Functional characterization of alanine racemase from Schizosaccharomyces pombe: A eucaryotic counterpart to bacterial alanine racemase

T. Uo, T. Yoshimura, N. Tanaka, K. Takegawa, N. Esaki

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59 Citations (Scopus)

Abstract

Schizosaccharomyces pombe has an open reading frame, which we named alr1+, encoding a putative protein similar to bacterial alanine racemase. We cloned the alr1+ gene in Escherichia coli and purified the gene product (Alr1p), with an Mr of 41,590, to homogeneity. Alr1p contains pyridoxal 5′-phosphate as a coenzyme and catalyzes the racemization of alanine with apparent Km and Vmax values as follows: for L-alanine, 5.0 mM and 670 μmol/min/mg, respectively, and for D-alanine, 2.4 mM and 350 μmol/min/mg, respectively. The enzyme is almost specific to alanine, but L-serine and L-2-aminobutyrate are racemized slowly at rates 3.7 and 0.37% of that of L-alanine, respectively. S. pombe uses D-alanine as a sole nitrogen source, but deletion of the alr1+ gene resulted in retarded growth on the same medium. This indicates that S. pombe has catabolic pathways for both enantiomers of alanine and that the pathway for L-alanine coupled with racemization plays a major role in the catabolism of D-alanine. Saccharomyces cerevisiae differs markedly from S. pombe: S. cerevisiae uses L-alanine but not D-alanine as a sole nitrogen source. Moreover, D-alanine is toxic to S. cerevisiae. However, heterologous expression of the alr1+ gene enabled S. cerevisiae to grow efficiently on D-alanine as a sole nitrogen source. The recombinant yeast was relieved from the toxicity of D-alanine.

Original languageEnglish
Pages (from-to)2226-2233
Number of pages8
JournalJournal of bacteriology
Volume183
Issue number7
DOIs
Publication statusPublished - 2001
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Molecular Biology

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