Teufel Robin, Stull Frederick, Meehan Michael J, Michaudel Quentin, Dorrestein Pieter C, Palfey Bruce, Moore Bradley S
†Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California, San Diego, California 92093, United States.
‡Program in Chemical Biology, University of Michigan, Ann Arbor, Michigan 48109, United States.
J Am Chem Soc. 2015 Jul 1;137(25):8078-85. doi: 10.1021/jacs.5b03983. Epub 2015 Jun 19.
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.
普遍存在的黄素依赖性单加氧酶通常通过瞬时的C4a-过氧黄素催化氧化反应。然而，最近的一项研究表明，一种前所未有的黄素氧化物种，被认为是黄素-N5-氧化物（Fl(N5[O])），是细菌聚酮类抗生素肠菌素生物合成中氧化Favorskii型重排的关键。这种稳定的超氧化黄素与酶EncM共价连接，并在底物周转过程中转化为FADH2（Fl(red)）。随后，Fl(red)与分子氧反应，通过未知途径恢复假定的Fl(N5[O])。在这里，我们通过对EncM进行同位素标记、蛋白酶消化和高分辨率串联质谱分析，为Fl(N5[O])物种提供了直接证据。我们提出，该物种的形成是通过从Fl(red)向分子氧的氢转移实现的，使得形成的质子化超氧化物和N5处的阴离子黄素半醌发生自由基偶联，然后消除水生成Fl(N5[O])辅因子。进一步的生化和光谱研究揭示了Fl(N5[O])物种和EncM催化机制的重要特征。我们推测，黄素-N5-氧化物可能是其他黄素蛋白中的中间体或催化活性物种，这些黄素蛋白形成阴离子半醌并促进氧进入N5。
