Semirationally Engineering an Efficient P450 Peroxygenase for Regio- and Enantioselective Hydroxylation of Steroids

• 全文链接

半合理地设计一种用于类固醇区域和对映选择性羟基化的高效P450过氧酶

关键词：

来源：

ACS Catalysis

全文链接：

//agri.nais.net.cn/topic/downloadFile/34cafa17-da59-43c1-8975-dd7f707cf0a4

来源地址：

https://pubs.acs.org/doi/10.1021/acscatal.4c06342

资源所属：

饲料用酶工程

类型：

学术文献

语种：

英语

原文发布日期：

2025-02-05

摘要：

Enzymatic direct hydroxylation of unactivated C–H bonds in steroids provides a promising approach to enrich their structural and functional diversity, together with higher physiological and pharmacological activity. Here, we construct an efficient peroxide-driven P450 hydroxylase for the regio- and enantioselective hydroxylation of steroids. The NADH-dependent CYP154C5 monooxygenase is smoothly transformed into its peroxygenase mode by combining the strategies of H2O2 tunnel engineering and the introduction of a catalytic aspartate residue, which avoids the use of expensive nicotinamide cofactors and redox partner proteins. The variant F92A/R114A/E282A/T248D (AAA/T248D) quantitatively converted testosterone and nandrolone into the corresponding 16α-hydroxylation products, showing the best catalytic efficiency (kcat/Km) for testosterone hydroxylation among all known natural and engineered P450 peroxygenases to date. Crystal structural analysis and molecular dynamics simulations suggest that H2O2 tunnel engineering plays a crucial role in promoting the flow of H2O2 into active centers, and the introduced aspartate residue may participate in the activation of H2O2. Moreover, the milligram-scale preparation of 16α-hydroxytestosterone by AAA/T248D gave a substrate conversion rate (>98%) and an isolated yield (90%), suggesting potential for synthetic application. This work not only establishes a feasible semirational approach to engineered non-natural P450 peroxygenases but also provides a potentially practical approach for the enzymatic synthesis of hydroxylated steroid compounds.