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Diversity of chemical mechanisms in thioredoxin catalysis revealed by single-molecule force spectroscopy

Nature Structural & Molecular Biology volume 16, pages 890–896 (2009)Cite this article

A Corrigendum to this article was published on 01 December 2009

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Abstract

Thioredoxins (Trxs) are oxidoreductase enzymes, present in all organisms, that catalyze the reduction of disulfide bonds in proteins. By applying a calibrated force to a substrate disulfide, the chemical mechanisms of Trx catalysis can be examined in detail at the single-molecule level. Here we use single-molecule force-clamp spectroscopy to explore the chemical evolution of Trx catalysis by probing the chemistry of eight different Trx enzymes. All Trxs show a characteristic Michaelis-Menten mechanism that is detected when the disulfide bond is stretched at low forces, but at high forces, two different chemical behaviors distinguish bacterial-origin from eukaryotic-origin Trxs. Eukaryotic-origin Trxs reduce disulfide bonds through a single-electron transfer reaction (SET), whereas bacterial-origin Trxs show both nucleophilic substitution (SN2) and SET reactions. A computational analysis of Trx structures identifies the evolution of the binding groove as an important factor controlling the chemistry of Trx catalysis.

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Figure 1: Phylogeny of Trx homologs from representative species of the three domains of life.
Figure 2: Single-molecule force-clamp detection of disulfide bond–reduction events catalyzed by Trx enzymes.
Figure 3: Force-dependency of the rate of disulfide reduction by Trx enzymes from different species.
Figure 4: The three chemical mechanisms of disulfide reduction detected by force-clamp spectroscopy.
Figure 5: Structural analysis and molecular dynamics simulations of the binding groove in Trx enzymes.

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  • 18 November 2009

    In the version of this article initially published, Eric A Gaucher’s middle initial was omitted. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank S. Garcia-Manyes and J. Morrone assistance in writing the manuscript and all the Fernandez laboratory members for helpful discussions. This work was supported by the US National Institutes of Health grants HL66030 and HL61228 to J.M.F., grant GMO55090 to J.B., grant GM43340 to B.J.B., grant I.C.E. (Columbia University) to B.J.B. and J.M.F., grant BIO2006-07332 from the Spanish Ministry of Education and Science and FEDER Funds to J.M.S.-R.

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Authors and Affiliations

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

    Raul Perez-Jimenez, Pallav Kosuri, Arun P Wiita & Julio M Fernandez

  2. Department of Chemistry, and Columbia University, New York, New York, USA

    Jingyuan Li & Bruce J Berne

  3. Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York, USA

    Pallav Kosuri

  4. Departamento de Química-Fisica, Facultad de Ciencias, Universidad de Granada, Granada, Spain

    Inmaculada Sanchez-Romero, David Rodriguez-Larrea & Jose M Sanchez-Ruiz

  5. Estación Experimental del Zaidin, Granada, CSIC, Spain

    Ana Chueca

  6. Department of Medical Biochemistry and Biophysics, Medical Nobel Institute for Biochemistry, Karolinska Institutet, Stockholm, Sweden

    Arne Holmgren

  7. Departamento de Fisiología, Centro Andaluz de Biología del Desarrollo (CABD-CSIC), Anatomía y Biología Celular, Universidad Pablo de Olavide, Sevilla, Spain

    Antonio Miranda-Vizuete

  8. Interdisciplinary Research Center, Justus Liebig University, Giessen, Germany

    Katja Becker

  9. Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts, USA

    Seung-Hyun Cho & Jon Beckwith

  10. Nancy University, IFR110 GEEF, UMR 1136 Interactions Arbres Microorganismes, Faculte des Sciences, Vandoeuvre, Cedex, France

    Eric Gelhaye & Jean P Jacquot

  11. Georgia Institute of Technology, School of Biology, Atlanta, Georgia, USA

    Eric A Gaucher

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  1. Raul Perez-Jimenez
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Contributions

R.P.-J., J.L. and J.M.F. designed the research; R.P.-J., J.L., P.K., I.S.-R. and A.P.W. carried out the experiments; R.P.-J., J.L., P.K., I.S.-R. and J.M.F. conducted the data analysis; D.R.-L. and J.M.S.-R. provided Trx from E. coli; A.C. provided Trxm from pea; A.H. provided human TRX1; A.M.-V. provided human TRX2; K.B. provided Trx1 from P. falciparum; S.-H.C. and J.B. provided Trx2 from E. coli; E. Gelhaye and J.P.J. provided Trxh1 and Trxh3 from poplar; R.P.-J. and E. Gaucher performed the phylogenetic analysis; J.L. and B.J.B. performed computational analysis and molecular dynamics simulations; R.P.-J., J.L., P.K. and J.M.F. wrote the paper; all authors have actively participated in revising the manuscript.

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Correspondence to Raul Perez-Jimenez or Julio M Fernandez.

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Perez-Jimenez, R., Li, J., Kosuri, P. et al. Diversity of chemical mechanisms in thioredoxin catalysis revealed by single-molecule force spectroscopy. Nat Struct Mol Biol 16, 890–896 (2009). https://doi.org/10.1038/nsmb.1627

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