Unexpected Reactions of α,β-Unsaturated Fatty Acids Provide Insight into the Mechanisms of CYP152 Peroxygenases

Abstract

CYP152 peroxygenases catalyze decarboxylation and hydroxylation of fatty acids using H₂ O₂ as cofactor. To understand the molecular basis for the chemo- and regioselectivity of these unique P450 enzymes, we analyze the activities of three CYP152 peroxygenases (OleT_JE , P450_SPα , P450_BSβ ) towards cis- and trans-dodecenoic acids as substrate probes. The unexpected 6S-hydroxylation of the trans-isomer and 4R-hydroxylation of the cis-isomer by OleT_JE , and molecular docking results suggest that the unprecedented selectivity is due to OleT_JE 's preference of C2-C3 cis-configuration. In addition to the common epoxide products, undecanal is the unexpected major product of P450_SPα and P450_BSβ regardless of the cis/trans-configuration of substrates. The combined H₂¹⁸ O₂ tracing experiments, MD simulations, and QM/MM calculations unravel an unusual mechanism for Compound I-mediated aldehyde formation in which the active site water derived from H₂ O₂ activation is involved in the generation of a four-membered ring lactone intermediate. These findings provide new insights into the unusual mechanisms of CYP152 peroxygenases.

Keywords: CYP152 peroxygenases; QM/MM calculations; molecular dynamics simulations; substrate probes; α,β-unsaturated fatty acids.