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Hokkaido University Collection of Scholarly and Academic Papers >
Graduate School of Agriculture / Faculty of Agriculture >
Peer-reviewed Journal Articles, etc >
Gene Cloning and Expression of Pyridoxal 5'-Phosphate-Dependent L-threo-3-Hydroxyaspartate Dehydratase from Pseudomonas sp. T62, and Characterization of the Recombinant Enzyme
| 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 |
| Authors: | Murakami, Tomoko Browse this author | | Maeda, Takayuki Browse this author | | Yokota, Atsushi Browse this author →KAKEN DB | | Wada, Masaru Browse this author →KAKEN DB |
| Keywords: | L-threo-3-hydroxyaspartate dehydratase | | serine racemase | | pyridoxal 5'-phosphate | | Pseudomonas sp. T62 | | serine/threonine dehydratase |
| Issue Date: | May-2009 |
| Publisher: | Oxford University Press |
| Journal Title: | Journal of Biochemistry |
| Volume: | 145 |
| Issue: | 5 |
| Start Page: | 661 |
| End Page: | 668 |
| Publisher DOI: | 10.1093/jb/mvp023 |
| PMID: | 19193709 |
| 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 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. |
| Rights: | 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 |
| Type: | article (author version) |
| URI: | http://hdl.handle.net/2115/43033 |
| Appears in Collections: | 農学院・農学研究院 (Graduate School of Agriculture / Faculty of Agriculture) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)
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Submitter: 和田 大
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