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 ( Bt DAPDH) 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 Bt DAPDH 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.