Abstract
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.
Highlights
The human cytochrome P450 superfamily includes 57 isoforms of heme-enclosed enzymes that catalyze the redox reactions of a variety of endogenous and exogenous compounds.[1]
Hydroxylation reactions catalyzed by P450s are important in phase I metabolism.[1b]. Studying the mechanisms of the regio- and stereoselectivities of the hydroxylation reactions is beneficial for understanding the occurrence of reactive phase I metabolites and for finding ways to avoid the occurrence.[1b]. For instance, by studying the regioselectivity of tienilic acid hydroxylation, which occurs at site 5, the mechanism for the occurrence of the reactive metabolites was disclosed.[6]
From the quantum mechanics (QM) calculations, we found that all the b sites are more reactive than the corresponding a sites, which explains the stereo-selectivity of TES hydroxylation by CYP3A4
Summary
Mechanism leading to regio- and stereoselective P450 mediated hydroxylation are still not clear.[4d,5]. As an important case,[3c,8] the mechanism of selective hydroxylation reactions of steroid substrates catalyzed by two P450 isoforms, CYP3A4 and CYP19A1, is still unclear. We focus on the hydroxylation of two important steroid molecules, testosterone (TES) and dihydrotestosterone (DHT), catalyzed by CYP3A4 and CYP19A1. We used a multi-scale modeling approach, which involves molecular dockings, molecular dynamics (MD) simulations, quantum mechanics (QM) calculations, and quantum mechanics/molecular mechanics (QM/MM) calculations, to investigate the mechanism underlying the selective hydroxylation of TES and DHT by CYP3A4 and CYP19A1. The accessibility of each potential site of metabolism (SOM) to an enzyme’s reaction center was evaluated by 100-ns long MD simulations. Together with the QM/MM calculations, our results unravel the mechanism underlying the selectivity of hydroxylation of TES and DHT by CYP3A4/CYP19A1
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