Directed evolution of cytochrome P450DA hydroxylase activity for stereoselective biohydroxylation. Issue 18 (16th August 2022)

• Record Type: Journal Article
• Authors: Wan, Nan-Wei; Cui, Hai-Bo; Zhao, Ling; Shan, Jing; Chen, Ke; Wang, Zhong-Qiang; Zhou, Xiao-Jian; Cui, Bao-Dong; Han, Wen-Yong; Chen, Yong-Zheng
• Is Part Of: Catalysis science & technology. Volume 12:Issue 18(2022)
• Page Start: 5703
• Page End: 5708
• Publication Date: 2022-08-16
• Subjects: Catalysis -- Periodicals; 541.395
• DOI: 10.1039/d2cy00164k
• Languages: English
• ISSNs: 2044-4753

Abstract

Abstract : A colorimetric high throughput screening method was developed based on a dual-enzyme cascade and used for the directed evolution of cytochrome P450 hydroxylase activity. Abstract : Engineering of a hydroxylase for highly active and stereoselective biohydroxylation of C(sp 3 )–H bonds attracts keen interest in synthetic chemistry. Herein, we report the development of a colorimetric high throughput screening assay for the directed evolution of cytochrome P450DA hydroxylase. The best triple-mutant P450DA-M3 (N190F/V356L/A486E) was identified with improvements in the turnover frequency (TOF) and total turnover number (TTN). P450DA-M3 exhibited good catalytic efficiency (up to 6750 TTNs) and stereoselectivity (up to 98% ee) in the biohydroxylation of diverse substrates. The heme domain structure of the P450DA was also solved for understanding the possible influence of these positive mutations.