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.
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 (β) -OH of L-EHA occupied a position close to the active-site Mg(2+), clearly indicating a possibility of metal-assisted C (β) -OH elimination from the substrate. In contrast, the C (β) -OH of an inhibitor was bound far from the active-site Mg(2+). This suggests that the substrate specificity of D-THA DH is determined by the orientation of the C (β) -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 β-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.
authors.affiliation: Laboratory of Microbial Physiology, Research Faculty of Agriculture, Hokkaido University, Kita-9, Nishi-9, Kita-ku, Sapporo, 060-8589, Japan.
authors.forename: Yu
authors.initials: Y
authors.lastname: Matsumoto
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Yasutake
authors.forename: Yuki
authors.initials: Y
authors.lastname: Takeda
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Tamura
authors.forename: Atsushi
authors.initials: A
authors.lastname: Yokota
authors.forename: Masaru
authors.initials: M
authors.lastname: Wada
mesh_terms: D001224:Aspartic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D042742:Delftia / Q000201:enzymology* / Q000235:genetics; D006836:Hydro-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: C011274:3-hydroxyaspartic acid; D001224:Aspartic Acid; D006836:Hydro-Lyases