 M sodium formate in 100 mM Tris–HCl buffer pH 8.2. Diffraction data were collected under cryogenic conditions to a spacing of 2.5 Å. The crystals belonged to the orthorhombic space group C222{sub 1}, with unit-cell parameters a = 111.0, b = 308.0, c = 56.0 Å. The estimated Matthews coefficient was 3.23 Å{sup 3} Da{sup −1}, corresponding to 62% solvent content. The structure has been solved using molecular-replacement methods with B. sphaericus penicillin V acylase (PDB code 2pva) as the search model.

  6. Enzymatic cycling method using creatine kinase to measure creatine by real-time detection.

    Science.gov (United States)

    Ueda, Shigeru; Sakasegawa, Shin-Ichi

    2016-08-01

    We have developed a novel enzymatic cycling method that uses creatine kinase (CK) to measure creatine. The method takes advantage of the reversibility of the CK reaction in which the forward (creatine phosphate forming) and reverse reactions are catalyzed in the presence of an excess amount of ATP and IDP, respectively. Real-time detection was accomplished using ADP-dependent glucokinase (ADP-GK) together with glucose-6-phosphate dehydrogenase. ADP, one of the cycling reaction products, was distinguished from IDP by using the nucleotide selectivity of the ADP-GK. The increasing level of ADP was measured from the level of reduced NADP at 340 nm. The method is appropriate for an assay that requires high sensitivity because the rate of increase in absorbance at 340 nm is proportional to the amount of CK present in the reaction mix. We reasoned that the method with CK in combination with creatinine amidohydrolase could be used to assay creatinine, an important marker of kidney function. Our results confirmed the quantitative capability of the assay. PMID:27173608

  7. Immobilization of cells for use as biocatalysts

    Energy Technology Data Exchange (ETDEWEB)

    Vojtisek, V.; Jirku, V.; Krumphanzl, V.; Culik, K.

    1983-07-21

    Bacterial cells and cells of higher organisms are immobilized on polymers, either as whole cells, cell fragments, or subcellular components. This immobilization is used for stabilization of their various enzymic activities, which are of commercial interest, e.g. for the enzymes themselves, for alkaloid production, for hormone transformations, or for various fermentations. Thus, Sedipur CL-930 was polymerized in the presence of glutaraldehyde and the polymer was incubated with Alcaligenes metalcaligenes cells for immobilization. The nonimmobilized cells contained an aspartate ammonia-lyase activity of 550 mumol L-aspartate converted/min/g, and the immobilized cells contained an activity of 420 or 500 mumol aspartate/min/g when the polymer used was made with 2 different ratios of Sedipur to glutaraldehyde. The immobilized cell product had the form of defined platelets (lamellae) with a diameter of 100-600 mum, depending on the Sedipur/glutaraldehyde ratio. In other procedures, cells were permeabilized with tensides and/or organic solvents after the immobilization. Other cells immobilized included yeast, fungi, and plant cells. The activities which were examined included glycolytic enzymes, penicillin acylase, L-asparagine amidohydrolase and production of alkaloids and phytosterols from Solanum aviculare.

  8. 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

    The soil bacterium Bacillus subtilis has developed a highly controlled system for the utilization of a diverse array of low molecular-weight compounds as a nitrogen source when the preferred nitrogen sources, e.g., glutamate plus ammonia, are exhausted. We have identified such a system for the...... 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...... by pucF. In a pucR mutant, the level of expression was low for all genes tested, indicating that PucR is a positive regulator of puc gene expression. All 14 genes except pucI are located in a gene cluster at 284 to 285 degrees on the chromosome and are contained in six transcription units, which are...

  9. Construction of expression systems for Escherichia coli asparaginase II and two-step purification of the recombinant enzyme from periplasmic extracts.

    Science.gov (United States)

    Harms, E; Wehner, A; Jennings, M P; Pugh, K J; Beacham, I R; Röhm, K H

    1991-01-01

    Isoenzyme II of Escherichia coli L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) is among the few enzymes of major therapeutic importance, being used in the treatment of acute lymphoblastic leukemia. We have constructed several inducible expression systems that overproduce asparaginase II from recombinant plasmids. The most efficient of these systems consists of plasmid pTWE1, a derivative of pT7-7, and an ansB- strain of E. coli, CU1783. These cells produce and secrete amounts of asparaginase II that account for 10-15% of the total cellular protein. Most of the active recombinant enzyme can be released from the periplasmic space by a simple osmotic shock procedure. From the resulting material homogeneous asparaginase II was obtained by a two-step procedure. Overall yields of purified asparaginase were 10-15 mg asparaginase II per liter of E. coli culture. The recombinant enzyme appeared identical to conventionally purified preparations. PMID:1821783

  10. 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. PMID:25664524

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

    Directory of Open Access Journals (Sweden)

    Maristella Maggi

    2015-03-01

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

  12. Meta-analysis of organ-specific differences in the structure of the immune infiltrate in major malignancies.

    Science.gov (United States)

    Stoll, Gautier; Bindea, Gabriela; Mlecnik, Bernhard; Galon, Jérôme; Zitvogel, Laurence; Kroemer, Guido

    2015-05-20

    Anticancer immunosurveillance is one of the major endogenous breaks of tumor progression. Here, we analyzed gene expression pattern indicative of the presence of distinct leukocyte subtypes within four cancer types (breast cancer, colorectal carcinoma, melanoma, and non-small cell lung cancer) and 20 different microarray datasets corresponding to a total of 3471 patients. Multiple metagenes reflecting the presence of such immune cell subtypes were highly reproducible across distinct cohorts. Nonetheless, there were sizable differences in the correlation patterns among such immune-relevant metagenes across distinct malignancies. The reproducibility of the correlations among immune-relevant metagenes was highest in breast cancer (followed by colorectal cancer, non-small cell lung cancer and melanoma), reflecting the fact that mammary carcinoma has an intrinsically better prognosis than the three other malignancies. Among breast cancer patients, we found that the expression of a lysosomal enzyme-related metagene centered around ASAH1 (which codes for N-acylsphingosine amidohydrolase-1, also called acid ceramidase) exhibited a higher correlation with multiple immune-relevant metagenes in patients that responded to neoadjuvant chemotherapy than in non-responders. Altogether, this meta-analysis revealed novel organ-specific features of the immune infiltrate in distinct cancer types, as well as a strategy for defining new prognostic biomarkers. PMID:26059437

  13. Cloning, nucleotide sequence and expression of a new L-N-carbamoylase gene from Arthrobacter aurescens DSM 3747 in E. coli.

    Science.gov (United States)

    Wilms, B; Wiese, A; Syldatk, C; Mattes, R; Altenbuchner, J; Pietzsch, M

    1999-02-19

    An L-N-carbamoyl amino acid amidohydrolase (L-N-carbamoylase) from Arthrobacter aurescens DSM 3747 was cloned in E. coli and the nucleotide sequence was determined. After expression of the gene in E. coli the enzyme was purified to homogeneity and characterized. The enzyme was shown to be strictly L-specific and exhibited the highest activity in the hydrolysis of beta-aryl substituted N alpha-carbamoyl-alanines as e.g. N-carbamoyl-tryptophan. Carbamoyl derivatives of beta-alanine and charged aliphatic amino acids were not accepted as substrates. The N-carbamoylase of A. aurescens DSM 3747 differs from all known enzymes with respect to its substrate specificity although amino acid sequence identity scores of 35-38% to other N-carbamoylases have been detected. The enzyme consists of two subunits of 44,000 Da, and has an isoelectric point of 4.3. The optima of temperature and pH were determined to be 50 degrees C and pH 8.5 respectively. At 37 degrees C the enzyme was completely stable for several days. PMID:10194852

  14. A new protein structure representation for efficient protein function prediction.

    Science.gov (United States)

    Maghawry, Huda A; Mostafa, Mostafa G M; Gharib, Tarek F

    2014-12-01

    One of the challenging problems in bioinformatics is the prediction of protein function. Protein function is the main key that can be used to classify different proteins. Protein function can be inferred experimentally with very small throughput or computationally with very high throughput. Computational methods are sequence based or structure based. Structure-based methods produce more accurate protein function prediction. In this article, we propose a new protein structure representation for efficient protein function prediction. The representation is based on three-dimensional patterns of protein residues. In the analysis, we used protein function based on enzyme activity through six mechanistically diverse enzyme superfamilies: amidohydrolase, crotonase, haloacid dehalogenase, isoprenoid synthase type I, and vicinal oxygen chelate. We applied three different classification methods, naïve Bayes, k-nearest neighbors, and random forest, to predict the enzyme superfamily of a given protein. The prediction accuracy using the proposed representation outperforms a recently introduced representation method that is based only on the distance patterns. The results show that the proposed representation achieved prediction accuracy up to 98%, with improvement of about 10% on average. PMID:25343279

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

    International Nuclear Information System (INIS)

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

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

    Science.gov (United States)

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

    2012-01-01

    The antitumour enzyme L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1, ASNase), which catalyses the deamidation of L-asparagine (Asn) to L-aspartic acid and ammonia, has been used for many years in the treatment of acute lymphoblastic leukaemia. Also NK tumours, subtypes of myeloid leukaemias and T-cell lymphomas respond to ASNase, and ovarian carcinomas and other solid tumours have been proposed as additional targets for ASNase, with a potential role for its glutaminase activity. The increasing attention devoted to the antitumour activity of ASNase prompted us to analyse recent patents specifically concerning this enzyme. Here, we first give an overview of metabolic pathways affected by Asn and Gln depletion and, hence, potential targets of ASNase. We then discuss recent published patents concerning ASNases. In particular, we pay attention to novel ASNases, such as the recently characterised ASNase produced by Helicobacter pylori, and those presenting amino acid substitutions aimed at improving enzymatic activity of the classical Escherichia coli enzyme. We detail modifications, such as natural glycosylation or synthetic conjugation with other molecules, for therapeutic purposes. Finally, we analyse patents concerning biotechnological protocols and strategies applied to production of ASNase as well as to its administration and delivery in organisms. PMID:21854356

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2007-04-01

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

  18. Cloning, expression, purification, crystallization and preliminary X-ray characterization of allantoinase from Bacillus licheniformis ATCC 14580.

    Science.gov (United States)

    Conejero-Muriel, Mayte; Martínez-Gómez, Ana Isabel; Martínez-Rodríguez, Sergio; Gavira, Jose A

    2014-11-01

    Allantoinase, a member of the amidohydrolase superfamily, exists in a wide variety of organisms, including bacteria, fungi, plants and a few animals, such as fishes and amphibians. Allantoinase catalyzes the reversible hydrolysis of allantoin into allantoate by hydrolytic cleavage of the N1-C2 amide bond of the five-membered hydantoin ring. Allantoinase from Bacillus licheniformis (AllBali) presents an inverted enantioselectivity towards allantoin (R-enantioselective), which is a distinguishable feature that is not observed for other allantoinases. In this work, B. licheniformis ATCC 14580 allantoinase (AllBali) containing a C-terminal His6 tag was overproduced in Escherichia coli and purified to homogeneity. Crystals of AllBali were obtained by the vapour-diffusion method using 0.1 M potassium thiocyanate, 20%(w/v) polyethylene glycol 3350 as a crystallization solution. X-ray diffraction data were collected to a resolution of 3.5 Å with an Rmerge of 29.2% from a crystal belonging to space group P12₁1, with unit-cell parameters a=54.93, b=164.74, c=106.89 Å, β=98.49°. There are four molecules in the asymmetric unit with a solvent content of 47% as estimated from the Matthews coefficient (VM=2.34 Å3 Da(-1)). PMID:25372819

  19. 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. PMID:24413890

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

    Science.gov (United States)

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

    2015-10-01

    Hepatic injury undergoes significant increases in endocannabinoidsand infiltrations of macrophages, yet the concrete mechanisms of changes in endocannabinoids and the functions of macrophage-expressed cannabinoid receptors (CBs) are unclear. Biosynthetic and degradative enzymes of endocannabinoids revealed a significant change in human fibrotic liver. Meanwhile, we showed dynamic changes of these enzymes and CBs (CB1 and CB2) from 1 to 56 d in carbon tetrachloride-induced murine liver injury. Biosynthetic enzymes (N-acylphosphatidyl-ethanolamine selective phospholipase D and diacylglycerol lipase-α) and CBs were markedly increased, whereas degradative enzymes (fatty acid amidohydrolase and monoacylglycerol lipase) were downregulated. Moreover, these enzymes intimately correlated with the fibrosis parameter [procollagen α1(III)]. Bone marrow-derived monocytes/macrophages (BMM) expressed CBs. Interestingly, CB1 but not CB2 mediated BMM migration through a Boyden chambers assay, and the effect depended on the G(α)i/o/RhoA/ROCK signaling pathway. ICR mice were lethally irradiated and received BM transplants from enhanced GFP transgenic mice. Four weeks later, mice of BM reconstruction were subjected to carbon tetrachloride-induced liver injury. In the chimeric murine model, we found that blockade of CB1 by administration of a CB1 antagonist inhibited the recruitment of BMM into injured liver using immunofluorescence staining and FACS, but it did not have effects on migration of T cells and dendritic cells without CB1 expression. Furthermore, activation of CB1 enhanced cytokine expression of BMM. In vivo, inhibition of CB1 attenuated the inflammatory cytokine level through real-time RT-PCR and cytometric bead array, ameliorating hepatic inflammation and fibrosis. In this study, we identify inactivation of BMM-expressed CB1 as a therapeutic strategy for reducing hepatic inflammation and fibrosis. PMID:26320250

  1. Molecular biology of microbial ureases.

    Science.gov (United States)

    Mobley, H L; Island, M D; Hausinger, R P

    1995-09-01

    Urease (urea amidohydrolase; EC 3.5.1.5) catalyzes the hydrolysis of urea to yield ammonia and carbamate. The latter compound spontaneously decomposes to yield another molecule of ammonia and carbonic acid. The urease phenotype is widely distributed across the bacterial kingdom, and the gene clusters encoding this enzyme have been cloned from numerous bacterial species. The complete nucleotide sequence, ranging from 5.15 to 6.45 kb, has been determined for five species including Bacillus sp. strain TB-90, Klebsiella aerogenes, Proteus mirabilis, Helicobacter pylori, and Yersinia enterocolitica. Sequences for selected genes have been determined for at least 10 other bacterial species and the jack bean enzyme. Urease synthesis can be nitrogen regulated, urea inducible, or constitutive. The crystal structure of the K. aerogenes enzyme has been determined. When combined with chemical modification studies, biophysical and spectroscopic analyses, site-directed mutagenesis results, and kinetic inhibition experiments, the structure provides important insight into the mechanism of catalysis. Synthesis of active enzyme requires incorporation of both carbon dioxide and nickel ions into the protein. Accessory genes have been shown to be required for activation of urease apoprotein, and roles for the accessory proteins in metallocenter assembly have been proposed. Urease is central to the virulence of P. mirabilis and H. pylori. Urea hydrolysis by P. mirabilis in the urinary tract leads directly to urolithiasis (stone formation) and contributes to the development of acute pyelonephritis. The urease of H. pylori is necessary for colonization of the gastric mucosa in experimental animal models of gastritis and serves as the major antigen and diagnostic marker for gastritis and peptic ulcer disease in humans. In addition, the urease of Y. enterocolitica has been implicated as an arthritogenic factor in the development of infection-induced reactive arthritis. The significant

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

    Directory of Open Access Journals (Sweden)

    Sabine Marie Genicot

    2014-08-01

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

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

    Science.gov (United States)

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

    2014-08-01

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

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

    Directory of Open Access Journals (Sweden)

    Emilie Widemann

    2016-01-01

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

  5. The Hunt for 8-Oxoguanine Deaminase

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-01-01

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

  6. Discovery and Structure Determination of the Orphan Enzyme Isoxanthopterin Deaminase

    Energy Technology Data Exchange (ETDEWEB)

    Hall, R.S.; Swaminathan, S.; Agarwal, R.; Hitchcock, D.; Sauder, J. M.; Burley, S. K.; Raushel, F. M.

    2010-05-25

    Two previously uncharacterized proteins have been identified that efficiently catalyze the deamination of isoxanthopterin and pterin 6-carboxylate. The genes encoding these two enzymes, NYSGXRC-9339a (gi|44585104) and NYSGXRC-9236b (gi|44611670), were first identified from DNA isolated from the Sargasso Sea as part of the Global Ocean Sampling Project. The genes were synthesized, and the proteins were subsequently expressed and purified. The X-ray structure of Sgx9339a was determined at 2.7 {angstrom} resolution (Protein Data Bank entry 2PAJ). This protein folds as a distorted ({beta}/{alpha}){sub 8} barrel and contains a single zinc ion in the active site. These enzymes are members of the amidohydrolase superfamily and belong to cog0402 within the clusters of orthologous groups (COG). Enzymes in cog0402 have previously been shown to catalyze the deamination of guanine, cytosine, S-adenosylhomocysteine, and 8-oxoguanine. A small compound library of pteridines, purines, and pyrimidines was used to probe catalytic activity. The only substrates identified in this search were isoxanthopterin and pterin 6-carboxylate. The kinetic constants for the deamination of isoxanthopterin with Sgx9339a were determined to be 1.0 s{sup -1}, 8.0 {micro}M, and 1.3 x 10{sup 5} M{sup -1} s{sup -1} (k{sub cat}, K{sub m}, and k{sub cat}/K{sub m}, respectively). The active site of Sgx9339a most closely resembles the active site for 8-oxoguanine deaminase (Protein Data Bank entry 2UZ9). A model for substrate recognition of isoxanthopterin by Sgx9339a was proposed on the basis of the binding of guanine and xanthine in the active site of guanine deaminase. Residues critical for substrate binding appear to be conserved glutamine and tyrosine residues that form hydrogen bonds with the carbonyl oxygen at C4, a conserved threonine residue that forms hydrogen bonds with N5, and another conserved threonine residue that forms hydrogen bonds with the carbonyl group at C7. These conserved active site

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

    Directory of Open Access Journals (Sweden)

    Donovan David M

    2011-06-01

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

  8. Structure, Mechanism, and Substrate Profile for Sco3058: The Closest Bacterial Homologue to Human Renal Dipeptidase

    Energy Technology Data Exchange (ETDEWEB)

    Cummings, J.; Nguyen, T; Fedorov, A; Kolb, P; Xu, C; Fedorov, E; Shoichet, B; Barondeau, D; Almo, S; Raushel, F

    2010-01-01

    Human renal dipeptidase, an enzyme associated with glutathione metabolism and the hydrolysis of {beta}-lactams, is similar in sequence to a cluster of 400 microbial proteins currently annotated as nonspecific dipeptidases within the amidohydrolase superfamily. The closest homologue to the human renal dipeptidase from a fully sequenced microbe is Sco3058 from Streptomyces coelicolor. Dipeptide substrates of Sco3058 were identified by screening a comprehensive series of L-Xaa-L-Xaa, L-Xaa-D-Xaa, and D-Xaa-L-Xaa dipeptide libraries. The substrate specificity profile shows that Sco3058 hydrolyzes a broad range of dipeptides with a marked preference for an l-amino acid at the N-terminus and a d-amino acid at the C-terminus. The best substrate identified was L-Arg-D-Asp (k{sub cat}/K{sub m} = 7.6 x 10{sup 5} M{sup -1} s{sup -1}). The three-dimensional structure of Sco3058 was determined in the absence and presence of the inhibitors citrate and a phosphinate mimic of L-Ala-D-Asp. The enzyme folds as a ({beta}/{alpha}){sub 8} barrel, and two zinc ions are bound in the active site. Site-directed mutagenesis was used to probe the importance of specific residues that have direct interactions with the substrate analogues in the active site (Asp-22, His-150, Arg-223, and Asp-320). The solvent viscosity and kinetic effects of D{sub 2}O indicate that substrate binding is relatively sticky and that proton transfers do not occurr during the rate-limiting step. A bell-shaped pH-rate profile for k{sub cat} and k{sub cat}/K{sub m} indicated that one group needs to be deprotonated and a second group must be protonated for optimal turnover. Computational docking of high-energy intermediate forms of L/D-Ala-L/D-Ala to the three-dimensional structure of Sco3058 identified the structural determinants for the stereochemical preferences for substrate binding and turnover.

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

    Directory of Open Access Journals (Sweden)

    Bovenberg Roel AL

    2009-05-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  11. 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

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

    Indian Academy of Sciences (India)

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

    2010-03-01

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

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

    Science.gov (United States)

    König, Stefanie; Brodhun, Florian

    2016-01-01

    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 that may be

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

    Institute of Scientific and Technical Information of China (English)

    崔凤娟; 李立军; 刘景辉; 高婕; 王永强

    2011-01-01

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