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

J Biochem. 2009 May;145(5):661-8. doi: 10.1093/jb/mvp023. Epub 2009 Feb 4.


L-threo-3-Hydroxyaspartate dehydratase (L-THA DH, 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 [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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acids / metabolism
  • Base Sequence
  • Catalytic Domain
  • Cloning, Molecular
  • Electrophoresis, Polyacrylamide Gel
  • Genes, Bacterial
  • Hydro-Lyases / chemistry
  • Hydro-Lyases / genetics*
  • Hydro-Lyases / isolation & purification
  • Hydro-Lyases / metabolism
  • Hydrogen-Ion Concentration
  • Lysine / metabolism
  • Metals / metabolism
  • Molecular Sequence Data
  • Molecular Weight
  • Nucleotides / metabolism
  • Phylogeny
  • Pseudomonas / enzymology*
  • Pseudomonas / genetics
  • Pyridoxal Phosphate / metabolism*
  • Recombinant Proteins / metabolism*
  • Sequence Alignment
  • Spectrum Analysis
  • Substrate Specificity
  • Temperature


  • Amino Acids
  • Metals
  • Nucleotides
  • Recombinant Proteins
  • Pyridoxal Phosphate
  • Hydro-Lyases
  • L-threo-3-hydroxyaspartate dehydratase
  • Lysine