One of the hallmark reactions catalyzed by flavin-dependent enzymes is the incorporation of an oxygen atom derived from dioxygen into organic substrates. For many decades, these flavin monooxygenases were assumed to use exclusively the flavin-C4a-(hydro)peroxide as their oxygen-transferring intermediate. We demonstrate that flavoenzymes may instead employ a flavin-N5-peroxide as a soft α-nucleophile for catalysis, which enables chemistry not accessible to canonical monooxygenases. This includes, for example, the redox-neutral cleavage of carbon-hetero bonds or the dehalogenation of inert environmental pollutants via atypical oxygenations. We furthermore identify a shared structural motif for dioxygen activation and N5-functionalization, suggesting a conserved pathway that may be operative in numerous characterized and uncharacterized flavoenzymes from diverse organisms. Our findings show that overlooked flavin-N5-oxygen adducts are more widespread and may facilitate versatile chemistry, thus upending the notion that flavin monooxygenases exclusively function as nature’s equivalents to organic peroxides in synthetic chemistry.
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All data generated or analyzed during this study are included in this published article (and its Supplementary information files). The structures for proteins described in this paper have been deposited in the PDB. The PDB accession codes are 6TEE for RutA under anaerobic conditions, 6TEF for RutA pressurized with 5 bars O2, 6SGG for RutA pressurized with 15 bars O2, 6SGL for RutA in complex with uracil, 6SGM for RutA in complex with 4-thiouracil, 6SGN for RutA in complex with 2,4-dimethoxy pyrimidine and 6TEG for RutA in complex with uracil pressurized with 15 bars O2.
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This work was supported by the Deutsche Forschungsgemeinschaft (DFG) by grant nos. TE 931/2–1 (awarded to R.T.) and 235777276/GRK1976 (R.T.) as well as the National Institute of General Medical Sciences and of the National Institutes of Health under Award Number F32GM122218 (J.N.S.). Computational resources were provided by the UCLA Institute for Digital Research and Education. We thank M. Boll, B. Palfey, C. Fanhi, B. Kammerer and S. Lagies for technical support.
The authors declare no competing interests.
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Matthews, A., Saleem-Batcha, R., Sanders, J.N. et al. Aminoperoxide adducts expand the catalytic repertoire of flavin monooxygenases. Nat Chem Biol 16, 556–563 (2020). https://doi.org/10.1038/s41589-020-0476-2
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