Abstract
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.
MeSH terms
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Aspartic Acid / analogs & derivatives
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Aspartic Acid / metabolism
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Aspartic Acid / pharmacology
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Cations, Divalent / pharmacology
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Drug Resistance, Fungal
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Edetic Acid / pharmacology
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Enzyme Inhibitors / pharmacology
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Escherichia coli / genetics
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Hydro-Lyases / antagonists & inhibitors
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Hydro-Lyases / chemistry
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Hydro-Lyases / genetics
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Hydro-Lyases / metabolism*
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Hydroxylamine / pharmacology
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Molecular Weight
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Mutagenesis, Insertional
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Racemases and Epimerases / chemistry
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Racemases and Epimerases / isolation & purification
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Racemases and Epimerases / metabolism
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Recombinant Proteins / metabolism
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Saccharomyces cerevisiae / drug effects
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Saccharomyces cerevisiae / enzymology*
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae / growth & development
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Serine / pharmacology
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Spectrum Analysis
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Stereoisomerism
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Substrate Specificity
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Transformation, Genetic
Substances
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Cations, Divalent
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Enzyme Inhibitors
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Recombinant Proteins
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3-hydroxyaspartic acid
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Hydroxylamine
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Aspartic Acid
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Serine
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Edetic Acid
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Hydro-Lyases
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L-threo-3-hydroxyaspartate dehydratase
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Racemases and Epimerases
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serine racemase