Biochemical Establishment and Characterization of EncM’s Flavin-N5-oxide Cofactor

Abstract

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.

Oxidative Cyclization in Natural Product Biosynthesis

“…Mechanistic and structural examination of the key EncM catalyst revealed the first example of an N 5 -oxide of FAD ( 548 ) as an on pathway oxygen transfer agent (Scheme 77B). 395,396 …”

The Enzymology of Organic Transformations: A Survey of Name Reactions in Biological Systems

“…Interestingly, spectroscopic data suggests that flavin-N 5 -oxide, rather than flavin C 4a -peroxide, is the oxygen donor adding oxygen to C-4 of the substrate. [273] …”

Sweating the assets of flavin cofactors: new insight of chemical versatility from knowledge of structure and mechanism

“…The unexpected oxidative chemistry catalyzed by EncM and the methylene transfer chemistry proposed to occur in TrmFO and ThyX serve to illustrate that the flavin N5 position is an equal asset (compared to the C4a locus) in terms of enzyme catalysis. In EncM, the superoxidized N5-oxide species is proposed to be formed by hydrogen transfer from the reduced flavin to molecular oxygen, followed by radical coupling of the anionic semiquinone N5 with the protonated superoxide [20 ]. Elimination of water from the resultant N5-peroxide is postulated to lead to the N5-oxide species.…”