Mechanism of Regio- and Enantioselective Hydroxylation of Arachidonic Acid Catalyzed by Human CYP2E1: A Combined Molecular Dynamics and Quantum Mechanics/Molecular Mechanics Study

Regio- and enantioselective hydroxylation of free fatty acids by human cytochrome P450 2E1 (CYP2E1) plays an important role in metabolic regulation and has significant pathological implications. Despite extensive research, the detailed hydroxylation mechanism of CYP2E1 remains incompletely understood. To clarify the origins of regioselectivity and enantioselectivity observed for CYP2E1-mediated fatty acid hydroxylation, molecular dynamics (MD) simulations and quantum mechanics/molecular mechanics (QM/MM) calculations were performed. MD simulations provided key insights into the proximity of arachidonic acid’s carbon atoms to the reactive iron(IV)-oxo moiety in compound I (Cpd I), with the ω-1 position being closest, indicating higher reactivity at this site. QM/MM calculations identified hydrogen abstraction as the rate-determining step, with the ω-1S transition state exhibiting the lowest energy barrier, consistent with experimentally observed enantioselectivity. Energy decomposition analysis revealed that variations in quantum mechanical energy (ΔEQM) significantly influence reaction barriers, with the most efficient hydrogen abstraction occurring at the ω-1S and ω-2R positions. These findings underscore the importance of substrate positioning within the active site in determining product selectivity. Comparisons with two related P450s, P450BM3 and P450SPα, further highlighted the critical role of active site architecture and substrate positioning in modulating selectivity. While surrounding residues do not directly dictate product selectivity, they shape the active site environment and influence substrate positioning. Furthermore, our analysis revealed a previously unrecognized catalytic role of Ala299. These findings provide a deeper mechanistic understanding of human CYP2E1 and offer valuable insights for its precise engineering in targeted C–H functionalization.