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Tohru Yoshimura - One of the best experts on this subject based on the ideXlab platform.
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Lysine Racemase from a lactic acid bacterium, Oenococcus oeni: structural basis of substrate specificity
The Journal of Biochemistry, 2012Co-Authors: Shiro Kato, Hisashi Hemmi, Tohru YoshimuraAbstract:Oenococcus oeni, a lactic acid bacterium, possesses a lysine Racemase, which has a specific activity towards basic amino acids. A comparison of amino acid residues around the active site suggested that Ile222 and Tyr354 of the Geobacillus stearothermophilus alanine Racemase, which shares 60% sequence similarity with lysine Racemase, were replaced by Thr224 and Trp355 in the O. oeni lysine Racemase. T224I/W355Y double mutations significantly decreased the activity of lysine Racemase, whereas I222T/Y354W double mutations endowed alanine Racemase with lysine racemization activity. These results suggest that the two residues play an important role in lysine racemization.
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d amino acids in the brain structure and function of pyridoxal phosphate dependent amino acid Racemases
FEBS Journal, 2008Co-Authors: Tohru Yoshimura, Masaru GotoAbstract:D-serine serves as a co-agonist of the N-methyl D-aspartate receptor in mammalian brains, and its behavior is probably related to neurological disorders such as schizophrenia, Alzheimer's disease and amyotrophic lateral sclerosis. D-Serine is synthesized by a pyridoxal 5'-phosphate (PLP)-dependent serine Racemase. In this minireview, we provide a detailed discussion on the reaction mechanism of the PLP-dependent amino acid Racemase on the basis of its 3D structure. We compared the eukaryotic serine Racemase with bacterial alanine Racemase, the best-studied enzyme among the PLP-dependent amino acid Racemases, and thus suggested a putative reaction mechanism for mammalian D-serine synthesis.
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d‐Amino acids in the brain: structure and function of pyridoxal phosphate‐dependent amino acid Racemases
The FEBS journal, 2008Co-Authors: Tohru Yoshimura, Masaru GotoAbstract:D-serine serves as a co-agonist of the N-methyl D-aspartate receptor in mammalian brains, and its behavior is probably related to neurological disorders such as schizophrenia, Alzheimer's disease and amyotrophic lateral sclerosis. D-Serine is synthesized by a pyridoxal 5'-phosphate (PLP)-dependent serine Racemase. In this minireview, we provide a detailed discussion on the reaction mechanism of the PLP-dependent amino acid Racemase on the basis of its 3D structure. We compared the eukaryotic serine Racemase with bacterial alanine Racemase, the best-studied enzyme among the PLP-dependent amino acid Racemases, and thus suggested a putative reaction mechanism for mammalian D-serine synthesis.
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functional characterization of alanine Racemase from schizosaccharomyces pombe a eucaryotic counterpart to bacterial alanine Racemase
Journal of Bacteriology, 2001Co-Authors: Takuma Uo, Naotaka Tanaka, Kaoru Takegawa, Tohru Yoshimura, Nobuyoshi EsakiAbstract:d-Alanine is an essential component of bacterial peptidoglycans and is produced by alanine Racemase (EC 5.1.1.1), a pyridoxal 5′-phosphate (PLP)-dependent enzyme (22, 29). However, d-alanine also occurs in various natural compounds produced by other organisms. For example, cyclosporin A contains d-alanine as a component and is produced by a fungus, Tolypocladium niveum (10). Alanine Racemase was shown to be involved in the biosynthesis of d-alanine in this fungus and was later purified and characterized. Even though it differs markedly in primary structure from bacterial alanine Racemase, it rather resembles yeast threonine aldolase (4, 10). d-Serine occurs in mammalian brains and serves as an endogenous ligand of the glycine site of the N-methyl-d-aspartate receptor (14). Serine Racemase, a PLP-dependent enzyme, was purified from rat brain to homogeneity (35), and a cDNA clone for mouse brain enzyme was obtained (36); this enzyme is distinct from both bacterial and fungal alanine Racemases but similar to bacterial threonine dehydratase in primary structure (36). Serine Racemase occurs also in vancomycin-resistant Enterococcus gallinarum (1). However, it resembles bacterial alanine Racemases but not brain serine Racemase. Therefore, alanine Racemases and serine Racemases are classified into three groups: bacterial alanine Racemases plus serine Racemase from E. gallinarum, fungal alanine Racemase, and serine Racemase of mammalian brain. Similar structural divergence has been found between other types of PLP enzymes, as exemplified by ornithine decarboxylases. Bacterial and eucaryotic ornithine decarboxylases differ markedly from each other, and the eucaryotic one shares the same protein fold with bacterial alanine Racemase (9). Contrary to a long-standing belief, various d-amino acids, including d-alanine and d-serine, have been found in yeasts in a peptide-bound form (15). Although no evidence has been obtained for the occurrence of a free form of d-amino acids, they are most probably produced in yeast cells upon proteolytic cleavage of the peptides. Therefore, it is reasonable to assume that amino acid Racemases occur in yeasts. In fact, we have found that fission yeast, Schizosaccharomyces pombe, has a gene encoding a putative amino acid Racemase similar to bacterial alanine Racemases and the serine Racemase of E. gallinarum. We have named the gene alr1+ because it shows alanine Racemase activity upon cloning and expression in E. coli. We report here the gene cloning, purification, and characterization of the gene product, alanine Racemase ALR1p. Furthermore, we show that the Alr1p protein is involved in the catabolism of d-alanine in S. pombe, which we have confirmed through construction of a deletion mutation of the gene in S. pombe and heterologous expression of the gene in Saccharomyces cerevisiae.
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Stereochemistry of the hydrogen abstraction from pyridoxamine phosphate catalyzed by alanine Racemase of Bacillus stearothermophilus
Journal of Molecular Catalysis B-enzymatic, 2001Co-Authors: Akira Watanabe, Yoichi Kurokawa, Tohru Yoshimura, Kenji Soda, Nobuyoshi EsakiAbstract:Abstract Alanine Racemase of Bacillus stearothermophilus catalyzes transamination as a side reaction. Stereospecificity for the hydrogen abstraction from C-4′ of pyridoxamine 5′-phosphate occurring in the latter half transamination was examined. Both apo-wild-type and apo-fragmentary alanine Racemases abstracted approximately 20 and 80% of tritium from the stereospecifically-labeled (4′ S )- and (4′ R )-[4′- 3 H ]PMP, respectively, in the presence of pyruvate. Alanine Racemase catalyzes the abstraction of both 4′ S - and 4′ R -hydrogen like amino acid Racemase with broad substrate specificity. However, R -isomer preference is a characteristic property of alanine Racemase.
Kenji Soda - One of the best experts on this subject based on the ideXlab platform.
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alanine Racemase of alfalfa seedlings medicago sativa l first evidence for the presence of an amino acid Racemase in plants
Phytochemistry, 2006Co-Authors: Kazuki Yanagida, Tadao Oikawa, Tadashi Ogawa, Kenji SodaAbstract:Abstract We demonstrated several kinds of d -amino acids in plant seedlings, and moreover alanine Racemase (E.C.5.1.1.1) in alfalfa ( Medicago sativa L.) seedlings. This is the first evidence for the presence of amino acid Racemase in plant. The enzyme was effectively induced by the addition of l - or d -alanine, and we highly purified the enzyme to show enzymological properties. The enzyme exclusively catalyzed racemization of l - and d -alanine. The K m and V max values of enzyme for l -alanine were 29.6 × 10 −3 M and 1.02 mol/s/kg, and those for d -alanine are 12.0 × 10 −3 M and 0.44 mol/s/kg, respectively. The K eq value was estimated to be about 1 and indicated that the enzyme catalyzes a typical racemization of both enantiomers of alanine. The enzyme was inactivated by hydroxylamine, phenylhydrazine and some other pyridoxal 5′-phosphate enzyme inhibitors. Accordingly, the enzyme required pyridoxal 5′-phosphate as a coenzyme, and enzymologically resembled bacterial alanine Racemases studied so far.
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Glutamate Racemase is an endogenous DNA gyrase inhibitor.
Journal of Biological Chemistry, 2002Co-Authors: Makoto Ashiuchi, Kenji Soda, Eriko Kuwana, Takashi Yamamoto, Kazuya Komatsu, Haruo MisonoAbstract:Abstract Almost all bacteria possess glutamate Racemase to synthesize d-glutamate as an essential component of peptidoglycans in the cell walls. The enforced production of glutamate Racemase, however, resulted in suppression of cell proliferation. In the Escherichia coli JM109/pGR3 clone, the overproducer of glutamate Racemase, the copy number (i.e. replication efficiency) of plasmid DNA declined dramatically, whereas the E. coli WM335 mutant that is defective in the gene of glutamate Racemase showed little genetic competency. The comparatively low and high activities for DNA supercoiling were contained in the E. coli JM109/pGR3 and WM335 cells, respectively. Furthermore, we found that the DNA gyrase ofE. coli was modulated by the glutamate Racemase of E. coli in the presence of UDP-N-acetylmuramyl-l-alanine, which is a peptidoglycan precursor and functions as an absolute activator for the Racemase. This is the first finding of the enzyme protein participating in both d-amino acid metabolism and DNA processing.
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Gene cloning and characterization of alanine Racemases from Shigella dysenteriae, Shigella boydii, Shigella flexneri, and Shigella sonnei
Biochemical and biophysical research communications, 2001Co-Authors: Kumio Yokoigawa, Reiko Hirasawa, Hiroshi Ueno, Yoko Okubo, Seiichi Umesako, Kenji SodaAbstract:Alanine Racemase genes (alr) from Shigella dysenteriae, Shigella boydii, Shigella flexneri, and Shigella sonnei were cloned and expressed in Escherichia coli JM109. All genes encoded a polypeptide of 359 amino acids, and showed more than 99% sequence identities with each other. In particular, the S. dysenteriae alr was identical with the S. flexneri alr. Differences in the amino acid sequences between the four Shigella enzymes were only two residues: Gly138 in S. dysenteriae and S. flexneri (Glu138 in the other) and Ile225 in S. sonnei (Thr225 in the other). The S. boydii enzyme was identical with the E. coli K12 alr enzyme. Each Shigella alr enzyme purified to homogeneity has an apparent molecular mass about 43,000 by SDS-gel electrophoresis, and about 46,000 by gel filtration. However, all enzymes showed an apparent molecular mass about 60,000 by gel filtration in the presence of a substrate, 0.1 M l-alanine. These results suggest that the Shigella alr enzymes having an ordinary monomeric structure interact with other monomer in the presence of the substrate. The enzymes were almost identical in the enzymological properties, and showed lower catalytic activities (about 210 units/mg) than those of homodimeric alanine Racemases reported.
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Stereochemistry of the hydrogen abstraction from pyridoxamine phosphate catalyzed by alanine Racemase of Bacillus stearothermophilus
Journal of Molecular Catalysis B-enzymatic, 2001Co-Authors: Akira Watanabe, Yoichi Kurokawa, Tohru Yoshimura, Kenji Soda, Nobuyoshi EsakiAbstract:Abstract Alanine Racemase of Bacillus stearothermophilus catalyzes transamination as a side reaction. Stereospecificity for the hydrogen abstraction from C-4′ of pyridoxamine 5′-phosphate occurring in the latter half transamination was examined. Both apo-wild-type and apo-fragmentary alanine Racemases abstracted approximately 20 and 80% of tritium from the stereospecifically-labeled (4′ S )- and (4′ R )-[4′- 3 H ]PMP, respectively, in the presence of pyruvate. Alanine Racemase catalyzes the abstraction of both 4′ S - and 4′ R -hydrogen like amino acid Racemase with broad substrate specificity. However, R -isomer preference is a characteristic property of alanine Racemase.
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properties of glutamate Racemase from bacillus subtilis ifo 3336 producing poly γ glutamate
Journal of Biochemistry, 1998Co-Authors: Makoto Ashiuchi, Kenji Soda, Kazuhiko Tani, Haruo MisonoAbstract:We found glutamate Racemase activity in cell extracts of Bacillus subtilis IFO 3336, which abundantly produces poly-gamma-glutamate. The highest activity was obtained in the early stationary phase of growth. The Racemase was purified to homogeneity. The enzyme was a monomer with a molecular mass of about 30 kDa and required no cofactor. It almost exclusively catalyzed the racemization of glutamate; other amino acids, including alanine and aspartate but not homocysteinesulfinate, were inactive as either substrates or inhibitors. Although the Vmax value of the enzyme for L-glutamate is 21-fold higher than that for D-glutamate, the Vmax/Km value for L-glutamate is almost equal to that for the D-enantiomer. The Racemase gene, glr, was cloned into Escherichia coli cells and sequenced. The Racemase was overproduced in the soluble fraction of the E. coli clone cells with the substitution of ATG for TTG, the initial codon of the glr gene. D-Amino acid aminotransferase activity was not detected in Bacillus subtilis IFO 3336 cells. B. subtilis CU741, a leuC7 derivative of B. subtilis 168, showed lower glutamate Racemase activity and lower productivity of poly-gamma-glutamate than B. subtilis IFO 3336. These results suggest that the glutamate Racemase is mainly concerned in D-glutamate synthesis for poly-gamma-glutamate production in B. subtilis IFO 3336.
Herman Wolosker - One of the best experts on this subject based on the ideXlab platform.
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Modulation of D-serine levels via ubiquitin-dependent proteasomal degradation of serine Racemase.
Journal of Biological Chemistry, 2006Co-Authors: Elena Dumin, Veronika N. Foltyn, Inna Bendikov, Elena Kartvelishvily, Yoshio Misumi, Yukio Ikehara, Herman WoloskerAbstract:Abstract Mammalian serine Racemase is a brain-enriched enzyme that converts l- into d-serine in the nervous system. d-Serine is an endogenous co-agonist at the “glycine site” of N-methyl d-aspartate (NMDA) receptors that is required for the receptor/channel opening. Factors regulating the synthesis of d-serine have implications for the NMDA receptor transmission, but little is known on the signals and events affecting serine Racemase levels. We found that serine Racemase interacts with the Golgin subfamily A member 3 (Golga3) protein in yeast two-hybrid screening. The interaction was confirmed in vitro with the recombinant proteins in co-transfected HEK293 cells and in vivo by co-immunoprecipitation studies from brain homogenates. Golga3 and serine Racemase co-localized at the cytosol, perinuclear Golgi region, and neuronal and glial cell processes in primary cultures. Golga3 significantly increased serine Racemase steady-state levels in co-transfected HEK293 cells and primary astrocyte cultures. This observation led us to investigate mechanisms regulating serine Racemase levels. We found that serine Racemase is degraded through the ubiquitin-proteasomal system in a Golga3-modulated manner. Golga3 decreased the ubiquitylation of serine Racemase both in vitro and in vivo and significantly increased the protein half-life in pulse-chase experiments. Our results suggest that the ubiquitin system is a main regulator of serine Racemase and d-serine levels. Modulation of serine Racemase degradation, such as that promoted by Golga3, provides a new mechanism for regulating brain d-serine levels and NMDA receptor activity.
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Human serine Racemase: moleular cloning, genomic organization and functional analysis.
Gene, 2000Co-Authors: J De Miranda, A Santoro, S Engelender, Herman WoloskerAbstract:High levels of D-serine are found in mammalian brain, where it is an endogenous agonist of the strichinine-insensitive site of N-methyl D-aspartate type of glutamate receptors. D-serine is enriched in protoplasmic astrocytes that occur in gray matter areas of the brain and was shown to be synthesized from L-serine. We now report cloning and expression of human serine Racemase, an enzyme that catalyses the synthesis of D-serine from L-serine. The enzyme displays a high homology to the murine serine Racemase. It contains a pyridoxal 5'-phosphate attachment sequence similar to bacterial biosynthetic threonine dehydratase. Northern-blot analysis show high levels of human serine Racemase in areas known to contain large amounts of endogenous D-serine, such as hippocampus and corpus callosum. Robust synthesis of D-serine was detected in cells transfected with human serine Racemase, demonstrating the conservation of D-amino acid metabolism in humans. Serine Racemase may be a therapeutic target in psychiatric diseases as supplementation of D-serine greatly improves schizophrenia symptoms. We identify the human serine Racemase genomic structure and show that the gene encompasses seven exons and localizes to chromosome 17q13.3. Identification of the intron-exon boundaries of the human serine Racemase gene will be useful to search for mutations in neuropsychiatric disorders.
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serine Racemase a glial enzyme synthesizing d serine to regulate glutamate n methyl d aspartate neurotransmission
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Herman Wolosker, Seth Blackshaw, Solomon H. SnyderAbstract:Although d amino acids are prominent in bacteria, they generally are thought not to occur in mammals. Recently, high levels of d-serine have been found in mammalian brain where it activates glutamate/N-methyl-d-aspartate receptors by interacting with the “glycine site” of the receptor. Because amino acid Racemases are thought to be restricted to bacteria and insects, the origin of d-serine in mammals has been puzzling. We now report cloning and expression of serine Racemase, an enzyme catalyzing the formation of d-serine from l-serine. Serine Racemase is a protein representing an additional family of pyridoxal-5′ phosphate-dependent enzymes in eukaryotes. The enzyme is enriched in rat brain where it occurs in glial cells that possess high levels of d-serine in vivo. Occurrence of serine Racemase in the brain demonstrates the conservation of d-amino acid metabolism in mammals with implications for the regulation of N-methyl-d-aspartate neurotransmission through glia-neuronal interactions.
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Purification of serine Racemase: Biosynthesis of the neuromodulator d-serine
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Herman Wolosker, Christopher D. Ferris, Jean-pierre Mothet, Kevin N Sheth, Masaaki Takahashi, Roscoe O. Brady, Solomon H. SnyderAbstract:High levels of d-serine occur in mammalian brain, where it appears to be an endogenous ligand of the glycine site of N-methyl-d-aspartate receptors. In glial cultures of rat cerebral cortex, d-serine is enriched in type II astrocytes and is released upon stimulation with agonists of non-N-methyl-d-aspartate glutamate receptors. The high levels of d-serine in discrete areas of rat brain imply the existence of a biosynthetic pathway. We have purified from rat brain a soluble enzyme that catalyzes the direct racemization of l-serine to d-serine. Purified serine Racemase has a molecular mass of 37 kDa and requires pyridoxal 5′-phosphate for its activity. The enzyme is highly selective toward l-serine, failing to racemize any other amino acid tested. Properties such as pH optimum, Km values, and the requirement for pyridoxal phosphate resemble those of bacterial Racemases, suggesting that the biosynthetic pathway for d-amino acids is conserved from bacteria to mammalian brain.
Nicolas Coatnoan - One of the best experts on this subject based on the ideXlab platform.
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Proline Racemases are conserved mitogens: characterization of a Trypanosoma vivax proline Racemase.
Molecular and biochemical parasitology, 2009Co-Authors: Nathalie Chamond, Alain Cosson, Nicolas Coatnoan, Paola MinoprioAbstract:Trypanosoma cruzi proline Racemases (TcPRAC) are the only eukaryotic proline Racemases described so far. Except their role in the interconversion of free l- and d-proline enantiomers, parasite TcPRACs are involved in major T. cruzi biological pathways. These essential enzymes are implicated in the process of parasite differentiation and the acquisition of virulence during metacyclogenesis and are currently considered as key targets for drug development against Chagas' disease. In this study, we searched for the presence of TcPRAC gene homologues among other trypanosomatid genomes. Despite the high degree of gene synteny observed in Kinetoplastidae genomes, PRAC genes are missing in Trypanosoma brucei, Trypanosoma congolense and Leishmania spp. genomes. Interestingly, we identified a hypothetical PRAC gene in Trypanosoma vivax that is the major hemoparasite responsible for livestock trypanosomiasis, a serious economical impact for most of African and South American countries. We report here that the product of this T. vivax gene is bona fide a proline Racemase with an activity comparable to the one we described previously for TcPRAC. Inhibition studies using the pyrrole-2-carboxylic acid confirmed that this compound is a competitive inhibitor for both TcPRAC and TvPRAC enzymes. Similarly to TcPRAC and all members of the Racemase family studied so far in other pathogenic and nosocomial bacteria, our results show that TvPRAC is a T-cell-independent B-cell mitogen. Therefore the product of the novel TvPRAC gene identified in T. vivax and reported herein has the potential to be used as a drug target for this parasite-based trypanosomiasis.
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Proline Racemases: insights into Trypanosoma cruzi peptides containing D-proline
Memórias do Instituto Oswaldo Cruz., 2009Co-Authors: Nicolas Coatnoan, Nathalie Chamond, Armand Berneman, Paola MinoprioAbstract:Trypanosoma cruzi proline Racemases (TcPRAC) are homodimeric enzymes that interconvert the L and D-enantiomers of proline. At least two paralogous copies of proline Racemase (PR) genes are present per parasite haploid genome and they are differentially expressed during T. cruzi development. Non-infective epimastigote forms that overexpress PR genes differentiate more readily into metacyclic infective forms that are more invasive to host cells, indicating that PR participates in mechanisms of virulence acquisition. Using a combination of biochemical and enzymatic methods, we show here that, in addition to free D-amino acids, non-infective epimastigote and infective metacyclic parasite extracts possess peptides composed notably of D-proline. The relative contribution of TcPRAC to D-proline availability and its further assembly into peptides was estimated through the use of wild-type parasites and parasites over-expressing TcPRAC genes. Our data suggest that D-proline-bearing peptides, similarly to the mucopeptide layer of bacterial cell walls, may be of benefit to T. cruzi by providing resistance against host proteolytic mechanisms.
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Crystal structure, catalytic mechanism, and mitogenic properties of Trypanosoma cruzi proline Racemase
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Alejandro Buschiazzo, Alain Cosson, Wim Degrave, Armand Berneman, Nathalie Chamond, Christophe Grégoire, Maira Goytia, Francis Schaeffer, William Shepard, Nicolas CoatnoanAbstract:Amino acid Racemases catalyze the stereoinversion of the chiral C alpha to produce the d-enantiomers that participate in biological processes, such as cell wall construction in prokaryotes. Within this large protein family, bacterial proline Racemases have been extensively studied as a model of enzymes acting with a pyridoxal-phosphate-independent mechanism. Here we report the crystal structure of the proline Racemase from the human parasite Trypanosoma cruzi (TcPRACA), a secreted enzyme that triggers host B cell polyclonal activation, which prevents specific humoral immune responses and is crucial for parasite evasion and fate. The enzyme is a homodimer, with each monomer folded in two symmetric alpha/beta subunits separated by a deep crevice. The structure of TcPRACA in complex with a transition-state analog, pyrrole-2-carboxylic acid, reveals the presence of one reaction center per monomer, with two Cys residues optimally located to perform acid/base catalysis through a carbanion stabilization mechanism. Mutation of the catalytic Cys residues abolishes the enzymatic activity but preserves the mitogenic properties of the protein. In contrast, inhibitor binding promotes the closure of the interdomain crevice and completely abrogates B cell proliferation, suggesting that the mitogenic properties of TcPRACA depend on the exposure of transient epitopes in the ligand-free enzyme.
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crystal structure catalytic mechanism and mitogenic properties of trypanosoma cruzi proline Racemase
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Alejandro Buschiazzo, Alain Cosson, Wim Degrave, Armand Berneman, Nathalie Chamond, Christophe Grégoire, Maira Goytia, Francis Schaeffer, William Shepard, Nicolas CoatnoanAbstract:Amino acid Racemases catalyze the stereoinversion of the chiral Cα to produce the d-enantiomers that participate in biological processes, such as cell wall construction in prokaryotes. Within this large protein family, bacterial proline Racemases have been extensively studied as a model of enzymes acting with a pyridoxal-phosphate-independent mechanism. Here we report the crystal structure of the proline Racemase from the human parasite Trypanosoma cruzi (TcPRACA), a secreted enzyme that triggers host B cell polyclonal activation, which prevents specific humoral immune responses and is crucial for parasite evasion and fate. The enzyme is a homodimer, with each monomer folded in two symmetric α/β subunits separated by a deep crevice. The structure of TcPRACA in complex with a transition-state analog, pyrrole-2-carboxylic acid, reveals the presence of one reaction center per monomer, with two Cys residues optimally located to perform acid/base catalysis through a carbanion stabilization mechanism. Mutation of the catalytic Cys residues abolishes the enzymatic activity but preserves the mitogenic properties of the protein. In contrast, inhibitor binding promotes the closure of the interdomain crevice and completely abrogates B cell proliferation, suggesting that the mitogenic properties of TcPRACA depend on the exposure of transient epitopes in the ligand-free enzyme.
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Biochemical Characterization of Proline Racemases from the Human Protozoan Parasite Trypanosoma cruzi and Definition of Putative Protein Signatures
Journal of Biological Chemistry, 2003Co-Authors: Nathalie Chamond, Wim Degrave, Christophe Grégoire, Nicolas Coatnoan, Catherine Rougeot, Lucio H Freitas-junior, José Franco Da Silveira, Paola MinoprioAbstract:Proline Racemase catalyzes the interconversion of L- and D-proline enantiomers and has to date been described in only two species. Originally found in the bacterium Clostridium sticklandii, it contains cysteine residues in the active site and does not require co-factors or other known coenzymes. We recently described the first eukaryotic amino acid (proline) Racemase, after isolation and cloning of a gene from the pathogenic human parasite Trypanosoma cruzi. Although this enzyme is intracellularly located in replicative non-infective forms of T. cruzi, membrane-bound and secreted forms of the enzyme are present upon differentiation of the parasite into non-dividing infective forms. The secreted form of proline Racemase is a potent host B-cell mitogen supporting parasite evasion of specific immune responses. Here we describe that the TcPRAC genes in T. cruzi encode functional intracellular or secreted versions of the enzyme exhibiting distinct kinetic properties that may be relevant for their relative catalytic efficiency. Although the Km of the enzyme isoforms were of a similar order of magnitude (29-75 mM), Vmax varied between 2 x 10(-4 )and 5.3 x 10(-5) mol of L-proline/s/0.125 microM of homodimeric recombinant protein. Studies with the enzyme-specific inhibitor and abrogation of enzymatic activity by site-directed mutagenesis of the active site Cys330 residue reinforced the potential of proline Racemase as a critical target for drug development against Chagas' disease. Finally, we propose a protein signature for proline Racemases and suggest that the enzyme is present in several other pathogenic and non-pathogenic bacterial genomes of medical and agricultural interest, yet absent in mammalian host, suggesting that inhibition of proline Racemases may have therapeutic potential.
Nobuyoshi Esaki - One of the best experts on this subject based on the ideXlab platform.
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functional characterization of alanine Racemase from schizosaccharomyces pombe a eucaryotic counterpart to bacterial alanine Racemase
Journal of Bacteriology, 2001Co-Authors: Takuma Uo, Naotaka Tanaka, Kaoru Takegawa, Tohru Yoshimura, Nobuyoshi EsakiAbstract:d-Alanine is an essential component of bacterial peptidoglycans and is produced by alanine Racemase (EC 5.1.1.1), a pyridoxal 5′-phosphate (PLP)-dependent enzyme (22, 29). However, d-alanine also occurs in various natural compounds produced by other organisms. For example, cyclosporin A contains d-alanine as a component and is produced by a fungus, Tolypocladium niveum (10). Alanine Racemase was shown to be involved in the biosynthesis of d-alanine in this fungus and was later purified and characterized. Even though it differs markedly in primary structure from bacterial alanine Racemase, it rather resembles yeast threonine aldolase (4, 10). d-Serine occurs in mammalian brains and serves as an endogenous ligand of the glycine site of the N-methyl-d-aspartate receptor (14). Serine Racemase, a PLP-dependent enzyme, was purified from rat brain to homogeneity (35), and a cDNA clone for mouse brain enzyme was obtained (36); this enzyme is distinct from both bacterial and fungal alanine Racemases but similar to bacterial threonine dehydratase in primary structure (36). Serine Racemase occurs also in vancomycin-resistant Enterococcus gallinarum (1). However, it resembles bacterial alanine Racemases but not brain serine Racemase. Therefore, alanine Racemases and serine Racemases are classified into three groups: bacterial alanine Racemases plus serine Racemase from E. gallinarum, fungal alanine Racemase, and serine Racemase of mammalian brain. Similar structural divergence has been found between other types of PLP enzymes, as exemplified by ornithine decarboxylases. Bacterial and eucaryotic ornithine decarboxylases differ markedly from each other, and the eucaryotic one shares the same protein fold with bacterial alanine Racemase (9). Contrary to a long-standing belief, various d-amino acids, including d-alanine and d-serine, have been found in yeasts in a peptide-bound form (15). Although no evidence has been obtained for the occurrence of a free form of d-amino acids, they are most probably produced in yeast cells upon proteolytic cleavage of the peptides. Therefore, it is reasonable to assume that amino acid Racemases occur in yeasts. In fact, we have found that fission yeast, Schizosaccharomyces pombe, has a gene encoding a putative amino acid Racemase similar to bacterial alanine Racemases and the serine Racemase of E. gallinarum. We have named the gene alr1+ because it shows alanine Racemase activity upon cloning and expression in E. coli. We report here the gene cloning, purification, and characterization of the gene product, alanine Racemase ALR1p. Furthermore, we show that the Alr1p protein is involved in the catabolism of d-alanine in S. pombe, which we have confirmed through construction of a deletion mutation of the gene in S. pombe and heterologous expression of the gene in Saccharomyces cerevisiae.
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Stereochemistry of the hydrogen abstraction from pyridoxamine phosphate catalyzed by alanine Racemase of Bacillus stearothermophilus
Journal of Molecular Catalysis B-enzymatic, 2001Co-Authors: Akira Watanabe, Yoichi Kurokawa, Tohru Yoshimura, Kenji Soda, Nobuyoshi EsakiAbstract:Abstract Alanine Racemase of Bacillus stearothermophilus catalyzes transamination as a side reaction. Stereospecificity for the hydrogen abstraction from C-4′ of pyridoxamine 5′-phosphate occurring in the latter half transamination was examined. Both apo-wild-type and apo-fragmentary alanine Racemases abstracted approximately 20 and 80% of tritium from the stereospecifically-labeled (4′ S )- and (4′ R )-[4′- 3 H ]PMP, respectively, in the presence of pyruvate. Alanine Racemase catalyzes the abstraction of both 4′ S - and 4′ R -hydrogen like amino acid Racemase with broad substrate specificity. However, R -isomer preference is a characteristic property of alanine Racemase.
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occurrence of pyridoxal 5 phosphate dependent serine Racemase in silkworm bombyx mori
Biochemical and Biophysical Research Communications, 1998Co-Authors: Tohru Yoshimura, Susumu Shimizu, Nobuyoshi EsakiAbstract:D-Serine is known to occur in the silkwormBombyx morias well as in the mammalian central nervous systems. We found that serine Racemase occurs in the insect, catalyzing the conversion of L-serine to its antipode. The enzyme was partially purified from pupae of the insect, and was inactivated by treatment with hydroxylamine and reactivated with pyridoxal 5′-phosphate (PLP). L-Alanine was racemized slowly by the enzyme at a rate of only about 6% of that of L-serine, and L-arginine and L-glutamine were inert as substrates. Therefore, the enzyme is a member of PLP-dependent amino acid Racemases, and is distinct from alanine Racemase (EC 5.1.1.1) and amino acid Racemase with low substrate specificity (EC 5.1.1.10). This is the first report of the occurrence of serine Racemase in eukaryotes producing D-serine.
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Nonstereospecific Transamination Catalyzed by Pyridoxal Phosphate-dependent Amino Acid Racemases of Broad Substrate Specificity
Journal of Biological Chemistry, 1998Co-Authors: Young Hee Lim, Yoichi Kurokawa, Nobuyoshi Esaki, Tohru Yoshimura, Kenji SodaAbstract:Abstract Pyridoxal 5′-phosphate-dependent amino acid Racemases of broad substrate specificity catalyze transamination as a side reaction. We studied the stereospecificities for hydrogen abstraction from C-4′ of the bound pyridoxamine 5′-phosphate during transamination from pyridoxamine 5′-phosphate to pyruvate catalyzed by three amino acid Racemases of broad substrate specificity. When the enzymes were incubated with (4′S)- or (4′R)-[4′-3H]pyridoxamine 5′-phosphate in the presence of pyruvate, tritium was released into the solvent from both pyridoxamine 5′-phosphates. Thus, these enzymes abstract a hydrogen nonstereospecifically from C-4′ of the coenzyme in contrast to the other pyridoxal 5′-phosphate-dependent enzymes so far studied, which catalyze the stereospecific hydrogen removal. Amino acid Racemase of broad substrate specificity from Pseudomonas putidaproduced d- and l-glutamate from α-ketoglutarate through the transamination withl-ornithine. Because glutamate does not serve as a substrate for racemization, the enzyme catalyzed the nonstereospecific overall transamination between l-ornithine and α-ketoglutarate. The cleavage and formation of the C–H bond at C-4′ of the coenzyme and C-2 of the substrate thus occurs nonstereospecifically on both sides of the plane of the coenzyme-substrate complex intermediate. Amino acid Racemase of broad substrate specificity is the first example of a pyridoxal enzyme catalyzing nonstereospecific transamination.
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Cloning and expression of the glutamate Racemase gene of Bacillus pumilus.
Journal of biochemistry, 1997Co-Authors: Lidong Liu, Nobuyoshi Esaki, Tohru Yoshimura, Keiji Endo, Kenji SodaAbstract:A glutamate Racemase gene (murI) was found in Bacillus pumilus cells and cloned into Escherichia coli WM335, a D-glutamate auxotroph, by means of a genetic complement method. MurI of B. pumilus encodes a 272-amino acid protein with an unusual initiation codon, TTG. The deduced amino acid sequence shows significant similarity with those of glutamate Racemases from E. coli (ratio of identical residues, 28%), Pediococcus pentosaceus (44%), and Staphylococcus haemolyticus (49%). B. pumilus MurI was expressed as a fusion protein connected to the N-terminal 12 residues of beta-galactosidase; the fusion protein showed glutamate Racemase activity, and resembled the enzyme of P. pentosaceus in physicochemical and enzymological properties.
