文献类型：期刊文献

英文题名：Rational hinge engineering of carboxylic acid reductase from Mycobacterium smegmatis enhances its catalytic efficiency in biocatalysis

作者：Wang, Liuzhu[1];Sun, Yangyang[1];Diao, Shiqing[1];Jiang, Shuiqin[1];Wang, Hualei[1];Wei, Dongzhi[1]

机构：[1]East China Univ Sci & Technol, New World Inst Biotechnol, State Key Lab Bioreactor Engn, 130 Meilong Rd, Shanghai 200237, Peoples R China

年份：2022

卷号：17

期号：2

外文期刊名：BIOTECHNOLOGY JOURNAL

收录：;EI(收录号：20215111368521);WOS:【SCI-EXPANDED(收录号:WOS:000730345100001)】；

基金：This work was supported by the National Natural Science Foundation of China (No. 21776084).

语种：英文

外文关键词：carboxylic acid reductases; hinge region; protein engineering; rational design; virtual mutation

摘要：Background: Carboxylic acid reductases (CARs) represent useful tools for the production of aldehydes from ubiquitous organic carboxylic acids. However, the low catalytic efficiency of these enzymes hampers their application. Methods: Herein, a CAR originating from Mycobacterium smegmatis was redesigned through rational hinge engineering to enhance the catalytic efficiency. Results: Based on the unique domain architecture of CARs and their superfamily, a mutagenesis library of the hinge region was designed. The best mutant R505I/N506K showed a 6.57-fold improved catalytic efficiency. Molecular dynamics simulations showed the increased catalytic efficiency was due to the strong binding of the acyl-AMP complex with it. Meanwhile, the epsilon-nitrogen atom of Lys610 frequently interacted with the ribose-ring oxygen atom of the complex, the distance (d1) between them represents a great indicator for that. The d1 value was used as a nimble indicator to evaluate unexplored mutants of that region for enhanced activity by in silico mutational experiments. Overall, eight mutants were identified to show higher enhanced activity compared with wild-type enzyme and R505F/N506G showed the highest catalytic efficiency. Conclusion: Altogether, the two-step strategy used here provided useful references for the engineering of CARs and other similar multiple-domain enzymes.