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Journal of Biochemistry Advance Access published online on February 4, 2009
Journal of Biochemistry, doi:10.1093/jb/mvp023
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© The authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Gene cloning and expression of pyridoxal 5'-phosphate-dependent L-threo-3-hydroxyaspartate dehydratase from Pseudomonas sp. T62, and characterization of the recombinant enzyme

Tomoko Murakami, Takayuki Maeda, Atsushi Yokota and Masaru Wada*

Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Kita-9, Nishi-9, Kita-ku, Sapporo, 060-8589, Japan

*Correspondence to Masaru Wada, Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Kita-9, Nishi-9, Kita-ku, Sapporo, 060-8589, Japan Tel: +81-11-706-4185; Fax: +81-11-706-4961; E-mail: wada{at}chem.agr.hokudai.ac.jp

Received October 31, 2008; Accepted January 27, 2009


  Abstract

L-threo-3-Hydroxyaspartate dehydratase (L-THA DH, EC 4.3.1.16 [EC] ), which catalyzes 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.

Key Words: L-threo-3-hydroxyaspartate dehydratase, serine racemase, pyridoxal 5'-phosphate, Pseudomonas sp. T62, serine/threonine dehydratase


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