文献类型：期刊文献

英文题名：An Engineered meso-Diaminopimelate Dehydrogenase Enables the Biocatalytic Synthesis of Bulky β-Substituted d-Amino Acids

作者：Wei, Yan[1];Zheng, Yu-Cong[1];Liu, Hai-Ping[1];Geng, Qiang[1];Wang, Zheng[1];Wang, Yu-Qing[1];Yu, Hui-Lei[1];Xu, Jian-He[1];Zhang, Zhi-Jun[1]

机构：[1]East China Univ Sci & Technol, Shanghai Collaborat Innovat Ctr Biomfg, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China

年份：2025

卷号：15

期号：10

起止页码：7720

外文期刊名：ACS CATALYSIS

收录：;EI(收录号：20251718313388);WOS:【SCI-EXPANDED(收录号:WOS:001475202200001)】；

基金：The authors are grateful to the National Natural Science Foundation of China (32071475), The National Key Research and Development Program of China (2022YFC2105900), and Shanghai Commission of Science and Technology (23HC1400200). The authors are also grateful for the access to beamline BL02U1 at Shanghai Synchrotron Radiation Facility and thank the beamline staff for their technical help.

语种：英文

外文关键词：reductive amination; asymmetric synthesis; d-amino acid; meso-diaminopimelate dehydrogenase; protein engineering

摘要：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 beta-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.