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

Abstract

The hydroxylation of nonreactive CÀHb onds can be easily catalyzed by av ariety of metalloenzymes, especially cytochrome P450s (P450s). The mechanism of P450 mediated hydroxylation has been intensively studied, both experimentally and theoretically.H owever,u nderstandingt he regio-and stereoselectivities of substrates hydroxylated by P450s remains ag reat challenge. Herein, we use am ultiscale modeling approach to investigatet he selectivity of testosterone (TES) and dihydrotestosterone (DHT)h ydroxylation catalyzed by two important P450s,C YP3A4 and CYP19A1. For CYP3A4, two distinct binding modes for TES/DHTw ere predicted by dockings and molecular dynamics simulations, in whicht he experimentally identified sites of metabolism of TES/DHTc an access to the catalytic center.T he regio-and stereoselectivities of TES/DHTh ydroxylation were further evaluated by quantum mechanical andO NIOM calculations. For CYP19A1, we found that sites 1b,2 b and 19 can access the catalytic center, with the intrinsic reactivity 2b > 1b > 19. However, ourO NIOM calculations indicate that the hydroxylation is favored at site 19 for both TES and DHT,w hich is consistentw ith the experiments and reflects the importance of the catalytic environment in determining the selectivity. Our study unravels the mechanism underlyingt he selectivity of TES/DHT hydroxylationm ediated by CYP3A4 and CYP19A1a nd is helpful for understanding the selectivity of other substrates that are hydroxylatedb yP450s.[a] J.

Homotropic Cooperativity of Midazolam Metabolism by Cytochrome P450 3A4: Insight from Computational Studies

“…The protein models of CYP3A4 were selected from 11 crystal structures, including 1TQN, 40 2V0M, 17 3NXU, 17 3UA1, 41 4D78, 19 4I4G, 42 4K9T, 18 4K9V, 18 4K9W, 18 5TE8, 27 and 5VC0, 43 which were prepared in our previous study. 44 For the docking of the first MDZ molecule, the oxo atom in Cpd I was adopted as the center of a sphere with radius of 15 Å for defining the binding pocket. For the docking of the second MDZ, the centroid of the first docked MDZ was deemed as the center of the binding pocket.…”

“…The initial binding modes of MDZ at the productive site of CYP3A4 were obtained using the CCDC GOLD Suite 5.2.2 program. The protein models of CYP3A4 were selected from 11 crystal structures, including 1TQN, 2V0M, 3NXU, 3UA1, 4D78, 4I4G, 4K9T, 4K9V, 4K9W, 5TE8, and 5VC0, which were prepared in our previous study . For the docking of the first MDZ molecule, the oxo atom in Cpd I was adopted as the center of a sphere with radius of 15 Å for defining the binding pocket.…”

Regio‐ and stereoselective steroid hydroxylation by CYP109A2 from Bacillus megaterium explored by X‐ray crystallography and computational modeling

“…The second, more typical set of relaxed NAC criteria was the same as reported by Li et al . [36]. Like in most sets of published NAC criteria for P450s, the allowed ranges of angles in the second set are wider and the allowed distances significantly exceed the van der Waals contact distances (see also for instance [37]).…”

Investigation of the molecular and mechanistic basis for the regioselective metabolism of midazolam by cytochrome P450 3A4

“…31 Finally, the compound I (Cpd I) form of heme was manually prepared with a single oxygen atom bound to the heme iron at B1.65 Å. 14,32 2.1.2. Single-and multiple-ligand complexes.…”

“…CYP3A4 often exhibits unusual kinetic characteristics, and both cooperativity (homotropic and heterotropic cooperativity) and allosteric effects have been reported to stimulate or inhibit metabolism, cause regiospecific oxidation and drugdrug interaction. [10][11][12][13][14] The metabolic characteristic of CYP3A4 is one of the most concerning but still controversial subjects in P450 research. Midazolam (MDZ) is a sensitive molecular probe for CYP3A4, and it is one of the most lipid soluble of benzodiazepines at physiologic pH.…”