open access

https://hdl.handle.net/2115/43033

Gene Cloning and Expression of Pyridoxal 5'-Phosphate-Dependent L-threo-3-Hydroxyaspartate Dehydratase from Pseudomonas sp. T62, and Characterization of the Recombinant Enzyme

英語

serine/threonine dehydratase

2009-05

Oxford University Press

Journal of Biochemistry

145(5)

661-668

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 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.

学術雑誌論文

Accepted Manuscript

This is a pre-copy-editing, author-produced PDF of an article accepted for publication in Journal of Biochemistry following peer review. The definitive publisher-authenticated version, Journal of Biochemistry 2009 145(5):661-668, is available online at: http://dx.doi.org/10.1093/jb/mvp023

0021-924X