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

<p>An enzyme catalyzing the ammonia-lyase reaction for the conversion of d-<i>erythro</i>-3-hydroxyaspartate to oxaloacetate was purified from the cell-free extract of a soil-isolated bacterium <i>Pseudomonas</i> sp. N99. The enzyme exhibited ammonia-lyase activity toward l-<i>threo</i>-3-hydroxyaspartate and d-<i>erythro</i>-3-hydroxyaspartate, but not toward other 3-hydroxyaspartate isomers. The deduced amino acid sequence of the enzyme, which belongs to the serine/threonine dehydratase family, shows similarity to the sequence of l-<i>threo</i>-3-hydroxyaspartate ammonia-lyase (EC from <i>Pseudomonas</i> sp. T62 (74%) and <i>Saccharomyces cerevisiae</i> (64%) and serine racemase from <i>Schizosaccharomyces pombe</i> (65%). These results suggest that the enzyme is similar to l-<i>threo</i>-3-hydroxyaspartate ammonia-lyase from <i>Pseudomonas</i> sp. T62, which does not act on d-<i>erythro</i>-3-hydroxyaspartate. We also then used the recombinant enzyme expressed in <i>Escherichia coli</i> to produce optically pure l-<i>erythro</i>-3-hydroxyaspartate and d-<i>threo</i>-3-hydroxyaspartate from the corresponding dl-racemic mixtures. The enzymatic resolution reported here is one of the simplest and the first enzymatic method that can be used for obtaining optically pure l-<i>erythro</i>-3-hydroxyaspartate.</p> <p>A novel enzyme “d-<i>erythro</i>-3-hydroxyaspartate dehydratase” was applied to optically active l-<i>erythro</i>-3-hydroxyaspartate production.</p>