Rationally Controlling Selective Steroid Hydroxylation via Scaffold Sampling of a P450 Family

被引：96

作者：

Zhang, Xiaodong ^{[1
]}

Shen, Panpan ^{[1
]}

Zhao, Jing ^{[1
]}

Chen, Yueyue ^{[1
]}

Li, Xian ^{[1
]}

Huang, Jian-Wen ^{[1
]}

Zhang, Lilan ^{[1
]}

Li, Qian ^{[1
]}

Gao, Chenghua ^{[1
]}

Xing, Qiong ^{[1
]}

Chen, Chun-Chi ^{[1
]}

Guo, Rey-Ting ^{[1
]}

Li, Aitao ^{[1
]}

机构：

[1] Hubei Univ, Sch Life Sci, State Key Lab Biocatalysis & Enzyme Engn, Hubei Key Lab Ind Biotechnol, Wuhan 430062, Peoples R China

来源：

ACS CATALYSIS | 2023年 / 13卷 / 02期

基金：

中国国家自然科学基金;

关键词：

P450; monooxygenase; steroids; directed evolution; iterative saturation mutagenesis; regioselectivity; stereoselectivity;

D O I：

10.1021/acscatal.2c04906

中图分类号：

O64 [物理化学（理论化学）、化学物理学];

学科分类号：

070304 ; 081704 ;

摘要：

Many steroids are important pharmaceutically active compounds, while cytochrome P450 monooxygenases (CYPs) are attractive enzymes for applications in steroidal drug synthesis. However, the catalytic efficiency of existing P450s is not routinely high enough, as well as the molecular basis for selectivity control is unclear, which severely restrict their real applications. Here, a 16 beta steroid-hydroxylase CYP109B4 from Bacillus sonorensis is identified with excellent selectivity and activity. The crystallization and structural analysis of CYP109B4 reveal potential three "hotspot" residues (V84, V292, and S387) responsible for selectivity control. Then, guided by the sequence-function relationships revealed from the mutability landscape construction on the three residues, focused rational iterative site-specific mutagenesis (FRISM) and limited iterative saturation mutagenesis were performed, which provide variant B4-M7 (L240V/S387F/V84L/V292S/I291T/M290F/F294I) with completely switched regioselectivity from 16 beta to 15 beta. The subsequent computational analysis uncovers insights into the substrate binding modes in CYP109B4 and its variants, which further confirms the critical role of the "hotspot" residues for selectivity control. Finally, the generality of conserved-"hotspots"-mediated selectivity control is demonstrated by performing scaffold sampling between a panel of CYP109B members. Overall, in addition to the present chemical results, our study provides guidance in rationally designing more excellent P450 biocatalysts for potential practical (industrial) applications.

引用

页码：1280 / 1289

页数：10

共 40 条

[1] P450-Catalyzed Regio- and Diastereoselective Steroid Hydroxylation: Efficient Directed Evolution Enabled by Mutability Landscaping [J].