Serine racemase homologue of Saccharomyces cerevisiae has L-threo-3-hydroxyaspartate dehydratase activity.
The NH(2)-terminal amino acid sequence of L-threo-3-hydroxyaspartate dehydratase from Pseudomonas sp. T62 showed significant similarity to that of the SRY1 gene product of Saccharomyces cerevisiae (serine racemase in yeast). SRY1 was cloned and expressed in Escherichia coli, and the gene product was purified and partially characterized. The SRY1 gene product exhibited dehydratase activity specific for L-threo-3-hydroxyaspartate (K(m)=3.9 mM, V(max)=110 micromol min(-1) (mg protein)(-1)) but not for D-threo- or DL-erythro-3-hydroxyaspartate. The purified enzyme showed no detectable serine racemase activity. The activity of the enzyme was inhibited by hydroxylamine and EDTA, and was activated by Mg(2+), Ca(2+), and Mn(2+), suggesting that pyridoxal-5'-phosphate and divalent cations participate in the enzyme reaction. Gene disruption and overexpression indicated that SRY1 is responsible for the 3-hydroxyaspartate resistance of S. cerevisiae. To our knowledge, this is the first report of 3-hydroxyaspartate dehydratase activity in eukaryotic cells.
Wada
Masaru
M
Department of Bioscience, Fukui Prefectural University, 4-1-1 Kenjojima, Matsuoka-cho, Fukui 910-1195, Japan. wada@chem.agr.hokudai.ac.jp
Nakamori
Shigeru
S
Takagi
Hiroshi
H
D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology; D025141:Drug Resistance, Fungal; D004492:Edetic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D006836:Hydro-Lyases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019811:Hydroxylamine / Q000494:pharmacology; D008970:Molecular Weight; D016254:Mutagenesis, Insertional; D019998:Racemases and Epimerases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D012694:Serine / Q000494:pharmacology; D013057:Spectrum Analysis; D013237:Stereoisomerism; D013379:Substrate Specificity; D014170:Transformation, Genetic; D000099300:Serine Racemase
D001224:Aspartic Acid; D002413:Cations, Divalent; D004492:Edetic Acid; D004791:Enzyme Inhibitors; D006836:Hydro-Lyases; D019811:Hydroxylamine; D019998:Racemases and Epimerases; D011994:Recombinant Proteins; D012694:Serine; D000099300:Serine Racemase; C011274:3-hydroxyaspartic acid; C122119:L-threo-3-hydroxyaspartate dehydratase