Temperature-dependent conformational dynamics govern regioselectivity in a CYP152 decarboxylase

Peroxygenases from the CYP152 family catalyze oxidative transformations of fatty acids using hydrogen peroxide and bypass the canonical redox partners. A subset of these enzymes performs fatty acid decarboxylation to generate terminal alkenes, which serve as key intermediates for renewable fuels and petrochemicals. A central unanswered question is how CYP152 enzymes orchestrate the branching between decarboxylation and hydroxylation, a balance that ultimately defines their utility as selective biocatalysts. Here, we uncover a temperature-dependent switch in the peroxygenase from Nosocomiicoccus massiliensis (OleT_NS) that governs its regioselectivity. OleT_NS displays a distinctive temperature-dependent catalytic profile, with enhanced β-regioselectivity at milder temperatures, a behavior not previously reported for other members of the family. Our analyses show that its enhanced chemoselectivity arises from coordinated motions between the F-G loop and catalytic His85, which promote deeper substrate burial and selectively favor decarboxylation. At elevated temperatures, this coordination is disrupted, leading to rearranged hydrogen-bonding networks and decreased alkene yields. The combination of loop flexibility and a charged surface typical of cold-active enzymes provides a layer of catalytic control. OleT_NS establishes a new model for understanding how structural dynamics and thermodynamic adaptation shape the reactivity of P450 enzymes, highlighting principles for engineering selective biocatalysts for sustainable alkene production.