文献类型：期刊文献

英文题名：Hierarchical Engineering of meso-Diaminopimelate Dehydrogenase for Efficient Synthesis of Bulky D-Amino Acids

作者：Wei, Yan[1];Geng, Qiang[1];Liu, Hai-Ping[1];Wang, Yu-Qing[1];Zhang, Guo-Feng[1];Qian, Xiao-Long[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：2024

卷号：14

期号：15

起止页码：11447

外文期刊名：ACS CATALYSIS

收录：;EI(收录号：20243016732953);WOS:【SCI-EXPANDED(收录号:WOS:001272032100001)】；

基金：This work was financially supported by the National Natural Science Foundation of China (32071475), the National Key Research and Development Program of China (2019YFA0905000 and 2022YFC2105900), and the Shanghai Commission of Science and Technology (23HC1400200). We are also grateful for the access to beamline BL02U1 at the Shanghai Synchrotron Radiation Facility and thank the beamline staff for their technical help.

语种：英文

外文关键词：meso-diaminopimelate dehydrogenase; asymmetric synthesis; < sc > d -amino acids; proteinengineering; catalytic efficiency

摘要：Asymmetric reductive amination of alpha-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 alpha-ketoacids, which limits their widespread applications. In this work, the activity of a DAPDH from Bacillus thermozeamaize (BtDAPDH) toward a number of alpha-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 alpha-ketoacids.