Preparation and Characterization of the Favorskiiase Flavoprotein EncM and Its Distinctive Flavin-N5-Oxide Cofactor

Flavoenzymes often function as oxygenases and have been extensively studied for many decades. Commonly, oxygenation reactions are mediated by a transient C4a-peroxyflavin formed from reaction of reduced flavin with O₂. EncM, however, employs a previously unrecognized flavin-oxygenating species, the flavin-N5-oxide, which is key to a complex oxidative Favorskii-type rearrangement and cyclization cascade in the biosynthesis of the bacterial polyketide antibiotic enterocin produced by the marine bacterium Streptomyces maritimus. Here, the methodology and key experiments are described that led to the discovery of this novel flavin redox species and granted insight into one of the most astounding single-enzyme-catalyzed reaction cascades in natural product biosynthesis.