open access

https://hdl.handle.net/2115/47109

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

英語

pyridoxal 5'-phosphate

2010-12

Oxford University Press

The Journal of Biochemistry

148(6)

705-712

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.

学術雑誌論文

Accepted Manuscript

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

0021-924X