文献类型：期刊文献

英文题名：Semirational Engineering of a Distal Loop Region to Enhance the Catalytic Activity and Stability of Leucine Dehydrogenase

作者：Li, Rui[1,2];Xu, Minglu[1];Xu, Meng[1];Zhou, Yiquan[1];Wen, Tao[1];Liu, Qinghai[1];Yi, Dong[2];Wang, Hualei[1];Wei, Dongzhi[1]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]China State Inst Pharmaceut Ind, Res Ctr Syst Biosynth, Natl Key Lab Lead Druggabil Res, Shanghai 201203, Peoples R China

年份：2025

卷号：73

期号：5

起止页码：2961

外文期刊名：JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY

收录：;EI(收录号：20250517785328);WOS:【SCI-EXPANDED(收录号:WOS:001408456200001)】；

基金：This study was supported by the National Key Research and Development Program of China (Grant Number: 2021YFC2102100) and the National Natural Science Foundation of China (No. 22078097).

语种：英文

外文关键词：l-phenylglycine; distant modulation; loop engineering; benzoylformicacid; enantiomericexcess; triple-point mutation; formate dehydrogenase; molecular dynamics simulations

摘要：Enzymatic asymmetric synthesis of l-phenylglycine by amino acid dehydrogenases has potential for industrial applications; however, this is hindered by their low catalytic efficiency toward high-concentration substrates. We identified and characterized a novel leucine dehydrogenase (MsLeuDH) with a high catalytic efficiency for benzoylformic acid via directed metagenomic approaches. Further, we obtained a triple-point mutant MsLeuDH-EER (D332E/G333E/L334R) with improved stability and catalytic efficiency through the rational design of distal loop 13. A coexpression system of MsLeuDH-EER and formate dehydrogenase completely converted a 300 mM substrate within 4 h with >99.9% enantiomeric excess. Molecular dynamics simulations revealed that mutations on loop 13 enhanced the overall structural rigidity of the protein to improve its stability but also stabilized the "closed" conformation through rigidifying the hinge region loop by distant modulation. Our results show that distal loop 13 can serve as a new hotspot region for enhancing the catalytic performance of leucine dehydrogenases.