文献类型：期刊文献

英文题名：Attenuated substrate inhibition of a haloketone reductase via structure-guided loop engineering

作者：Shang, Yue-Peng[1];Chen, Qi[1];Li, Ai-Tao[2];Quan, Shu[1];Xu, Jian-He[1];Yu, Hui-Lei[1]

机构：[1]East China Univ Sci & Technol, Shanghai Collaborat Innovat Ctr Biomfg, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]Hubei Univ, Coll Life Sci, Hubei Key Lab Ind Biotechnol, Hubei Collaborat Innovat Ctr Green Transformat Bi, 368 Youyi Rd, Wuhan 430062, Hubei, Peoples R China

年份：2020

卷号：308

起止页码：141

外文期刊名：JOURNAL OF BIOTECHNOLOGY

收录：;EI(收录号：20195207899961);WOS:【SCI-EXPANDED(收录号:WOS:000504828500017)】；

基金：This work was financially supported by the National Natural Science Foundation of China (Nos. 21536004, 21922804 & 21776085), Shanghai Science and Technology Program (No. 15JC1400403), Natural Science Foundation of Shanghai (No. 19ZR1472900) and the National Key R&D Program of China (No. 2019YFA09005000). All diffraction data of protein or complex crystals were collected at beamline BL17U of the Shanghai Synchrotron Radiation Facility. We thank Dr. Zhi-Jun Zhang and Dr. Xu-Dong Kong at East China University of Science and Technology for their insightful discussions. We thank Liwen Bianji, Edanz Editing China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.

语种：英文

外文关键词：Haloketone reductase; Substrate inhibition; Protein engineering; X-ray crystal structure; Asymmetric reduction

摘要：Substrate inhibition of enzymes is one of the main obstacles encountered frequently in industrial biocatalysis. Haloketone reductase SsCR was seriously inhibited by substrate 2,2',4'-trichloroacetophenone. In this study, two essential loops were found that have a relationship with substrate binding by conducting X-ray crystal structure analysis. Three key residues were selected from the tips of the loops and substituted with amino acids with lower hydrophobicity to weaken the hydrophobic interactions that bridge the two loops, resulting in a remarkable reduction of substrate inhibition. Among these variants, L211H showed a significant attenuation of substrate inhibition, with a K-i of 16 mM, which was 16 times that of the native enzyme. The kinetic parameter k(cat)/K-m of L211H was 3.1 x 10(3) s(-1) mM(-1), showing the comparable catalytic efficiency to that of the wild-type enzyme (WT). At the substrate loading of 100 mM, the space time yield of variant L211H in asymmetric reduction of the haloketone was 3-fold higher than that of the WT.