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Structural insights into the substrate stereospecificity of D-threo-3-hydroxyaspartate dehydratase from Delftia sp HT23: a useful enzyme for the synthesis of optically pure L-threo- and D-erythro-3-hydroxyaspartate

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Title: Structural insights into the substrate stereospecificity of D-threo-3-hydroxyaspartate dehydratase from Delftia sp HT23: a useful enzyme for the synthesis of optically pure L-threo- and D-erythro-3-hydroxyaspartate
Authors: Matsumoto, Yu Browse this author
Yasutake, Yoshiaki Browse this author
Takeda, Yuki Browse this author
Tamura, Tomohiro Browse this author →KAKEN DB
Yokota, Atsushi Browse this author →KAKEN DB
Wada, Masaru Browse this author →KAKEN DB
Keywords: D-threo-3-Hydroxyaspartate dehydratase
Delftia sp HT23
Alanine racemase
Pyridoxal 5 '-phosphate
Enzymatic optical resolution
Issue Date: Sep-2015
Publisher: Springer
Journal Title: Applied microbiology and biotechnology
Volume: 99
Issue: 17
Start Page: 7137
End Page: 7150
Publisher DOI: 10.1007/s00253-015-6479-3
PMID: 25715785
Abstract: d-threo-3-Hydroxyaspartate dehydratase (d-THA DH) is a fold-type III pyridoxal 5'-phosphate-dependent enzyme, isolated from a soil bacterium of Delftia sp. HT23. It catalyzes the dehydration of d-threo-3-hydroxyaspartate (d-THA) and l-erythro-3-hydroxyaspartate (l-EHA). To elucidate the mechanism of substrate stereospecificity, crystal structures of d-THA DH were determined in complex with various ligands, such as an inhibitor (d-erythro-3-hydroxyaspartate (d-EHA)), a substrate (l-EHA), and the reaction intermediate (2-amino maleic acid). The C (beta) -OH of l-EHA occupied a position close to the active-site Mg2+, clearly indicating a possibility of metal-assisted C (beta) -OH elimination from the substrate. In contrast, the C (beta) -OH of an inhibitor was bound far from the active-site Mg2+. This suggests that the substrate specificity of d-THA DH is determined by the orientation of the C (beta) -OH at the active site, whose spatial arrangement is compatible with the 3R configuration of 3-hydroxyaspartate. We also report an optically pure synthesis of l-threo-3-hydroxyaspartate (l-THA) and d-EHA, promising intermediates for the synthesis of beta-benzyloxyaspartate, by using a purified d-THA DH as a biocatalyst for the resolution of racemic dl-threo-3-hydroxyaspartate (dl-THA) and dl-erythro-3-hydroxyaspartate (dl-EHA). Considering 50 % of the theoretical maximum, efficient yields of l-THA (38.9 %) and d-EHA (48.9 %) as isolated crystals were achieved with > 99 % enantiomeric excess (e.e.). The results of nuclear magnetic resonance signals verified the chemical purity of the products. We were directly able to isolate analytically pure compounds by the recrystallization of acidified reaction mixtures (pH 2.0) and thus avoiding the use of environmentally harmful organic solvents for the chromatographic purification.
Type: article (author version)
URI: http://hdl.handle.net/2115/62880
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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