caa a22 4500 605503036 CHVBK 20210128100608.0 cr unu---uuuuu 210128e20150901xx s 000 0 eng 10.1007/s00253-015-6479-3 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6479-3 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 [Elektronische Daten] [Yu Matsumoto, Yoshiaki Yasutake, Yuki Takeda, Tomohiro Tamura, Atsushi Yokota, Masaru Wada] 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 Mg2+, 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 Mg2+. 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 (pH2.0) and thus avoiding the use of environmentally harmful organic solvents for the chromatographic purification. Springer-Verlag Berlin Heidelberg, 2015 d - threo -3-Hydroxyaspartate dehydratase nationallicence Delftia sp. HT23 nationallicence Alanine racemase nationallicence Pyridoxal 5′-phosphate nationallicence Enzymatic optical resolution nationallicence Matsumoto Yu Laboratory of Microbial Physiology, Research Faculty of Agriculture, Hokkaido University, Kita-9, Nishi-9, 060-8589, Sapporo, Kita-ku, Japan aut Yasutake Yoshiaki Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, 062-8517, Sapporo, Toyohira-ku, Japan aut Takeda Yuki Laboratory of Microbial Physiology, Research Faculty of Agriculture, Hokkaido University, Kita-9, Nishi-9, 060-8589, Sapporo, Kita-ku, Japan aut Tamura Tomohiro Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, 062-8517, Sapporo, Toyohira-ku, Japan aut Yokota Atsushi Laboratory of Microbial Physiology, Research Faculty of Agriculture, Hokkaido University, Kita-9, Nishi-9, 060-8589, Sapporo, Kita-ku, Japan aut Wada Masaru Laboratory of Microbial Physiology, Research Faculty of Agriculture, Hokkaido University, Kita-9, Nishi-9, 060-8589, Sapporo, Kita-ku, Japan aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/17(2015-09-01), 7137-7150 0175-7598 99:17<7137 2015 99 253 https://doi.org/10.1007/s00253-015-6479-3 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6479-3 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Matsumoto Yu Laboratory of Microbial Physiology, Research Faculty of Agriculture, Hokkaido University, Kita-9, Nishi-9, 060-8589, Sapporo, Kita-ku, Japan aut NATIONALLICENCE 700 1- Yasutake Yoshiaki Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, 062-8517, Sapporo, Toyohira-ku, Japan aut NATIONALLICENCE 700 1- Takeda Yuki Laboratory of Microbial Physiology, Research Faculty of Agriculture, Hokkaido University, Kita-9, Nishi-9, 060-8589, Sapporo, Kita-ku, Japan aut NATIONALLICENCE 700 1- Tamura Tomohiro Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, 062-8517, Sapporo, Toyohira-ku, Japan aut NATIONALLICENCE 700 1- Yokota Atsushi Laboratory of Microbial Physiology, Research Faculty of Agriculture, Hokkaido University, Kita-9, Nishi-9, 060-8589, Sapporo, Kita-ku, Japan aut NATIONALLICENCE 700 1- Wada Masaru Laboratory of Microbial Physiology, Research Faculty of Agriculture, Hokkaido University, Kita-9, Nishi-9, 060-8589, Sapporo, Kita-ku, Japan aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/17(2015-09-01), 7137-7150 0175-7598 99:17<7137 2015 99 253