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

Citation
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
Citations number
38
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Microbiology
Journal title
JOURNAL OF BACTERIOLOGY
ISSN journal
0021-9193 → ACNP
Volume
183
Issue
7
Year of publication
2001
Pages
2226 - 2233
Database
ISI
SICI code
0021-9193(200104)183:7<2226:FCOARF>2.0.ZU;2-2
Abstract
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.