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Two Fe-S clusters catalyze sulfur insertion by radical-SAM methylthiotransferases

Nature Chemical Biology volume 9, pages 333–338 (2013)Cite this article

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Abstract

How living organisms create carbon-sulfur bonds during the biosynthesis of critical sulfur-containing compounds is still poorly understood. The methylthiotransferases MiaB and RimO catalyze sulfur insertion into tRNAs and ribosomal protein S12, respectively. Both belong to a subgroup of radical–S-adenosylmethionine (radical-SAM) enzymes that bear two [4Fe-4S] clusters. One cluster binds S-adenosylmethionine and generates an Ado• radical via a well-established mechanism. However, the precise role of the second cluster is unclear. For some sulfur-inserting radical-SAM enzymes, this cluster has been proposed to act as a sacrificial source of sulfur for the reaction. In this paper, we report parallel enzymological, spectroscopic and crystallographic investigations of RimO and MiaB, which provide what is to our knowledge the first evidence that these enzymes are true catalysts and support a new sulfation mechanism involving activation of an exogenous sulfur cosubstrate at an exchangeable coordination site on the second cluster, which remains intact during the reaction.

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Figure 1: The methylthiolation reactions catalyzed by MiaB and RimO.
Figure 2: Enzymological characterization of MiaB.
Figure 3: HYSCORE spectra of variant MiaB3C.
Figure 4: Crystal structure of holo-TmRimO.

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Acknowledgements

We thank R. Abramowitz and J. Schwanof for assistance with synchrotron data collection, B. Gibney for advice on Fe-S reconstitution for crystallization and R. Breslow for discussion of the reaction mechanism. We thank O. Hamelin for GC/MS analysis and J.-M. Moulis for providing 77Se (both from Chemistry and Biology of Metals Laboratory, Grenoble). This work was supported by the US National Institutes of Health Protein Structure Initiative grants U54-GM074958 and U54-GM094597 to the NeSG (http://www.nesg.org/), a Groupement d′Intérêt Scientifique–CNRS fellowship to S.A., Agence Nationale de la Recherche–Blanc 2010 grant INSERAD and Région Rhône-Alpes grant CIBLE 2008-2011.

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Author notes

  1. Farhad Forouhar and Simon Arragain: These authors contributed equally to this work.

Authors and Affiliations

  1. Northeast Structural Genomics Consortium (NeSG),

    Farhad Forouhar, Munif Hussain, Rong Xiao, Jayaraman Seetharaman, Thomas B Acton, Gaetano T Montelione & John F Hunt

  2. Department of Biological Sciences, Columbia University, New York, New York, USA

    Farhad Forouhar, Munif Hussain, Jayaraman Seetharaman & John F Hunt

  3. Institut de Recherches en Technologie et Sciences pour le Vivant (IRTSV)–Laboratoire Chimie et Biologie des Métaux (LCBM), Unité Mixte de Recherche (UMR) 5249, Commissariat à l′Énergie Atomique et aux Énergies Alternatives (CEA)–Centre National de la Recherche Scientifique (CNRS)–Université Joseph Fourier (UJF), CEA-Grenoble, Grenoble, France

    Simon Arragain, Mohamed Atta, Etienne Mulliez & Marc Fontecave

  4. Direction des Sciences de la Matière (DSM)–Institute of Nanosciences and Cryogenics (INaC)–Service de Chimie Inorganique et Biologique (SCIB), UMR-E3 CEA-UJF, Laboratoire de Résonance Magnétique, CEA-Grenoble, Grenoble, France

    Serge Gambarelli & Jean-Marie Mouesca

  5. Department of Molecular Biology and Biochemistry, Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, New Jersey, USA

    Rong Xiao, Thomas B Acton & Gaetano T Montelione

  6. Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey, USA

    Rong Xiao, Thomas B Acton & Gaetano T Montelione

  7. IRTSV, Etude de la Dynamique des Protéomes, Laboratoire Biologie à Grande Echelle, U1038 Institut National de la Santé et de la Recherche Medicale (INSERM)–CEA–UJF, Grenoble, France

    Sylvie Kieffer-Jaquinod

  8. Collège de France, Paris, France

    Marc Fontecave

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  1. Farhad Forouhar
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Contributions

E.M., S.A., M.A. and M.F. designed the biochemical and enzymological experiments, which were conducted by E.M. and S.A. J.-M.M. and S.G. designed and conducted the EPR, HYSCORE and DFT experiments. S.K.-J. conducted the HPLC/MS experiments. T.B.A., R.X., J.F.H., J.S. and G.T.M. designed the target selection and protein purification–crystallization pipeline of the NeSG, which purified a wide variety of MTTases for this project. F.F., with advice from J.F.H., developed reconstitution methods for crystallization, which was performed by M.H. F.F. solved and refined related crystal structures. M.F., E.M., J.F.H., F.F., M.A. and G.T.M. interpreted the results, and M.F., E.M., J.F.H. and F.F. wrote the manuscript.

Corresponding authors

Correspondence to Etienne Mulliez, John F Hunt or Marc Fontecave.

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Forouhar, F., Arragain, S., Atta, M. et al. Two Fe-S clusters catalyze sulfur insertion by radical-SAM methylthiotransferases. Nat Chem Biol 9, 333–338 (2013). https://doi.org/10.1038/nchembio.1229

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