Purification and characterization of a novel enzyme, ?-threo-3-hydroxyaspartate dehydratase, from Pseudomonas sp. T62

Abstract

L-threo-3-Hydroxyaspartate dehydratase (L-threo-3-hydroxyaspartate hydro-lyase), which exhibited specificity for L-threo-3hydroxyaspartate (K m = 0.74 mM, V max = 37.5 Wmol min 31 (mg protein) 31 ) but not for D-threo or D,L-erythro-3hydroxyaspartate, was purified from a cell-free extract of Pseudomonas sp. T62. The activity of the enzyme was inhibited by hydroxylamine and EDTA, which suggests that pyridoxal 5P-phosphate and divalent cations participate in the enzyme reaction. The NH 2 -terminal amino acid sequence showed significant similarity to the Saccharomyces cerevisiae YKL218c gene product, a hypothetical threonine dehydratase. However, the purified enzyme showed no threonine dehydratase activity. ß

Serine racemase homologue ofSaccharomyces cerevisiaehas l-threo-3-hydroxyaspartate dehydratase activity

“…The enzyme was strongly inhibited by hydroxylamine (99% inhibition), suggesting that PLP participates in the enzyme reaction, as in the case of threo ‐3‐hydroxyaspartate dehydratase of Pseudomonas sp. T62 [5]. The enzyme was also strongly inhibited by EDTA (75% inhibition), suggesting that metal ions were involved in the enzyme reaction.…”

“…3‐Hydroxyaspartate dehydratase activity was determined by methods reported previously [5]. One unit of the enzyme was defined as the amount capable of catalyzing the oxidation of 1 μmol of NADH per min.…”

“…In our previous report, we showed that the NH 2 ‐terminal amino acid sequence of l ‐ threo ‐3‐hydroxyaspartate dehydratase from Pseudomonas sp. T62 showed significant similarity to that of the YKL218c gene product of Saccharomyces cerevisiae [5]. YKL218c is now annotated as SRY1 (serine racemase in yeast).…”

“…We recently found that an extract of a newly isolated bacterium, Pseudomonas sp. T62, exhibited dehydratase activity specific for l ‐ threo ‐3‐hydroxyaspartate but not for erythro ‐3‐hydroxyaspartate [5]. This novel enzyme catalyzes a deaminating reaction from l ‐ threo ‐3‐hydroxyaspartate to oxaloacetate and ammonia.…”

Isolation and amino acid sequence of a dehydratase acting on d-erythro-3-hydroxyaspartate from Pseudomonas sp. N99, and its application in the production of optically active 3-hydroxyaspartate

“…N99. Although 3-hydroxyaspartate dehydratases acting on L-THA or D-THA have been identified and characterized previously by our research group, 13,18,19,24) an enzyme acting on D-EHA has not yet been reported. To the best of our knowledge, the present study is the first to report an enzyme that catalyzes the deamination of D-EHA.…”