An Engineered <i>meso</i>-Diaminopimelate Dehydrogenase Enables the Biocatalytic Synthesis of Bulky β-Substituted <scp>d</scp>-Amino Acids

The strict stereoselectivity of meso-diaminopimelate dehydrogenase (DAPDH) promises a direct synthetic pathway for d-amino acids via the asymmetric reductive amination of keto acids with ammonium. However, the low activity and limited substrate scope of the wild-type and previously engineered DAPDH variants have posed significant challenges for their practical applications. In this study, a structure-guided engineering strategy based on triple code saturation mutagenesis (TCSM) was employed for the DAPDH from Bacillus thermozeamaize (BtDAPDH). A BtDAPDH-M9 variant, which demonstrates a remarkable up to 450-fold improvement in catalytic activity toward a series of bulky β-substituted ketoacids, was identified. The synthetic applicability of the newly engineered variant was evaluated through the gram-scale synthesis of previously difficult to obtain d-biphenylalanine as well as several bulky pharmaceutically relevant d-amino acids with yields ranging from 53% to 69% and a stereoselectivity >99% ee. Structural analysis and molecular dynamics simulations provide mechanistic insights into the enhanced catalytic activity of BtDAPDH-M9, revealing how mutagenesis optimizes the substrate-binding cleft. This study establishes an efficient biocatalytic route for the synthesis of sterically bulky d-amino acids and offers a valuable strategy for the engineering of enzymes to access unnatural amino acids.