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Biochemical establishment and characterization of EncM's flavin-N5-oxide cofactor

TitleBiochemical establishment and characterization of EncM's flavin-N5-oxide cofactor
Publication TypeJournal Article
Year of Publication2015
AuthorsTeufel R., Stull F., Meehan M.J, Michaudel Q., Dorrestein PC, Palfey B., Moore BS
JournalJournal of the American Chemical Society
Date Published2015/07
Type of ArticleArticle
ISBN Number0002-7863
Accession NumberWOS:000357436300028
Keywordsbiosynthesis; flavin-dependent monooxygenases; flavoproteins; oxidase; oxidation; oxygen; para-hydroxybenzoate hydroxylase; polyketide synthase; radiation-damage; rearrangement

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 (Fl(N5[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 FADH(2) (Fl(red)) during substrate turnover. Subsequent reaction of Flred with molecular oxygen restores the postulated Fl(N5[O]) via an unknown pathway. Here, we provide direct evidence for the Fl(N5[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 Fl(red) to molecular oxygen, allowing radical coupling of the formed protonated superoxide and anionic flavin semiquinone at N5, before elimination of water affords the Fl(N5[O]) cofactor. Further biochemical and spectroscopic investigations reveal important features of the Fl(N5[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.

Short TitleJ. Am. Chem. Soc.
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