T. Uo et al., Functional characterization of alanine racemase from Schizosaccharomyces pombe: a eucaryotic counterpart to bacterial alanine racemase, J BACT, 183(7), 2001, pp. 2226-2233
Schizosaccharomyces pombe has an open reading frame, which we named alr1(+)
, encoding a putative protein similar to bacterial alanine racemase. We clo
ned the alr1(+) gene in Escherichia coil and purified the gene product (Alr
1p), with an M-r of 41,590, to homogeneity. Alr1p contains pyridoxal 5'-pho
sphate as a coenzyme and catalyzes the racemization of alanine with apparen
t K-m and V-max values as follows: for L-alanine, 5.0 mM and 670 mu mol/min
/mg, respectively, and for D-alanine, 2.4 mM and 350 mu mol/min/mg, respect
ively. The enzyme is almost specific to alanine, but L-serine and L-2-amino
butyrate 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 deleti
on of the alr1(+) gene resulted in retarded growth on the same medium, This
indicates that S. pombe has catabolic pathways for both enantiomers of ala
nine and that the pathway for L-alanine coupled with racemization plays a m
ajor 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. Howev
er, heterologous expression of the alr1(+) gene enabled S. cerevisiae to gr
ow efficiently on D-alanine as a sole nitrogen source. The recombinant yeas
t was relieved from the toxicity of D-alanine.