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Alanine Racemase

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Jiansong Ju – 1st expert 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, Murtala Muhammad, Jiansong Ju, 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.

Dagmar Ringe – 2nd expert 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…

Baohua Zhao – 3rd expert 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, Murtala Muhammad, Jiansong Ju, 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.