Gene cloning and expression of pyridoxal 5'-phosphate-dependent L-threo-3-hydroxyaspartate dehydratase from Pseudomonas sp. T62, and characterization of the recombinant enzyme.
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 [K(m) = 0.54 mM, V(max) = 39.0 micromol 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.
Murakami
Tomoko
T
Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Sapporo, Japan.
Maeda
Takayuki
T
Yokota
Atsushi
A
Wada
Masaru
M
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D001483:Base Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005798:Genes, Bacterial; D006836:Hydro-Lyases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000378:metabolism; D008670:Metals / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009711:Nucleotides / Q000378:metabolism; D010802:Phylogeny; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D016415:Sequence Alignment; D013057:Spectrum Analysis; D013379:Substrate Specificity; D013696:Temperature
D000596:Amino Acids; D008670:Metals; D009711:Nucleotides; D011994:Recombinant Proteins; D011732:Pyridoxal Phosphate; D006836:Hydro-Lyases; C122119:L-threo-3-hydroxyaspartate dehydratase; D008239:Lysine