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.

Hiroyuki Nagano,Kana Shibano,Atsushi Yokota,Yu Matsumoto,Masaru Wada

doi:10.1080/09168451.2017.1295804

Hiroyuki Nagano, Kana Shibano + Show 3 more

Open Access

https://doi.org/10.1080/09168451.2017.1295804

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Abstract

An enzyme catalyzing the ammonia-lyase reaction for the conversion of d-erythro-3-hydroxyaspartate to oxaloacetate was purified from the cell-free extract of a soil-isolated bacterium Pseudomonas sp. N99. The enzyme exhibited ammonia-lyase activity toward l-threo-3-hydroxyaspartate and d-erythro-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-threo-3-hydroxyaspartate ammonia-lyase (EC 4.3.1.16) from Pseudomonas sp. T62 (74%) and Saccharomyces cerevisiae (64%) and serine racemase from Schizosaccharomyces pombe (65%). These results suggest that the enzyme is similar to l-threo-3-hydroxyaspartate ammonia-lyase from Pseudomonas sp. T62, which does not act on d-erythro-3-hydroxyaspartate. We also then used the recombinant enzyme expressed in Escherichia coli to produce optically pure l-erythro-3-hydroxyaspartate and d-threo-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-erythro-3-hydroxyaspartate.

Highlights

We recently reported the identification of a novel enzyme, D-threo-3-hydroxyaspartate ammonia-lyase (EC 4.3.1.27), from Delftia sp
These results suggested that enzymes having similar amino acid sequence to D-EHA
We isolated an enzyme acting on D-EHA and purified it to almost homogeneity from the newly isolated soil bacterium, Pseudomonas sp

Summary

Introduction

The deduced amino acid sequence of the enzyme, which belongs to the serine/threonine dehydratase family, shows similarity to the sequence of L-threo-3-hydroxyaspartate ammonia-lyase (EC 4.3.1.16) from. T62 (74%) and Saccharom yces cerevisiae (64%) and serine racemase from Schizosaccharom yces pom be (65%). These results suggest that the enzyme is similar to L-threo-3-hydroxyaspartate ammonia-lyase from Pseudom onas sp. T62, which does not act on D-erythro-3-hydroxyaspartate. We used the recombinant enzyme expressed in Escherichia coli to produce optically pure L-erythro-3-. The enzymatic resolution reported here is one of the simplest and the first enzymatic method that can be used for obtaining optically pure L-erythro-3-hydroxyaspartate. 3-Hydroxyaspartate is a non-proteinogenic amino acid; it was first found in human cerebrospinal fluid in the 1960s1) and has been known as an inhibitor of aspartate aminotransferase2)

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