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Purification, characterization and amino acid sequence of a novel enzyme, D-threo-3-hydroxyaspartate dehydratase, from Delftia sp. HT23

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Title: Purification, characterization and amino acid sequence of a novel enzyme, D-threo-3-hydroxyaspartate dehydratase, from Delftia sp. HT23
Authors: Maeda, Takayuki Browse this author
Takeda, Yuki Browse this author
Murakami, Tomoko Browse this author
Yokota, Atsushi Browse this author
Wada, Masaru Browse this author →KAKEN DB
Keywords: alanine racemase
Delftia sp. HT23
D-threo-3-hydroxyaspartate dehydratase
pyridoxal 5'-phosphate
Issue Date: Dec-2010
Publisher: Oxford University Press
Journal Title: The Journal of Biochemistry
Volume: 148
Issue: 6
Start Page: 705
End Page: 712
Publisher DOI: 10.1093/jb/mvq106
PMID: 20843822
Abstract: D-threo-3-hydroxyaspartate dehydratase (D-THA DH) was purified from the cell-free extract of the soil-isolated bacterium Delftia sp. HT23. The enzyme exhibited dehydratase activity towards D-threo-3-hydroxyaspartate, L-threo-3-hydroxyaspartate, L-erythro-3-hydroxyaspartate and D-serine. Absorption of the purified enzyme at 412 nm suggests that it contains pyridoxal 5'-phosphate (PLP) as a cofactor. The NH2-terminal and internal amino acid sequences showed significant similarity to hypothetical alanine racemase of genome-sequenced Delftia acidovorans SPH-1; however, the purified enzyme showed no alanine racemase activity. Using the sequence information of Delftia acidovorans SPH-1, the gene encoding D-THA DH was cloned. The deduced amino acid sequence, which belongs to the alanine racemase family, shows significant (26-36%) similarity to D-serine dehydratase of both Saccharomyces cerevisiae and chicken. In order to obtain purified D-THA DH efficiently, the gene was expressed in Escherichia coli. The recombinant enzyme was highly activated by divalent cations, such as Mn2+, Co2+ and Ni2+. Site-directed mutagenesis experiment revealed that lysine 43 is an important residue involved in PLP binding and catalysis. This is the first reported enzyme that acts on D-THA. In addition, this enzyme is the first example of a prokaryotic dehydratase belonging to the fold-type III PLP-dependent enzyme family.
Rights: This is a pre-copy-editing, author-produced PDF of an article accepted for publication in The Journal of Biochemistry following peer review. The definitive publisher-authenticated version, J Biochem (2010) 148 (6): 705-712, is available online at: http://jb.oxfordjournals.org/content/148/6/705
Type: article (author version)
URI: http://hdl.handle.net/2115/47109
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)

Submitter: 和田 大

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