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

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 (OleTJE , P450SPα , P450BSβ ) 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 OleTJE , and molecular docking results suggest that the unprecedented selectivity is due to OleTJE 's preference of C2-C3 cis-configuration. In addition to the common epoxide products, undecanal is the unexpected major product of P450SPα and P450BSβ 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.