AUTHOR=Akita Hironaga , Hayashi Junji , Sakuraba Haruhiko , Ohshima Toshihisa 

TITLE=Artificial Thermostable D-Amino Acid Dehydrogenase: Creation and Application

JOURNAL=Frontiers in Microbiology

VOLUME=9

YEAR=2018

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2018.01760

DOI=10.3389/fmicb.2018.01760

ISSN=1664-302X

ABSTRACT=<p>Many kinds of NAD(P)<sup>+</sup>-dependent <sc>L</sc>-amino acid dehydrogenases have been so far found and effectively used for synthesis of <sc>L</sc>-amino acids and their analogs, and for their sensing. By contrast, similar biotechnological use of <sc>D</sc>-amino acid dehydrogenase (<sc>D</sc>-AADH) has not been achieved because useful <sc>D</sc>-AADH has not been found from natural resources. Recently, using protein engineering methods, an NADP<sup>+</sup>-dependent <sc>D</sc>-AADH was created from <italic>meso</italic>-diaminopimelate dehydrogenase (<italic>meso</italic>-DAPDH). The artificially created <sc>D</sc>-AADH catalyzed the reversible NADP<sup>+</sup>-dependent oxidative deamination of <sc>D</sc>-amino acids to 2-oxo acids. The enzyme, especially thermostable one from thermophiles, was efficiently applicable to synthesis of <sc>D</sc>-branched-chain amino acids (<sc>D</sc>-BCAAs), with high yields and optical purity, and was useful for the practical synthesis of <sup>13</sup>C- and/or <sup>15</sup>N-labeled <sc>D</sc>-BCAAs. The enzyme also made it possible to assay <sc>D</sc>-isoleucine selectively in a mixture of isoleucine isomers. Analyses of the three-dimensional structures of <italic>meso</italic>-DAPDH and <sc>D</sc>-AADH, and designed mutations based on the information obtained made it possible to markedly enhance enzyme activity and to create <sc>D</sc>-AADH homologs with desired reactivity profiles. The methods described here may be an effective approach to artificial creation of biotechnologically useful enzymes.</p>