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Journal of Biochemistry Advance Access originally published online on February 4, 2009
Journal of Biochemistry 2009 145(5):661-668; 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

*To whom correspondence should be addressed. 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 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.

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

Abbreviations: ADP, adenosine 5'-diphosphate; AMP, adenosine 5'-monophosphate; ATP, adenosine 5'-triphosphate; DH, dehydratase; EDTA, ethylenediaminetetraacetic acid; GDP, guanosine 5'-diphosphate; LB, Luria–Bertani; L-THA, L-threo-3-hydroxyaspartate; MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; NADH, nicotinamide adenine dinucleotide; IPTG, isopropyl-β-D-thiogalactopyranoside; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; PLP, pyridoxal 5'-phosphate; SDS, sodium dodecyl sulphate; tdcB, the gene encoding biodegradative threonine dehydratase


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