Structural and functional studies reveal how the bacterial flavoenzyme EncM catalyses the oxygenation–dehydrogenation dual oxidation of a highly reactive substrate, and show that EncM maintains a stable flavin oxygenating species that promotes substrate oxidation and triggers a rarely seen Favorskii-type rearrangement. Flavoproteins, which contain either a flavin adenine dinucleotide or a flavin mononucleotide cofactor, are redox-active proteins involved in a broad range of biological processes including bioluminescence, photosynthesis and DNA repair. Here the authors undertook structural and functional studies to examine how the bacterial flavoenzyme EncM catalyses the oxygenation–dehydrogenation oxidation of a highly reactive substrate. They observed previously unknown flavin redox biochemistry: EncM maintains a stable flavin-oxygenating species that promotes substrate oxidation and triggers a rarely seen, Favorskii-type rearrangement that is central to the biosynthesis of the marine antibiotic enterocin. Flavoproteins catalyse a diversity of fundamental redox reactions and are one of the most studied enzyme families1,2. 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 substrate1,3,4. Here we report that the bacterial flavoenzyme EncM5,6 catalyses the peroxyflavin-independent oxygenation–dehydrogenation dual oxidation of a highly reactive poly(β-carbonyl). The crystal structure of EncM with bound substrate mimics and isotope labelling studies reveal previously unknown flavin redox biochemistry. We show that EncM maintains an unexpected 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.

Para -hydroxybenzoate hydroxylase inserts oxygen into substrates by means of the labile intermediate, flavin C(4a)-hydroperoxide. This reaction requires transient isolation of the flavin and substrate from the bulk solvent. Previous crystal structures have revealed the position of the substrate para -hydroxybenzoate during oxygenation but not how it enters the active site. In this study, enzyme structures with the flavin ring displaced relative to the protein were determined, and it was established that these or similar flavin conformations also occur in solution. Movement of the flavin appears to be essential for the translocation of substrates and products into the solvent-shielded active site during catalysis.