Regio- and Enantioselective Oxidation of Eicosapentaenoic and Arachidonic Acids by Human CYP2E1: A Comparative...

Human CYP2E1 oxidizes both eicosapentaenoic acid (EPA, an ω-3 polyunsaturated fatty acid [PUFA]) and arachidonic acid (AA, an ω-6 PUFA) to bioactive lipid mediators, yet the mechanistic basis for its regio- and enantioselective preferences remains incompletely understood. Here, we use a combined molecular dynamics (MD) and hybrid quantum mechanics/molecular mechanics (QM/MM) strategy to compare the oxidation of EPA and AA by human CYP2E1 at atomistic resolution. MD simulations indicate that EPA and AA occupy essentially the same CYP2E1 cavity but are stabilized through distinct interaction patterns. Compared with AA, EPA more frequently forms a hydrogen bond with A108 at the channel entrance and exhibits more uniform sampling of near-attack geometries in the ω-1, ω-2, and ω-3 regions. QM/MM free energy profiles show that ω-1S hydroxylation has the lowest barrier for both substrates, but that the barriers for the ω-2/ω-3 epoxidation pathways are close to that for ω-1S hydroxylation in EPA, rendering these pathways kinetically competitive. Energy decomposition analysis indicates that regioselectivity is largely determined by the QM energy term (ΔEQM), whereas enantioselectivity at ω-1 reflects small differences in packing and entropic cost between the pro-R and pro-S transition states in the confined active site. These results provide a coherent mechanistic picture of CYP2E1-catalyzed oxidation of ω-6 and ω-3 PUFAs and identify specific active-site residues as promising targets for tuning regio- and stereoselectivity in lipid oxidation.