Biochemical
Establishment and Characterization of
EncM’s Flavin-N5-oxide Cofactor
Robin Teufel
Frederick Stull
Michael J. Meehan
Quentin Michaudel
Pieter C. Dorrestein
Bruce Palfey
Bradley S. Moore
10.1021/jacs.5b03983.s001
https://acs.figshare.com/articles/journal_contribution/Biochemical_Establishment_and_Characterization_of_EncM_s_Flavin_N5_oxide_Cofactor/2154346
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<sub>N5[O]</sub>), 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<sub>2</sub> (Fl<sub>red</sub>) during substrate turnover. Subsequent
reaction of Fl<sub>red</sub> with molecular oxygen restores the postulated
Fl<sub>N5[O]</sub> via an unknown pathway. Here, we provide direct
evidence for the Fl<sub>N5[O]</sub> 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<sub>red</sub> to molecular oxygen, allowing radical coupling
of the formed protonated superoxide and anionic flavin semiquinone
at N5, before elimination of water affords the Fl<sub>N5[O]</sub> cofactor.
Further biochemical and spectroscopic investigations reveal important
features of the Fl<sub>N5[O]</sub> 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.
2015-07-01 00:00:00
C 4a
Flred
tandem mass spectrometry
flavin semiquinone
Subsequent reaction
spectroscopic investigations
FADH 2
enzyme EncM
Biochemical Establishment
superoxidized flavin
catalyze oxygenation reactions
FlN
protonated superoxide
species
substrate turnover
polyketide antibiotic enterocin