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A substrate radical intermediate in catalysis by the antibiotic resistance protein Cfr

Nature Chemical Biology volume 9, pages 422–427 (2013)Cite this article

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Abstract

Cfr-dependent methylation of C8 of A2503 in 23S ribosomal RNA confers bacterial resistance to an array of clinically important antibiotics that target the large subunit of the ribosome, including the synthetic oxazolidinone antibiotic linezolid. The key element of the proposed mechanism for Cfr, a radical S-adenosylmethionine enzyme, is the addition of a methylene radical, generated by hydrogen-atom abstraction from the methyl group of an S-methylated cysteine, onto C8 of A2503 to form a protein–nucleic acid crosslinked species containing an unpaired electron. Herein we use continuous-wave and pulsed EPR techniques to provide direct spectroscopic evidence for this intermediate, showing a spin-delocalized radical with maximum spin density at N7 of the adenine ring. In addition, we use rapid freeze-quench EPR to show that the radical forms and decays with rate constants that are consistent with the rate of formation of the methylated product.

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Figure 1: Mechanistic proposal for catalysis by Cfr.
Figure 2: EPR and ENDOR study of the substrate radical.
Figure 3: Spin density distribution in the A2503 radical species, as modeled by spin-unrestricted B3LYP (DFT) methods.
Figure 4: Kinetic competence of radical intermediate.

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Acknowledgements

We thank J.M. Bollinger Jr. and C. Krebs for a critical reading of the manuscript, and we are also grateful to J. Niklas (Argonne National Laboratory) for helpful discussions. This work was supported by US National Institutes of Health grants GM101957 (S.J.B.) and GM101390 (M.T.G.).

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Authors and Affiliations

  1. Department of Chemistry, Pennsylvania State University, University Park, Pennsylvania, USA

    Tyler L Grove, Jovan Livada, Erica L Schwalm, Michael T Green, Squire J Booker & Alexey Silakov

  2. Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania, USA

    Squire J Booker

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Contributions

T.L.G., S.J.B. and A.S. designed experiments, and T.L.G. and E.L.S. prepared samples and performed experiments; J.L. and A.S. acquired spectra and performed DFT calculations; A.S., T.L.G., S.J.B. and M.T.G. analyzed data; T.L.G., S.J.B. and A.S. prepared the manuscript. All of the authors discussed the results and commented on the manuscript.

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Correspondence to Squire J Booker or Alexey Silakov.

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Grove, T., Livada, J., Schwalm, E. et al. A substrate radical intermediate in catalysis by the antibiotic resistance protein Cfr. Nat Chem Biol 9, 422–427 (2013). https://doi.org/10.1038/nchembio.1251

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