Mechanistic Insights into the Regio‐ and Stereoselectivities of Testosterone and Dihydrotestosterone Hydroxylation Catalyzed by CYP3A4 and CYP19A1

The hydroxylation of nonreactive C−H bonds can be easily catalyzed by a variety of metalloenzymes, especially cytochrome P450s (P450s). The mechanism of P450 mediated hydroxylation has been intensively studied, both experimentally and theoretically. However, understanding the regio‐ and stereoselectivities of substrates hydroxylated by P450s remains a great challenge. Herein, we use a multi‐scale modeling approach to investigate the selectivity of testosterone (TES) and dihydrotestosterone (DHT) hydroxylation catalyzed by two important P450s, CYP3A4 and CYP19A1. For CYP3A4, two distinct binding modes for TES/DHT were predicted by dockings and molecular dynamics simulations, in which the experimentally identified sites of metabolism of TES/DHT can access to the catalytic center. The regio‐ and stereoselectivities of TES/DHT hydroxylation were further evaluated by quantum mechanical and ONIOM calculations. For CYP19A1, we found that sites 1β, 2β and 19 can access the catalytic center, with the intrinsic reactivity 2β>1β>19. However, our ONIOM calculations indicate that the hydroxylation is favored at site 19 for both TES and DHT, which is consistent with the experiments and reflects the importance of the catalytic environment in determining the selectivity. Our study unravels the mechanism underlying the selectivity of TES/DHT hydroxylation mediated by CYP3A4 and CYP19A1 and is helpful for understanding the selectivity of other substrates that are hydroxylated by P450s.