RRC ID 4975
Author Murakami T, Maeda T, Yokota A, Wada M.
Title Gene cloning and expression of pyridoxal 5'-phosphate-dependent L-threo-3-hydroxyaspartate dehydratase from Pseudomonas sp. T62, and characterization of the recombinant enzyme.
Journal J. Biochem.
Abstract 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.
Volume 145(5)
Pages 661-8
Published 2009-5
DOI 10.1093/jb/mvp023
PII mvp023
PMID 19193709
MeSH 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
IF 2.35
Times Cited 6
WOS Category BIOCHEMISTRY & MOLECULAR BIOLOGY
Resource
Prokaryotes E. coli NA