title: Identification, purification, and characterization of a novel amino acid racemase, isoleucine 2-epimerase, from Lactobacillus species.
abstract: Accumulation of d-leucine, d-allo-isoleucine, and d-valine was observed in the growth medium of a lactic acid bacterium, Lactobacillus otakiensis JCM 15040, and the racemase responsible was purified from the cells and identified. The N-terminal amino acid sequence of the purified enzyme was GKLDKASKLI, which is consistent with that of a putative γ-aminobutyrate aminotransferase from Lactobacillus buchneri. The putative γ-aminobutyrate aminotransferase gene from L. buchneri JCM 1115 was expressed in recombinant Escherichia coli and then purified to homogeneity. The enzyme catalyzed the racemization of a broad spectrum of nonpolar amino acids. In particular, it catalyzed at high rates the epimerization of l-isoleucine to d-allo-isoleucine and d-allo-isoleucine to l-isoleucine. In contrast, the enzyme showed no γ-aminobutyrate aminotransferase activity. The relative molecular masses of the subunit and native enzyme were estimated to be about 49 kDa and 200 kDa, respectively, indicating that the enzyme was composed of four subunits of equal molecular masses. The Km and Vmax values of the enzyme for l-isoleucine were 5.00 mM and 153 μmol·min(-1)·mg(-1), respectively, and those for d-allo-isoleucine were 13.2 mM and 286 μmol·min(-1)·mg(-1), respectively. Hydroxylamine and other inhibitors of pyridoxal 5'-phosphate-dependent enzymes completely blocked the enzyme activity, indicating the enzyme requires pyridoxal 5'-phosphate as a coenzyme. This is the first evidence of an amino acid racemase that specifically catalyzes racemization of nonpolar amino acids at the C-2 position.
authors.affiliation: Department of Biomedical Engineering, Faculty of Engineering, Osaka Institute of Technology, Omiya Asahi-ku, Osaka, Japan.
authors.forename: Yuta
authors.initials: Y
authors.lastname: Mutaguchi
authors.forename: Taketo
authors.initials: T
authors.lastname: Ohmori
authors.forename: Taisuke
authors.initials: T
authors.lastname: Wakamatsu
authors.forename: Katsumi
authors.initials: K
authors.lastname: Doi
authors.forename: Toshihisa
authors.initials: T
authors.lastname: Ohshima
mesh_terms: D000591:Amino Acid Isomerases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D003067:Coenzymes / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007532:Isoleucine / Q000378:metabolism*; D007700:Kinetics; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D055503:Protein Multimerization; D021122:Protein Subunits; D011732:Pyridoxal Phosphate / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity
chemical_list: D003067:Coenzymes; D021122:Protein Subunits; D011994:Recombinant Proteins; D007532:Isoleucine; D011732:Pyridoxal Phosphate; D000591:Amino Acid Isomerases
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