Akita, Hironaga National Institute of Advanced Industrial Science and Technology
Hayashi, Junji Ritsumeikan University Tokushima University Educator and Researcher Directory KAKEN Search Researchers
Sakuraba, Haruhiko Kagawa University
Ohshima, Toshihisa Osaka Institute of Technology Tokushima University Educator and Researcher Directory
thermostable D-amino acid dehydrogenase
stable isotope-labeled D-amino acid
crystal structure analysis
Many kinds of NAD(P)+-dependent L-amino acid dehydrogenases have been so far found and effectively used for synthesis of L-amino acids and their analogs, and for their sensing. By contrast, similar biotechnological use of D-amino acid dehydrogenase (D-AADH) has not been achieved because useful D-AADH has not been found from natural resources. Recently, using protein engineering methods, an NADP+-dependent D-AADH was created from meso-diaminopimelate dehydrogenase (meso-DAPDH). The artificially created D-AADH catalyzed the reversible NADP+-dependent oxidative deamination of D-amino acids to 2-oxo acids. The enzyme, especially thermostable one from thermophiles, was efficiently applicable to synthesis of D-branched-chain amino acids (D-BCAAs), with high yields and optical purity, and was useful for the practical synthesis of 13C- and/or 15N-labeled D-BCAAs. The enzyme also made it possible to assay D-isoleucine selectively in a mixture of isoleucine isomers. Analyses of the three-dimensional structures of meso-DAPDH and D-AADH, and designed mutations based on the information obtained made it possible to markedly enhance enzyme activity and to create D-AADH homologs with desired reactivity profiles. The methods described here may be an effective approach to artificial creation of biotechnologically useful enzymes.
Frontiers in Microbiology
Frontiers Media S.A.
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Bioscience and Bioindustry