文献类型：期刊文献

英文题名：Switching Cofactor Dependence of 7β-Hydroxysteroid Dehydrogenase for Cost-Effective Production of Ursodeoxycholic Acid

作者：You, Zhi-Neng[1];Chen, Qi[1,2];Shi, Shou-Cheng[1];Zheng, Ming-Min[1];Pan, Jiang[1,2];Qian, Xiao-Long[3];Li, Chun-Xiu[1,2];Xu, Jian-He[1,2]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, Lab Biocatalysis & Synthet Biotechnol, 130 Meilong Rd, Shanghai 200237, Peoples R China;[2]East China Univ Sci & Technol, Shanghai Collaborat Innovat Ctr Biomfg, Sch Biotechnol, 130 Meilong Rd, Shanghai 200237, Peoples R China;[3]Suzhou Bioforany EnzyTech Co Ltd, 8 Yanjiuyuan Rd, Changshu 215512, Jiangsu, Peoples R China

年份：2019

卷号：9

期号：1

起止页码：466

外文期刊名：ACS CATALYSIS

收录：;EI(收录号：20190206361900);WOS:【SCI-EXPANDED(收录号:WOS:000455286600048)】；

基金：This work was financially supported by The National Key Research and Development Program of China (No. SQ2018YFC170017), The National Natural Science Foundation of China (Nos. 21536004, 21871085, and 31500592), the Fundamental Research Funds for the Central Universities (No. 22221818014) and Shanghai Commission of Science and Technology (No. 11431921600). We thank Simon Partridge, Ph.D., from Liwen Bianji, Edanz Editing China, for editing the English text of this manuscript.

语种：英文

外文关键词：biocatalysis; cofactor dependence; small-and-smart library; 7 beta-hydroxysteroid dehydrogenase; molecular dynamics simulation; ursodeoxycholic acid

摘要：Dehydrogenases are widely employed as bio-catalysts for the production of optically pure chemicals under mild conditions. Most dehydrogenases are nicotinamide cofactor (NADPH or NADH)-dependent oxidoreductases. 7 beta-Hydroxysteroid dehydrogenase (7 beta-HSDH) is a key enzyme for the biochemical synthesis of ursodeoxycholic acid (UDCA). To date, all reported 7 beta-HSDHs are strictly NADPH-dependent enzymes. However, compared with NADPH, NADH is much more economical, making it the preferential cofactor for synthetic applications of dehydrogenases. In this work, a recombinant 7 beta-HSDH originating from Ruminococcus torques was rationally engineered to alter its cofactor dependence using a strategy referred to as Cofactor Specificity Reversal: Small and-Smart Library Design (CSR-SaSLiD), which is based on structural information and conservative sequence alignment. We rationally designed a small-and-smart library containing only five mutants that enabled the quick identification of target variants. Compared with the wild type, the resultant mutant, G39D, showed a 953 000-fold switch in cofactor specificity from NADPH to NADH, and another mutant, G39D/T17A, resulted in 223-fold enhanced activity with NADH. The structural mechanism regarding the effect of mutation on the reversal of cofactor preference and improvement of catalytic activity was elucidated with the aid of molecular dynamics simulation. Furthermore, it was confirmed that the CSR-SaSLiD strategy can be extended to other 7 beta-HSDHs. This work provides an efficient approach to altering cofactor preference and subsequently recovering the enzymatic activity of dehydrogenases for cost-effective biotechnical applications.