Computational Study on Key Factors Controlling the Regioselectivity of the CYP11A1-Catalyzed Sequential Hydroxylations of Cholesterol

Cytochrome P450 enzyme CYP11A1 is a key enzyme in the biosynthesis of pregnenolone; however, the detailed catalytic mechanism, including the underlying factors controlling regioselectivity in the highly sequential hydroxylation reactions, remains unclear. Here, we combined molecular dynamics (MD) simulations and quantum mechanical/molecular mechanical (QM/MM) calculations to investigate the regioselective hydroxylation reactions catalyzed by CYP11A1. Our results indicate that the enzyme controls the hydroxylation site at different stages of the catalytic cycle depending on the substrate. For cholesterol (CH) hydroxylation, the regioselectivity is determined in the Cpd 0/substrate complex rather than in the Cpd I/substrate complex. Meanwhile, for the hydroxylation of the generated 22R-hydroxycholesterol (22R-OHCH), the hydrogen-bonding interaction between the C22−OH of the substrate and the oxygen of Cpd I plays an important role in the selective hydroxylation of the C20 site and in promoting the adjustment of the substrate binding mode. In addition, a competing side reaction pathway for the hydroxylation of 22R-OHCH is observed, in which the H-abstraction from the C22−OH group initiates C22−C20 bond cleavage and is followed by oxygen rebound. Overall, these results provide insights into the reaction mechanism of CYP11A1-catalyzed CH hydroxylation reactions and highlight the importance of the Cpd 0 species in understanding the catalytic selectivity of P450s and in the rational design of selective P450 enzymes.