Hierarchical Engineering of meso-Diaminopimelate Dehydrogenase for Efficient Synthesis of Bulky d-Amino Acids

Asymmetric reductive amination of α-ketoacids by d-amino acid dehydrogenase is a straightforward and promising method for the synthesis of d-amino acids in the pharmaceutical and fine chemical industries. Since the naturally occurring d-amino acid dehydrogenases are scarce and mainly exist as membrane-bound proteins, only the engineered meso-diaminopimelate dehydrogenases (DAPDHs) can be applied for the desired reaction. However, previously reported DAPDH variants showed restricted activity toward bulky α-ketoacids, which limits their widespread applications. In this work, the activity of a DAPDH from Bacillus thermozeamaize (BtDAPDH) toward a number of α-ketoacids was improved by hierarchical engineering of the active pocket. The best variant M5 exhibits a specific activity of up to 1650 mU mg–1 toward bulky benzoylformic acid, which is 275-fold that of the wild type. Additionally, all variants preserve good thermostability of the wild type. Using M5 as a biocatalyst, three pharmaceutically relevant d-amino acids, d-phenylglycine, d-phenylalanine, and d-homophenylalanine, were prepared on a gram scale in up to 89% yield and >99% ee. These results suggest that the engineered BtDAPDH M5 is a promising biocatalyst for the asymmetric synthesis of d-amino acids. Structural analysis and molecular dynamics simulations provide insights into how the mutations in M5 improve the activity toward bulky α-ketoacids.