Artificial Thermostable D-Amino Acid Dehydrogenase: Creation and Application

作者：Akita Hironaga; Hayashi Junji; Sakuraba Haruhiko; Ohshima Toshihisa^*

来源：Frontiers in Microbiology, 2018, 9: 1760.

DOI：10.3389/fmicb.2018.01760

• D-amino acid
• thermostable D-amino acid dehydrogenase
• meso-diaminopimelate dehydrogenase
• stable isotope-labeled D-amino acid
• Ureibacillus thermosphaericus
• protein engineering
• 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 C-13- and/or N-15-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.

• 出版日期2018-8-3