Rationally engineering an H2O2-dependent P450 dihydroxylase for steroid functionalisation

Abstract

P450-catalysed steroid hydroxylation serves as both a fundamental biochemical pathway for in vivo steroid hormone biosynthesis and metabolism, and a pivotal tool for the biotechnological production of steroidal pharmaceuticals. Herein, we report the construction of an efficient H2O2-dependent P450 steroid dihydroxylase through rational engineering of the H2O2 tunnel, guided by molecular dynamics (MD) simulations and crystallographic analysis. The triple mutant F184A/F191A/E196A demonstrated an approximately 80-fold enhancement in catalytic efficiency (kcat/Km) for testosterone hydroxylation compared to wild-type CYP105D18, indicating a dramatic improvement in peroxygenase activity. Testosterone hydroxylation by this mutant predominantly yielded 2β-hydroxytestosterone (81%), with minor 16α-hydroxytestosterone (19%). Notably, the 2β-hydroxylated product could be quantitatively converted to 2β,15α-dihydroxytestosterone in the subsequent reaction. This study provides novel insights into the stepwise design of H2O/H2O2 tunnels in P450 enzymes through the integration of MD simulations and crystallographic data. Furthermore, it establishes a practical enzymatic approach for the regio- and stereoselective dihydroxylation of steroids, with potential applications in pharmaceutical synthesis.

Graphical abstract: Rationally engineering an H2O2-dependent P450 dihydroxylase for steroid functionalisation

Supplementary files

Article information

Article type
Paper
Submitted
28 Apr 2025
Accepted
26 May 2025
First published
05 Jun 2025

Catal. Sci. Technol., 2025, Advance Article

Rationally engineering an H2O2-dependent P450 dihydroxylase for steroid functionalisation

Q. Wang, M. Qin, Q. Wang, K. Wang and Z. Cong, Catal. Sci. Technol., 2025, Advance Article , DOI: 10.1039/D5CY00504C

To request permission to reproduce material from this article, please go to the Copyright Clearance Center request page.

If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given.

If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page.

Read more about how to correctly acknowledge RSC content.

Social activity

Spotlight

Advertisements