文献类型：期刊文献

英文题名：Rational Engineering of Formate Dehydrogenase Substrate/Cofactor Affinity for Better Performance in NADPH Regeneration

作者：Jiang, He-Wen[1];Chen, Qi[1];Pan, Jiang[1];Zheng, Gao-Wei[1];Xu, Jian-He[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：2020

卷号：192

期号：2

起止页码：530

外文期刊名：APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY

收录：;EI(收录号：20202108688217);WOS:【SCI-EXPANDED(收录号:WOS:000532635000001)】；

基金：This work was financially supported by the National Natural Science Foundation of China (Nos. 21536004, 21871085, and 21878085), the National Key Research and Development Program of China (No. 2018YFC1706200), and the Fundamental Research Funds for the Central Universities (No. 22221818014).

语种：英文

外文关键词：Binding affinity; Burkholderia stabilis; Formate dehydrogenase; NADPH regeneration; Rational engineering

摘要：Formate dehydrogenases are critical tools for nicotinamide cofactor regeneration, but their limited catalytic efficiency (k(cat)/K-m) is a major drawback. A formate dehydrogenase from Burkholderia stabilis 15516 (BstFDH) was the first native NADP(+)-dependent formate dehydrogenase reported and has the highest k(cat)/K-m toward NADP(+) (k(cat)/K-m(NADP+)) compared with other FDHs that can utilize NADP(+) as a hydrogen acceptor. However, the substrate and cofactor affinities of BstFDH are inferior to those of other FDHs, making its practical application difficult. Herein, we engineered recombinant BstFDH to enhance its HCOO- and NADP(+) affinities. Based on sequence information analysis and homologous modeling results, I124, G146, S262, and A287 were found to affect the binding affinity for HCOO- and NADP(+). By combining these mutations, we identified a BstFDH variant (G146M/A287G) that reduced K-m(NADP+) to 0.09 mM, with a concomitant decrease in K-m(HCOO-), and gave 1.6-fold higher k(cat)/K-m(NADP+) than the wild type (WT). Furthermore, BstFDH I124V/G146H/A287G, with the lowest K-m(HCOO-) of 8.51 mM, showed a catalytic efficiency that was 2.3-fold higher than that of the wild type and a decreased K-m(NADP+) of 0.11 mM. These results are beneficial for improving the performance of NADP(+)-dependent formate dehydrogenase in the NADPH regeneration of various bioreductive reactions and provide a useful guide for engineering of the substrate and cofactor affinity of other enzymes.