Alanine Racemase

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Jiansong Ju - One of the best experts on this subject based on the ideXlab platform.

  • Purification, Characterization and Inhibition of Alanine Racemase from a Pathogenic Strain of Streptococcus iniae
    Polish Journal of Microbiology, 2019
    Co-Authors: Murtala Muhammad, Jiansong Ju, Yangyang Li, Siyu Gong, Baohua Zhao
    Abstract:

    Streptococcus iniae is a pathogenic and zoonotic bacteria that impacted high mortality to many fish species as well as capable of causing serious disease to humans. Alanine Racemase (Alr, EC 5.1.1.1) is a pyridoxal-5'-phosphate (PLP)-containing homodimeric enzyme that catalyzes the racemization of L-Alanine and D-Alanine. In this study, we purified Alanine Racemase from S. iniae that was isolated from an infected Chinese sturgeon (Acipenser sinensis), as well as determined its biochemical characteristics and inhibitors. The alr gene has an open reading frame (ORF) of 1107 bp, encoding a protein of 369 amino acids, which has a molecular mass of 40 kDa. The enzyme has optimal activity at a temperature of 35°C and a pH of 9.5. It belongs to the PLP-dependent enzymes family and is highly specific to L-Alanine. S. iniae Alr (SiAlr) could be inhibited by some metal ions, hydroxylamine and dithiothreitol (DTT). The kinetic parameters K m and V max of the enzyme were 33.11 mM, 2426 units/mg for L-Alanine, and 14.36 mM, 963.6 units/mg for D-Alanine. Finally, the 50% inhibitory concentrations (IC50) values and antibiotic activity of two Alanine Racemase inhibitors (homogentisic acid and hydroquinone), were determined and found to be effective against both Gram-positive and Gram-negative bacteria employed in this study. Streptococcus iniae is a pathogenic and zoonotic bacteria that impacted high mortality to many fish species as well as capable of causing serious disease to humans. Alanine Racemase (Alr, EC 5.1.1.1) is a pyridoxal-5’-phosphate (PLP)-containing homodimeric enzyme that catalyzes the racemization of L-Alanine and D-Alanine. In this study, we purified Alanine Racemase from S. iniae that was isolated from an infected Chinese sturgeon (Acipenser sinensis), as well as determined its biochemical characteristics and inhibitors. The alr gene has an open reading frame (ORF) of 1107 bp, encoding a protein of 369 amino acids, which has a molecular mass of 40 kDa. The enzyme has optimal activity at a temperature of 35°C and a pH of 9.5. It belongs to the PLP-dependent enzymes family and is highly specific to L-Alanine. S. iniae Alr (SiAlr) could be inhibited by some metal ions, hydroxylamine and dithiothreitol (DTT). The kinetic parameters K m and V max of the enzyme were 33.11 mM, 2426 units/mg for L-Alanine, and 14.36 mM, 963.6 units/mg for D-Alanine. Finally, the 50% inhibitory concentrations (IC50) values and antibiotic activity of two Alanine Racemase inhibitors (homogentisic acid and hydroquinone), were determined and found to be effective against both Gram-positive and Gram-negative bacteria employed in this study.

  • Cloning, Biochemical Characterization and Inhibition of Alanine Racemase from Streptococcus iniae
    bioRxiv, 2019
    Co-Authors: Murtala Muhammad, Yangyang Li, Siyu Gong, Jiansong Ju
    Abstract:

    ABSTRACT Streptococcus iniae is a pathogenic and zoonotic bacteria that impacted high mortality to many fish species, as well as capable of causing serious disease to humans. Alanine Racemase (Alr, EC 5.1.1.1) is a pyridoxal-5′-phosphate (PLP)-containing homodimeric enzyme that catalyzes the racemization of L-Alanine and D-Alanine. In this study, we purified Alanine Racemase from the pathogenic strain of S. iniae, determined its biochemical characteristics and inhibitors. The alr gene has an open reading frame (ORF) of 1107 bp, encoding a protein of 369 amino acids, which has a molecular mass of 40 kDa. The optimal enzyme activity occurred at 35°C and a pH of 9.5. The enzyme belongs to the PLP dependent enzymes family and is highly specific to L-Alanine. S.iniae Alr can be inhibited by some metal ions, hydroxylamine and dithiothreitol (DTT). The kinetic parameters Km and Vmax of the enzyme were 33.11 mM, 2426 units/mg for L-Alanine and 14.36 mM, 963.6 units/mg for D-Alanine. Finally, the 50% inhibitory concentrations (IC50) values and antibiotic activity of two Alanine Racemase inhibitors, were determined and found to be effective against both gram positive and gram negative bacteria employed in this study. The important role of Alanine Racemase as a target of developing new antibiotics against S. iniae highlighted the usefulness of the enzyme for new antibiotics discovery.

  • Knockout of Alanine Racemase gene attenuates the pathogenicity of Aeromonas hydrophila
    BMC Microbiology, 2019
    Co-Authors: Ting Zhang, Jiansong Ju, Murtala Muhammad, Yaping Wang, Baohua Zhao
    Abstract:

    Aeromonas hydrophila is an opportunistic pathogen of poikilothermic and homoeothermic animals, including humans. In the present study, we described the role of Alanine Racemase (alr-2) in the virulence of A. hydrophila using an alr-2 knockout mutant (A.H.Δalr). In mouse and common carp models, the survival of animals challenged with A.H.Δalr was significantly increased compared with the wild-type (WT), and the mutant was also impaired in its ability to replicate in the organs and blood of infected mice and fish. The A.H.Δalr significantly increased phagocytosis by macrophages of the mice and fish. These attenuation effects of alr-2 could be complemented by the addition of D-Alanine to the A.H.Δalr strain. The histopathology results indicated that the extent of tissue injury in the WT-infected animals was more severe than in the A.H.Δalr-infected groups. The expression of 9 virulence genes was significantly down-regulated, and 3 outer membrane genes were significantly up-regulated in A.H.Δalr. Our data suggest that alr-2 is essential for the virulence of A. hydrophila. Our findings suggested Alanine Racemase could be applied in the development of new antibiotics against A. hydrophila.

  • Biochemical characterization and mutational analysis of Alanine Racemase from Clostridium perfringens.
    Journal of Bioscience and Bioengineering, 2019
    Co-Authors: Muhammad Israr, Baohua Zhao, Shujing Xu, Guoping Lv, Yunhe Li, Shengting Ding, Jiansong Ju
    Abstract:

    Clostridium perfringens is a gram-positive, anaerobic, pathogenic bacterium that can cause a wide range of diseases in humans, poultry and agriculturally important livestock. A pyridoxal-5-phosphate-dependent Alanine Racemase with a function in the racemization of d - and l -Alanine is an attractive drug target for C. perfringens and other pathogens due to its absence in animals and humans. In this study Alanine Racemase from C. perfringens (CPAlr) was successfully expressed and purified in Escherichia coli and biochemically characterized. The purified CPAlr protein was a dimeric PLP-dependent enzyme with high substrate specificity. The optimal racemization temperature and pH were 40°C and 8.0, respectively. The kinetic parameters Km and kcat of CPAlr, determined by HPLC at 40°C were 19.1 mM and 17.2 s−1 for l -Alanine, and 10.5 mM and 8.7 s−1 for d -Alanine, respectively. Gel filtration chromatographic analysis showed that the molecular weight of mutant Y359A was close to monomeric form, suggesting that the inner layer residue Tyr359 might play an essential role in dimer-formation. Furthermore, the mutation at residues Asp171 and Tyr359 resulted in a dramatic increase in Km value and/or decreased in kcat value, indicating that the middle and inner layer residues Asp171 and Tyr359 of CPAlr might have the key role in substrate binding, catalytic activity or oligomerization state through the hydrogen–bonding interaction with the pentagonal ring waters and/or PLP cofactor.

  • enzymatic characterization and crystal structure of biosynthetic Alanine Racemase from pseudomonas aeruginosa pao1
    Biochemical and Biophysical Research Communications, 2018
    Co-Authors: Hui Dong, Jiansong Ju, Shanshan Wang, Chao Yuan, Wei Long, Xin He, Shujing Xu, Sheng Li
    Abstract:

    Alanine Racemase is a pyridoxal-5'-phosphate (PLP)-dependent enzyme that reversibly catalyzes the conversion of L-Alanine to D-Alanine. D-Alanine is an essential constituent in many prokaryotic cell structures. Inhibition of Alanine Racemase is lethal to prokaryotes, creating an attractive target for designing antibacterial drugs. Here we report the crystal structure of biosynthetic Alanine Racemase (Alr) from a pathogenic bacteria Pseudomonas aeruginosa PAO1. Structural studies showed that P. aeruginosa Alr (PaAlr) adopts a conserved homodimer structure. A guest substrate D-lysine was observed in the active site and refined to dual-conformation. Two buffer ions, malonate and acetate, were bound in the proximity to D-lysine. Biochemical characterization revealed the optimal reaction conditions for PaAlr. (C) 2018 Elsevier Inc. All rights reserved.

Dagmar Ringe - One of the best experts on this subject based on the ideXlab platform.

  • effect of a y265f mutant on the transamination based cycloserine inactivation of Alanine Racemase
    Biochemistry, 2005
    Co-Authors: Timothy D Fenn, Geoffrey F Stamper, Todd Holyoak, Dagmar Ringe
    Abstract:

    The requirement for d-Alanine in the peptidoglycan layer of bacterial cell walls is fulfilled in part by Alanine Racemase (EC 5.1.1.1), a pyridoxal 5‘-phosphate (PLP)-assisted enzyme. The enzyme utilizes two antiparallel bases focused at the Cα position and oriented perpendicular to the PLP ring to facilitate the equilibration of Alanine enantiomers. Understanding how this two-base system is utilized and controlled to yield reaction specificity is therefore a potential means for designing antibiotics. Cycloserine is a known Alanine Racemase suicide substrate, although its mechanism of inactivation is based on transaminase chemistry. Here we characterize the effects of a Y265F mutant (Tyr265 acts as the catalytic base in the l-isomer case) of Bacillus stearothermophilus Alanine Racemase on cycloserine inactivation. The Y265F mutant reduces racemization activity 1600-fold [Watanabe, A., Yoshimura, T., Mikami, B., and Esaki, N. (1999) J. Biochem. 126, 781−786] and only leads to formation of the isoxazole end...

  • a side reaction of Alanine Racemase transamination of cycloserine
    Biochemistry, 2003
    Co-Authors: Timothy D Fenn, Geoffrey F Stamper, A A Morollo, Dagmar Ringe
    Abstract:

    Alanine Racemase (EC 5.1.1.1) catalyzes the interconversion of Alanine enantiomers, and thus represents the first committed step involved in bacterial cell wall biosynthesis. Cycloserine acts as a suicide inhibitor of Alanine Racemase and as such, serves as an antimicrobial agent. The chemical means by which cycloserine inhibits Alanine Racemase is unknown. Through spectroscopic assays, we show here evidence of a pyridoxal derivative (arising from either isomer of cycloserine) saturated at the C4‘ carbon position. We additionally report the l- and d-cycloserine inactivated crystal structures of Bacillus stearothermophilus Alanine Racemase, which corroborates the spectroscopy via evidence of a 3-hydroxyisoxazole pyridoxamine derivative. Upon the basis of the kinetic and structural properties of both the l- and d-isomers of the inhibitor, we propose a mechanism of Alanine Racemase inactivation by cycloserine. This pathway involves an initial transamination step followed by tautomerization to form a stable a...

  • structure of a michaelis complex analogue propionate binds in the substrate carboxylate site of Alanine Racemase
    Biochemistry, 1999
    Co-Authors: A A Morollo, Gregory A Petsko, Dagmar Ringe
    Abstract:

    The structure of Alanine Racemase from Bacillus stearothermophilus with the inhibitor propionate bound in the active site was determined by X-ray crystallography to a resolution of 1.9 A. The enzyme is a homodimer in solution and crystallizes with a dimer in the asymmetric unit. Both active sites contain a pyridoxal 5‘-phosphate (PLP) molecule in aldimine linkage to Lys39 as a protonated Schiff base, and the pH-independence of UV−visible absorption spectra suggests that the protonated PLP−Lys39 Schiff base is the reactive form of the enzyme. The carboxylate group of propionate bound in the active site makes numerous interactions with active-site residues, defining the substrate binding site of the enzyme. The propionate-bound structure therefore approximates features of the Michaelis complex formed between Alanine Racemase and its amino acid substrate. The structure also provides evidence for the existence of a carbamate formed on the side-chain amino group of Lys129, stabilized by interactions with one o...

  • Reaction of Alanine Racemase with 1-Aminoethylphosphonic Acid Forms a Stable External Aldimine†,‡
    Biochemistry, 1998
    Co-Authors: C. Geoffrey F. Stamper, And Anthony A Morollo, Dagmar Ringe
    Abstract:

    (R)-1-Aminoethylphosphonic acid (l-Ala-P), a synthetic l-Alanine analogue, has antibacterial activity and is a time-dependent inactivator of all purified Gram-positive bacterial Alanine Racemases that have been tested. l-Ala-P forms an external aldimine with the bound pyridoxal 5‘-phosphate (PLP) cofactor, but is neither racemized nor efficiently hydrolyzed. To understand the structural basis of the inactivation of the enzyme by l-Ala-P, we determined the crystal structure of the complex between l-Ala-P and Alanine Racemase at 1.6 A resolution. The cofactor derivative in the inhibited structure tilts outward from the protein approximately 20° relative to the internal aldimine. The phosphonate oxygens are within hydrogen bonding distance of four amino acid residues and two water molecules in the active site of the enzyme. l-Ala-P is an effective inhibitor of Alanine Racemase because, upon formation of the external aldimine, the phosphonate group interacts with putative catalytic residues, thereby rendering...

  • reaction of Alanine Racemase with 1 aminoethylphosphonic acid forms a stable external aldimine
    Biochemistry, 1998
    Co-Authors: Geoffrey C F Stamper, And Anthony A Morollo, Dagmar Ringe
    Abstract:

    (R)-1-Aminoethylphosphonic acid (l-Ala-P), a synthetic l-Alanine analogue, has antibacterial activity and is a time-dependent inactivator of all purified Gram-positive bacterial Alanine Racemases that have been tested. l-Ala-P forms an external aldimine with the bound pyridoxal 5‘-phosphate (PLP) cofactor, but is neither racemized nor efficiently hydrolyzed. To understand the structural basis of the inactivation of the enzyme by l-Ala-P, we determined the crystal structure of the complex between l-Ala-P and Alanine Racemase at 1.6 A resolution. The cofactor derivative in the inhibited structure tilts outward from the protein approximately 20° relative to the internal aldimine. The phosphonate oxygens are within hydrogen bonding distance of four amino acid residues and two water molecules in the active site of the enzyme. l-Ala-P is an effective inhibitor of Alanine Racemase because, upon formation of the external aldimine, the phosphonate group interacts with putative catalytic residues, thereby rendering...

Baohua Zhao - One of the best experts on this subject based on the ideXlab platform.

  • Purification, Characterization and Inhibition of Alanine Racemase from a Pathogenic Strain of Streptococcus iniae
    Polish Journal of Microbiology, 2019
    Co-Authors: Murtala Muhammad, Jiansong Ju, Yangyang Li, Siyu Gong, Baohua Zhao
    Abstract:

    Streptococcus iniae is a pathogenic and zoonotic bacteria that impacted high mortality to many fish species as well as capable of causing serious disease to humans. Alanine Racemase (Alr, EC 5.1.1.1) is a pyridoxal-5'-phosphate (PLP)-containing homodimeric enzyme that catalyzes the racemization of L-Alanine and D-Alanine. In this study, we purified Alanine Racemase from S. iniae that was isolated from an infected Chinese sturgeon (Acipenser sinensis), as well as determined its biochemical characteristics and inhibitors. The alr gene has an open reading frame (ORF) of 1107 bp, encoding a protein of 369 amino acids, which has a molecular mass of 40 kDa. The enzyme has optimal activity at a temperature of 35°C and a pH of 9.5. It belongs to the PLP-dependent enzymes family and is highly specific to L-Alanine. S. iniae Alr (SiAlr) could be inhibited by some metal ions, hydroxylamine and dithiothreitol (DTT). The kinetic parameters K m and V max of the enzyme were 33.11 mM, 2426 units/mg for L-Alanine, and 14.36 mM, 963.6 units/mg for D-Alanine. Finally, the 50% inhibitory concentrations (IC50) values and antibiotic activity of two Alanine Racemase inhibitors (homogentisic acid and hydroquinone), were determined and found to be effective against both Gram-positive and Gram-negative bacteria employed in this study. Streptococcus iniae is a pathogenic and zoonotic bacteria that impacted high mortality to many fish species as well as capable of causing serious disease to humans. Alanine Racemase (Alr, EC 5.1.1.1) is a pyridoxal-5’-phosphate (PLP)-containing homodimeric enzyme that catalyzes the racemization of L-Alanine and D-Alanine. In this study, we purified Alanine Racemase from S. iniae that was isolated from an infected Chinese sturgeon (Acipenser sinensis), as well as determined its biochemical characteristics and inhibitors. The alr gene has an open reading frame (ORF) of 1107 bp, encoding a protein of 369 amino acids, which has a molecular mass of 40 kDa. The enzyme has optimal activity at a temperature of 35°C and a pH of 9.5. It belongs to the PLP-dependent enzymes family and is highly specific to L-Alanine. S. iniae Alr (SiAlr) could be inhibited by some metal ions, hydroxylamine and dithiothreitol (DTT). The kinetic parameters K m and V max of the enzyme were 33.11 mM, 2426 units/mg for L-Alanine, and 14.36 mM, 963.6 units/mg for D-Alanine. Finally, the 50% inhibitory concentrations (IC50) values and antibiotic activity of two Alanine Racemase inhibitors (homogentisic acid and hydroquinone), were determined and found to be effective against both Gram-positive and Gram-negative bacteria employed in this study.

  • Knockout of Alanine Racemase gene attenuates the pathogenicity of Aeromonas hydrophila
    BMC Microbiology, 2019
    Co-Authors: Ting Zhang, Jiansong Ju, Murtala Muhammad, Yaping Wang, Baohua Zhao
    Abstract:

    Aeromonas hydrophila is an opportunistic pathogen of poikilothermic and homoeothermic animals, including humans. In the present study, we described the role of Alanine Racemase (alr-2) in the virulence of A. hydrophila using an alr-2 knockout mutant (A.H.Δalr). In mouse and common carp models, the survival of animals challenged with A.H.Δalr was significantly increased compared with the wild-type (WT), and the mutant was also impaired in its ability to replicate in the organs and blood of infected mice and fish. The A.H.Δalr significantly increased phagocytosis by macrophages of the mice and fish. These attenuation effects of alr-2 could be complemented by the addition of D-Alanine to the A.H.Δalr strain. The histopathology results indicated that the extent of tissue injury in the WT-infected animals was more severe than in the A.H.Δalr-infected groups. The expression of 9 virulence genes was significantly down-regulated, and 3 outer membrane genes were significantly up-regulated in A.H.Δalr. Our data suggest that alr-2 is essential for the virulence of A. hydrophila. Our findings suggested Alanine Racemase could be applied in the development of new antibiotics against A. hydrophila.

  • Biochemical characterization and mutational analysis of Alanine Racemase from Clostridium perfringens.
    Journal of Bioscience and Bioengineering, 2019
    Co-Authors: Muhammad Israr, Baohua Zhao, Shujing Xu, Guoping Lv, Yunhe Li, Shengting Ding, Jiansong Ju
    Abstract:

    Clostridium perfringens is a gram-positive, anaerobic, pathogenic bacterium that can cause a wide range of diseases in humans, poultry and agriculturally important livestock. A pyridoxal-5-phosphate-dependent Alanine Racemase with a function in the racemization of d - and l -Alanine is an attractive drug target for C. perfringens and other pathogens due to its absence in animals and humans. In this study Alanine Racemase from C. perfringens (CPAlr) was successfully expressed and purified in Escherichia coli and biochemically characterized. The purified CPAlr protein was a dimeric PLP-dependent enzyme with high substrate specificity. The optimal racemization temperature and pH were 40°C and 8.0, respectively. The kinetic parameters Km and kcat of CPAlr, determined by HPLC at 40°C were 19.1 mM and 17.2 s−1 for l -Alanine, and 10.5 mM and 8.7 s−1 for d -Alanine, respectively. Gel filtration chromatographic analysis showed that the molecular weight of mutant Y359A was close to monomeric form, suggesting that the inner layer residue Tyr359 might play an essential role in dimer-formation. Furthermore, the mutation at residues Asp171 and Tyr359 resulted in a dramatic increase in Km value and/or decreased in kcat value, indicating that the middle and inner layer residues Asp171 and Tyr359 of CPAlr might have the key role in substrate binding, catalytic activity or oligomerization state through the hydrogen–bonding interaction with the pentagonal ring waters and/or PLP cofactor.

  • selection and characterization of Alanine Racemase inhibitors against aeromonas hydrophila
    BMC Microbiology, 2017
    Co-Authors: Yaping Wang, Jiansong Ju, Chao Yang, Ting Zhang, Baohua Zhao
    Abstract:

    Combining experimental and computational screening methods has been of keen interest in drug discovery. In the present study, we developed an efficient screening method that has been used to screen 2100 small-molecule compounds for Alanine Racemase Alr-2 inhibitors. We identified ten novel non-substrate Alr-2 inhibitors, of which patulin, homogentisic acid, and hydroquinone were active against Aeromonas hydrophila. The compounds were found to be capable of inhibiting Alr-2 to different extents with 50% inhibitory concentrations (IC50) ranging from 6.6 to 17.7 μM. These compounds inhibited the growth of A. hydrophila with minimal inhibitory concentrations (MICs) ranging from 20 to 120 μg/ml. These compounds have no activity on horseradish peroxidase and d-amino acid oxidase at a concentration of 50 μM. The MTT assay revealed that homogentisic acid and hydroquinone have minimal cytotoxicity against mammalian cells. The kinetic studies indicated a competitive inhibition of homogentisic acid against Alr-2 with an inhibition constant (K i) of 51.7 μM, while hydroquinone was a noncompetitive inhibitor with a K i of 212 μM. Molecular docking studies suggested that homogentisic acid binds to the active site of Racemase, while hydroquinone lies near the active center of Alanine Racemase. Our findings suggested that combining experimental and computational methods could be used for an efficient, large-scale screening of Alanine Racemase inhibitors against A. hydrophila that could be applied in the development of new antibiotics against A. hydrophila.

  • knockout of the Alanine Racemase gene in aeromonas hydrophila hbnuah01 results in cell wall damage and enhanced membrane permeability
    Fems Microbiology Letters, 2015
    Co-Authors: Lu Zhang, Jiansong Ju, Yaping Wang, Baohua Zhao
    Abstract:

    This study focused on the Alanine Racemase gene ( alr -2), which is involved in the synthesis of d-Alanine that forms the backbone of the cell wall. A stable alr -2 knockout mutant of Aeromonas hydrophila HBNUAh01 was constructed. When the mutant was supplemented with d-Alanine, growth was unaffected; deprivation of d-Alanine caused the growth arrest of the starved mutant cells, but not cell lysis. No Alanine Racemase activity was detected in the culture of the mutant. Additionally, a membrane permeability assay showed increasing damage to the cell wall during d-Alanine starvation. No such damage was observed in the wild type during culture. Scanning and transmission electron microscopy analyses revealed deficiencies of the cell envelope and perforation of the cell wall. Leakage of UV-absorbing substances from the mutants was also observed. Thus, the partial viability of the mutants and their independence of d-Alanine for growth indicated that inactivation of alr -2 does not impose an auxotrophic requirement for d-Alanine.

Kouhei Ohnishi - One of the best experts on this subject based on the ideXlab platform.

  • characterization and preliminary mutation analysis of a thermostable Alanine Racemase from thermoanaerobacter tengcongensis mb4
    Extremophiles, 2013
    Co-Authors: Yi Hu, Kouhei Ohnishi, Jiansong Ju, Shujing Xu, Baohua Zhao
    Abstract:

    A thermostable Alanine Racemase from Thermoanaerobacter tengcongensis MB4 was successfully expressed in Escherichia coli and characterized. The full-length gene MBalr2 (1164 bp) encodes 388 amino acid residues including 6 out of 8 highly conserved amino acid residues at the entryway to the active site of Alanine Racemase. Recombinant MBAlr2 and three mutants (S171A, H359Y and double mutation S171A/H359Y) of MBAlr2 were purified by His6-tag affinity column and gel filtration chromatography. The purified protein MBAlr2 was a dimeric PLP-dependent enzyme with broad substrate specificity. The optimal racemization temperature and pH were 70–75 °C and 11.0, respectively. The kinetic parameters K m and V max of MBAlr2 at 70 °C, determined by HPLC, were 20.16 mM and 1414 μmol min−1 for l-Alanine, and 9.95 mM and 702.6 μmol min−1 for d-Alanine, respectively. Enzymatic assays showed that the activity of both mutants (S171A and H359Y) was lost, but the activity of mutant S171A/H359Y was recovered to 69.8 % of wild type, which suggested that residues Ser171 and His359 might be the important residues for catalytic mechanisms of MBAlr2.

  • characterization of endogenous pyridoxal 5 phosphate dependent Alanine Racemase from bacillus pseudofirmus of4
    Journal of Bioscience and Bioengineering, 2009
    Co-Authors: Shujing Xu, Jiansong Ju, Gang Li, Kouhei Ohnishi
    Abstract:

    Abstract An open reading frame of 1100 bp in the partially sequenced genome sequence of alkaliphilic Bacillus pseudofirmus OF4 was identified as a putative Alanine Racemase gene (dadXOF4), which was cloned and expressed in Escherichia coli BL21 (DE3). The encoded protein DadXOF4 was purified to homogeneity by His6-tag affinity column, gel filtration and ion-exchange chromatography. The amino acid sequence has highest identity with the known Alanine Racemase from Oceanobacillus iheyensis HTE831 (48%). The protein was a dimeric, endogenous PLP-dependent enzyme, which was demonstrated by absorption spectra and enzyme activity with or without PLP. The racemization temperature optimum was 40 °C and the optimal pH was 10.5. The kinetic parameters Km and Vmax at 40 °C of Alanine Racemase, determined by HPLC analysis, were 41.79 mM, 10,500 units/mg for L-Alanine and 14.91 mM, 3708 units/mg for D-Alanine, respectively.

  • crystallization and preliminary x ray study of alkaline Alanine Racemase from bacillus pseudofirmus of4
    Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2009
    Co-Authors: Jiansong Ju, Kouhei Ohnishi, Shujing Xu, Jianxun Qi, Michael J Benedik
    Abstract:

    Alanine Racemase (DadXOF4), a dimeric endogenous PLP-dependent alkaline enzyme from alkaliphilic Bacillus pseudofirmus OF4, was expressed in Escherichia coli and purified with a His6 tag in a form suitable for X-ray crystallographic analysis. Crystals were grown by the hanging-drop vapour-diffusion method at 291 K using a solution containing 1.4 M sodium/potassium phosphate pH 8.2. The protein crystallized in space group P212121, with two protein molecules in the asymmetric unit.

  • cloning of Alanine Racemase genes from pseudomonas fluorescens strains and oligomerization states of gene products expressed in escherichia coli
    Journal of Bioscience and Bioengineering, 2005
    Co-Authors: Jiansong Ju, Haruo Misono, Kumio Yokoigawa, Kouhei Ohnishi
    Abstract:

    Bacterial Alanine Racemase (EC 5.1.1.1) is a pyridoxal 5′-phosphate-dependent enzyme. Almost all eubacteria known to date possess a biosynthetic alr gene and some bacteria have an additional catabolic dadX gene. On the basis of the subunit structure, Alanine Racemases are classified into two types, monomeric and homodimeric. Alanine Racemase genes were cloned from two distinct Pseudomonas fluorescens strains, the psychrotrophic TM5-2 strain and the soil-borne LRB3W1 strain, by means of complementing an Escherichia coli Alanine Racemase-deficient mutant. From the cloning results, both strains are likely to possess only one Alanine Racemase gene, dadX, in the same manner as the other P. fluorescens strains. Gene organization surrounding the dadX gene is highly conserved among Pseudomonas strains. The gene for D -amino acid dehydrogenase is located adjacent to the dadX gene in both strains. The DadX Alanine Racemases were expressed in E. coli as C-terminal His-tagged fusion proteins and purified to homogeneity. The catalytic activity of LRB3W1 DadX was higher than that of TM5-2 DadX. The association states of P. fluorescens DadX subunits in the E. coli Alanine Racemase-deficient mutant were analyzed by gel filtration chromatography. Alanine Racemase subunits were demonstrated to exist as both monomers and dimers. The enzyme was in a monomer-dimer equilibrium, and the catalytic activity of the enzyme was proportional to the equilibrium association constant for dimerization.

  • molecular characterization of Alanine Racemase from bifidobacterium bifidum
    Journal of Molecular Catalysis B-enzymatic, 2003
    Co-Authors: Tatsuyuki Yamashita, Makoto Ashiuchi, Kouhei Ohnishi, Shinichiro Kato, Shinji Nagata, Haruo Misono
    Abstract:

    Abstract Bifidobacterium bifidum is a useful probiotic agent exhibiting health-promoting properties, and its peptidoglycans have the potential for applications in the fields of food science and medicine. We investigated the bifidobacterial Alanine Racemase, which is essential in the synthesis of d -Alanine as an essential component of the peptidoglycans. Alanine Racemase was purified to homogeneity from a crude extract of B. bifidum NBRC 14252. It consisted of two identical subunits with a molecular mass of 50 kDa. The enzyme required pyridoxal 5′-phosphate (PLP) as a coenzyme. The activity was lost in the presence of a thiol-modifying agent. The enzyme almost exclusively catalyzed the Alanine racemization; other amino acids tested, except for serine, were inactive as substrates. The kinetic parameters of the enzyme suggested that the B. bifidum Alanine Racemase possesses comparatively low affinities for both the coenzyme (9.1 μM for PLP) and substrates (44.3 mM for l -Alanine; 74.3 mM for d -Alanine). The alr gene encoding the Alanine Racemase was cloned and sequenced. The alr gene complemented the d -Alanine auxotrophy of Escherichia coli MB2795, and an abundant amount of the enzyme was produced in cells of the E. coli MB2795 clone. The enzymologic and kinetic properties of the purified recombinant enzyme were almost the same as those of the Alanine Racemase from B. bifidum NBRC 14252.

Nobuyoshi Esaki - One of the best experts on this subject based on the ideXlab platform.

  • conversion of the catalytic specificity of Alanine Racemase to a d amino acid aminotransferase activity by a double active site mutation
    Journal of Molecular Catalysis B-enzymatic, 2003
    Co-Authors: Akira Watanabe, Tohru Yoshimura, Nobuyoshi Esaki
    Abstract:

    Abstract Alanine Racemase depending on pyridoxal 5′-phosphate catalyzes the interconversion between d - and l -Alanine. The enzyme from Bacillus stearothermophilus catalyzes the transamination as a side reaction with both substrates once per 3×10 7 times of the racemization. In this work, we studied the effects of the mutation of Arg219, and that of Arg219 and Tyr265 on the catalysis of Bacillus Alanine Racemase. Arg219 interacting with pyridinium nitrogen of the cofactor is conserved in all Alanine Racemases. The corresponding residue of aminotransferases is an acidic residue, such as glutamate or aspartate. Mutation of Arg219 to a glutamyl residue resulted in a 5.4-fold increase in the forward half transamination activity with d -Alanine and a 10 3 -fold decrease in the Racemase activity. The double mutation, Arg219→Glu and Tyr265→Ala, completely abolished the Racemase activity and increased the forward half transaminase activity 6.6-fold. Arg219 is one of the structural determinants of the catalytic specificity of the Alanine Racemase.

  • functional characterization of Alanine Racemase from schizosaccharomyces pombe a eucaryotic counterpart to bacterial Alanine Racemase
    Journal of Bacteriology, 2001
    Co-Authors: Takuma Uo, Tohru Yoshimura, Naotaka Tanaka, Kaoru Takegawa, Nobuyoshi Esaki
    Abstract:

    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.

  • Stereochemistry of the hydrogen abstraction from pyridoxamine phosphate catalyzed by Alanine Racemase of Bacillus stearothermophilus
    Journal of Molecular Catalysis B-enzymatic, 2001
    Co-Authors: Akira Watanabe, Tohru Yoshimura, Kenji Soda, Yoichi Kurokawa, Nobuyoshi Esaki
    Abstract:

    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.

  • High catalytic activity of Alanine Racemase from psychrophilic Bacillus psychrosaccharolyticus at high temperatures in the presence of pyridoxal 5'-phosphate.
    Fems Microbiology Letters, 2000
    Co-Authors: Yoko Okubo, Nobuyoshi Esaki, Kumio Yokoigawa, Kenji Soda, Haruo Misono
    Abstract:

    We examined the effect of the pyridoxal 5′-phosphate (PLP) cofactor on the activity and stability of the psychrophilic Alanine Racemase, having a high catalytic activity at low temperature, from Bacillus psychrosaccharolyticus at high temperatures. The decrease in the enzyme activity at incubation temperatures over 40°C was consistent with the decrease in the amount of bound PLP. Unfolding of the enzyme at temperatures above 40°C was suppressed in the presence of PLP. In the presence of 0.125 mM PLP, the specific activity of the psychrophilic enzyme was higher than that of a thermophilic Alanine Racemase, having a high catalytic activity at high temperature, from Bacillus stearothermophilus even at 60°C.

  • characterization of psychrophilic Alanine Racemase from bacillus psychrosaccharolyticus
    Biochemical and Biophysical Research Communications, 1999
    Co-Authors: Yoko Okubo, Nobuyoshi Esaki, Kumio Yokoigawa, Kenji Soda, Hiroyasu Kawai
    Abstract:

    Abstract A psychrophilic Alanine Racemase gene fromBacillus psychrosaccharolyticuswas cloned and expressed inEscherichia coliSOLR with a plasmid pYOK3. The gene starting with the unusual initiation codon GTG showed higher preference for codons ending in A or T. The enzyme purified to homogeneity showed the high catalytic activity even at 0°C and was extremely labile over 35°C. The enzyme was found to have a markedly largeKm value (5.0 μM) for the pyridoxal 5′-phosphate (PLP) cofactor in comparison with other reported Alanine Racemases, and was stabilized up to 50°C in the presence of excess amounts of PLP. The low affinity of the enzyme for PLP may be related to the thermolability, and may be related to the high catalytic activity, initiated by the transaldimination reaction, at low temperature. The enzyme has a distinguishing hydrophilic region around the residue no. 150 in the deduced amino acid sequence (383 residues), whereas the corresponding regions of otherBacillusAlanine Racemases are hydrophobic. The position of the region in the three dimensional structure of Cαatoms of the enzyme was predicted to be in a surface loop surrounding the active site. The region may interact with solvent and reduce the compactness of the active site.

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