Journal Article

Gene Cloning and Expression of Pyridoxal 5′-Phosphate-Dependent L-<i>threo</i>-3-Hydroxyaspartate Dehydratase from <i>Pseudomonas</i> sp. T62, and Characterization of the Recombinant Enzyme

Tomoko Murakami, Takayuki Maeda, Atsushi Yokota and Masaru Wada

in The Journal of Biochemistry

Published on behalf of The Japanese Biochemical Society

Volume 145, issue 5, pages 661-668
Published in print May 2009 | ISSN: 0021-924X
Published online February 2009 | e-ISSN: 1756-2651 | DOI: http://dx.doi.org/10.1093/jb/mvp023
Gene Cloning and Expression of Pyridoxal 5′-Phosphate-Dependent L-threo-3-Hydroxyaspartate Dehydratase from Pseudomonas sp. T62, and Characterization of the Recombinant Enzyme

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L-threo-3-Hydroxyaspartate dehydratase (L-THA DH, EC 4.3.1.16), which catalyses the cleavage of L-threo-3-hydroxyaspartate (L-THA) to oxalacetate and ammonia, has been purified from the soil bacterium Pseudomonas sp. T62. In this report, the gene encoding L-THA DH was cloned and expressed in Escherichia coli, and the gene product was purified and characterized in detail. A 957-bp nucleotide fragment was confirmed to be the gene encoding L-THA DH, based on the agreement of internal amino acid sequences. The deduced amino acid sequence, which belongs to the serine/threonine dehydratase family, shows similarity to YKL218c from Saccharomyces cerevisiae (64%), serine racemase from Schizosaccharomyces pombe (64%) and Mus musculus (36%), and biodegradative threonine dehydratase from E. coli (38%). Site-directed mutagenesis experiments revealed that lysine at position 53 is an important residue for enzymatic activity. This enzyme exhibited dehydratase activity specific only to L-THA [Km = 0.54 mM, Vmax = 39.0 μmol min–1 (mg protein)−1], but not to other 3-hydroxyaspartate isomers, and exhibited no detectable serine/aspartate racemase activity. This is the first report of an amino acid sequence of the bacterial enzyme that acts on L-THA.

Keywords: L-threo-3-hydroxyaspartate dehydratase; serine racemase; pyridoxal 5′-phosphate; Pseudomonas sp. T62; serine/threonine dehydratase

Journal Article.  4982 words.  Illustrated.

Subjects: Biochemistry

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