Functional Characterization of Alanine Racemase from Schizosaccharomyces pombe : a Eucaryotic Counterpart to Bacterial Alanine Racemase

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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 M r of 41,590, to homogeneity. Alr1p contains pyridoxal 5′-phosphate as a coenzyme and catalyzes the racemization of alanine with apparent K m and V max 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.