Resurrecting Robust CYP152 Peroxygenases for Versatile Biocatalysis

ABSTRACT Cytochrome P450 CYP152s are attractive biocatalysts which catalyze decarboxylation and hydroxylation of fatty acids using H 2 O 2 as a co‐substrate (peroxygenase activity). Contemporary CYP152s have limited stability and substrate scope. Here we characterized eight reconstructed variants of CYP152 obtained using ancestral sequence reconstruction (ASR). The results showed that variants remote from Jeotgalicoccus sp. 8456 (CYP152L1) in the tree used for the ASR tended to catalyze hydroxylation, while nodes closer to CYP152L1 favored decarboxylation; this transition correlates with Q85H substitution and FG‐loop insertion alongside other structural features. Three reconstructed variants exhibit high T m (66°C–76°C), tolerance to solvents, H 2 O 2 , extreme pH, and use non‐native substrates. CYP152‐AncG2 can catalyze one‐pot chemoenzymatic C─C bond coupling reactions to synthesize methoxy stilbene and can serve as a template for directed evolution. X‐ray structure at 1.83 Å and molecular dynamics (MD) simulations indicate that loop structures, surface salt bridges, and optimized access tunnels in CYP152‐AncG2 are key for enabling its superior features. These findings provide insights into the structural and evolutionary features of robust peroxygenase templates for expanding peroxygenase biocatalysis.