RRC ID 29463
著者 Mutaguchi Y, Ohmori T, Wakamatsu T, Doi K, Ohshima T.
タイトル Identification, purification, and characterization of a novel amino acid racemase, isoleucine 2-epimerase, from Lactobacillus species.
ジャーナル J Bacteriol
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.
巻・号 195(22)
ページ 5207-15
公開日 2013-11-1
DOI 10.1128/JB.00709-13
PII JB.00709-13
PMID 24039265
PMC PMC3811583
MeSH Amino Acid Isomerases / chemistry Amino Acid Isomerases / genetics Amino Acid Isomerases / isolation & purification Amino Acid Isomerases / metabolism* Cloning, Molecular Coenzymes / metabolism Escherichia coli / genetics Gene Expression Isoleucine / metabolism* Kinetics Lactobacillus / enzymology* Lactobacillus / genetics Molecular Sequence Data Molecular Weight Protein Multimerization Protein Subunits Pyridoxal Phosphate / metabolism Recombinant Proteins / chemistry Recombinant Proteins / genetics Recombinant Proteins / isolation & purification Sequence Analysis, DNA Sequence Analysis, Protein Substrate Specificity
IF 3.006
引用数 27
WOS 分野 MICROBIOLOGY
リソース情報
一般微生物 JCM 1115 JCM 15040