A nucleobase-driven P450 peroxidase system enables regio- and stereo-specific formation of C─C and C─N bonds.

P450 peroxidase activities are valued for their ability to catalyze complex chemical transformations using economical HO; however, they have been largely underexplored compared to their monooxygenase and peroxygenase activities. In this study, we identified an unconventional P450 enzyme, PtmB, which catalyzes the dimerization of purine nucleobases and tryptophan-containing diketopiperazines (TDKPs), yielding C3-nucleobase pyrroloindolines and nucleobase-TDKP dimers. Unlike typical TDKP P450 enzymes reliant on NAD(P)H cofactors and electron transfer systems, PtmB, and its analogs exhibit remarkable peroxidase activity in synthesizing adenine and other modified 6-aminopurine nucleobase-TDKP dimers. Structural analysis of the PtmB-substrate complex, mutation assays, and computational investigations reveal adenine's dual role as both substrate and acid-base catalyst in activating HO to generate Compound I (Cpd I). This initiates a specific radical cascade reaction, facilitating the formation of precise C─C and C─N bonds. Biochemical assays and molecular dynamics simulations demonstrate that adenine's 6-NH hydrogen-bonding networks induce necessary conformational changes for HO activation, thereby driving peroxidase activity. This study unveils an unusual catalytic mechanism for the P450 peroxidase system and underscores the pivotal role of nucleobases in enzyme-mediated reactions, which offers different prospects for developing P450 peroxidases and nucleobase-based biocatalysts.