Biochemical Establishment and Characterization of EncM’s Flavin-N5-oxide Cofactor
2015-07-01T00:00:00Z (GMT) by
The ubiquitous flavin-dependent monooxygenases commonly catalyze oxygenation reactions by means of a transient C4a–peroxyflavin. A recent study, however, suggested an unprecedented flavin-oxygenating species, proposed as the flavin-N5-oxide (FlN5[O]), as key to an oxidative Favorskii-type rearrangement in the biosynthesis of the bacterial polyketide antibiotic enterocin. This stable superoxidized flavin is covalently tethered to the enzyme EncM and converted into FADH2 (Flred) during substrate turnover. Subsequent reaction of Flred with molecular oxygen restores the postulated FlN5[O] via an unknown pathway. Here, we provide direct evidence for the FlN5[O] species via isotope labeling, proteolytic digestion, and high-resolution tandem mass spectrometry of EncM. We propose that formation of this species occurs by hydrogen-transfer from Flred to molecular oxygen, allowing radical coupling of the formed protonated superoxide and anionic flavin semiquinone at N5, before elimination of water affords the FlN5[O] cofactor. Further biochemical and spectroscopic investigations reveal important features of the FlN5[O] species and the EncM catalytic mechanism. We speculate that flavin-N5-oxides may be intermediates or catalytically active species in other flavoproteins that form the anionic semiquinone and promote access of oxygen to N5.
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