Flavin-mediated dual oxidation controls an enzymatic Favorskii-type rearrangement

Flavoproteins catalyze a diversity of fundamental redox reactions and are one of the most studied enzyme families(1,2). As monooxygenases, they are universally thought to control oxygenation by means of a peroxyflavin species that transfers a single atom of molecular oxygen to an organic substrate(1,3,4). Here we report that the bacterial flavoenzyme EncM(5,6) catalyzes the peroxyflavin-independent oxygenation-dehydrogenation dual oxidation of a highly reactive poly(β-carbonyl). The crystal structure of EncM with bound substrate mimics coupled with isotope labeling studies reveal previously unknown flavin redox biochemistry. We show that EncM maintains an unanticipated stable flavin oxygenating species, proposed to be a flavin-N5-oxide, to promote substrate oxidation and trigger a rare Favorskii-type rearrangement that is central to the biosynthesis of the antibiotic enterocin. This work provides new insight into the fine-tuning of the flavin cofactor in offsetting the innate reactivity of a polyketide substrate to direct its efficient electrocyclization.