Identification, purification, and characterization of a novel amino acid racemase, isoleucine 2-epimerase, from Lactobacillus Species

Yuta Mutaguchi, Taketo Ohmori, Taisuke Wakamatsu, Katsumi Doi, Toshihisa Ohshima

Research output: Contribution to journalArticle

23 Citations (Scopus)

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 5.00mM and 153 μmol·min-1·mg-1, respectively, and those for D-allo-isoleucine were 13.2mM 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.

Original languageEnglish
Pages (from-to)5207-5215
Number of pages9
JournalJournal of bacteriology
Volume195
Issue number22
DOIs
Publication statusPublished - Nov 1 2013

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Amino Acid Isomerases
Racemases and Epimerases
Isoleucine
Lactobacillus
Enzymes
4-Aminobutyrate Transaminase
Pyridoxal Phosphate
Amino Acids
Hydroxylamine
Coenzymes
Valine
Leucine
Amino Acid Sequence
Lactic Acid

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Molecular Biology

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Identification, purification, and characterization of a novel amino acid racemase, isoleucine 2-epimerase, from Lactobacillus Species. / Mutaguchi, Yuta; Ohmori, Taketo; Wakamatsu, Taisuke; Doi, Katsumi; Ohshima, Toshihisa.

In: Journal of bacteriology, Vol. 195, No. 22, 01.11.2013, p. 5207-5215.

Research output: Contribution to journalArticle

Mutaguchi, Yuta ; Ohmori, Taketo ; Wakamatsu, Taisuke ; Doi, Katsumi ; Ohshima, Toshihisa. / Identification, purification, and characterization of a novel amino acid racemase, isoleucine 2-epimerase, from Lactobacillus Species. In: Journal of bacteriology. 2013 ; Vol. 195, No. 22. pp. 5207-5215.
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