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 H2O2 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(SP alpha), P450(BS beta)) 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(SP alpha) and P450(BS beta) regardless of the cis/trans-configuration of substrates. The combined (H2O2)-O-18 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 H2O2 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.

Automated summary

This study analyzed the activities of three CYP152 peroxygenases towards fatty acids, revealing unique selectivity mechanisms and unexpected product formation. Insights into the Compound I-mediated aldehyde formation mechanism provide new understanding of the unusual mechanisms of CYP152 peroxygenases.