title: 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 (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 FADH2 (Fl(red)) during substrate turnover. Subsequent reaction of Fl(red) 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.
authors.affiliation: †Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California, San Diego, California 92093, United States.
authors.forename: Robin
authors.initials: R
authors.lastname: Teufel
authors.affiliation: ‡Program in Chemical Biology, University of Michigan, Ann Arbor, Michigan 48109, United States.
authors.forename: Frederick
authors.initials: F
authors.lastname: Stull
authors.affiliation: §Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, San Diego, California 92093, United States.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Meehan
authors.affiliation: ⊥Department of Chemistry, The Scripps Research Institute, La Jolla, California 92037, United States.
authors.forename: Quentin
authors.initials: Q
authors.lastname: Michaudel
authors.affiliation: †Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California, San Diego, California 92093, United States.|§Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, San Diego, California 92093, United States.
authors.forename: Pieter C
authors.initials: PC
authors.lastname: Dorrestein
authors.affiliation: ‡Program in Chemical Biology, University of Michigan, Ann Arbor, Michigan 48109, United States.|∥Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States.
authors.forename: Bruce
authors.initials: B
authors.lastname: Palfey
authors.affiliation: †Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California, San Diego, California 92093, United States.|§Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, San Diego, California 92093, United States.
authors.forename: Bradley S
authors.initials: BS
authors.lastname: Moore
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D005415:Flavins / Q000737:chemistry / Q000378:metabolism*; D009602:Nitrosamines / Q000378:metabolism; D010084:Oxidation-Reduction; D010087:Oxides / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D013302:Streptomyces / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D005415:Flavins; D009602:Nitrosamines; D010087:Oxides; C041199:N-nitrosoallyl-2,3-dihydroxypropylamine