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Single-molecule paleoenzymology probes the chemistry of resurrected enzymes

Nature Structural & Molecular Biology volume 18, pages 592–596 (2011)Cite this article

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Abstract

It is possible to travel back in time at the molecular level by reconstructing proteins from extinct organisms. Here we report the reconstruction, based on sequence predicted by phylogenetic analysis, of seven Precambrian thioredoxin enzymes (Trx) dating back between ~1.4 and ~4 billion years (Gyr). The reconstructed enzymes are up to 32 °C more stable than modern enzymes, and the oldest show markedly higher activity than extant ones at pH 5. We probed the mechanisms of reduction of these enzymes using single-molecule force spectroscopy. From the force dependency of the rate of reduction of an engineered substrate, we conclude that ancient Trxs use chemical mechanisms of reduction similar to those of modern enzymes. Although Trx enzymes have maintained their reductase chemistry unchanged, they have adapted over 4 Gyr to the changes in temperature and ocean acidity that characterize the evolution of the global environment from ancient to modern Earth.

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Figure 1: Phylogenetic analysis of Trx enzymes and ancestral sequence reconstruction.
Figure 2: Single-molecule disulfide reduction assay.
Figure 3: Force dependence of disulfide reduction by ancestral Trx enzymes.
Figure 4: Rate constants of disulfide bond reduction at pH 5.

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Acknowledgements

Supported by the US National Institutes of Health (HL061228 and HL066030 to J.M.F.); the Spanish Ministry of Science and Innovation (J.M.S.-R.); NASA Astrobiology (Georgia Institute of Technology); NASA Exobiology (E.A.G.); Fundación Ibercaja and Fundación Caja Madrid (R.P.-J. and S.G.-M.); and Fundación Alfonso Martín Escudero (J.A.-C.). We thank B. Ibarra-Molero (University of Granada) for assistance with bulk enzymatic assays.

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Authors and Affiliations

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

    Raul Perez-Jimenez, Jorge Alegre-Cebollada, Pallav Kosuri, Sergi Garcia-Manyes & Julio M Fernandez

  2. Departamento de Química-Física, Facultad de Ciencias, Universidad de Granada, Granada, Spain

    Alvaro Inglés-Prieto, Inmaculada Sanchez-Romero & Jose M Sanchez-Ruiz

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

    Zi-Ming Zhao & Eric A Gaucher

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

    Pallav Kosuri

  5. Department of Biochemistry, Purdue University, West Lafayette, Indiana, USA

    T Joseph Kappock

  6. Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan

    Masaru Tanokura

  7. Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden

    Arne Holmgren

  8. Georgia Institute of Technology, School of Chemistry, Atlanta, Georgia, USA

    Eric A Gaucher

  9. Georgia Institute of Technology, Parker H. Petit Institute for Bioengineering and Bioscience, Atlanta, Georgia, USA

    Eric A Gaucher

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  1. Raul Perez-Jimenez
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Contributions

R.P.-J., J.M.S.-R., E.A.G. and J.M.F. designed the research; Z.-M.Z. and E.A.G. conducted the phylogenetic analysis and sequence reconstruction; A.I.-P. and J.M.S.-R. expressed and purified the ancestral enzymes and conducted the calorimetric measurements and analysis; T.J.K. provided A. aceti Trx; M.T. provided S. tokodaii Trx; A.H. provided human TRX; R.P.-J., I.S.-R., J.A.-C., P.K. and S.G.-M. performed AFM experiments; R.P.-J. and I.S.-R. analyzed AFM data; R.P.-J., E.A.G. and J.M.F. wrote the paper; all authors participated in revising the manuscript.

Corresponding authors

Correspondence to Raul Perez-Jimenez or Julio M Fernandez.

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The authors declare no competing financial interests.

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Perez-Jimenez, R., Inglés-Prieto, A., Zhao, ZM. et al. Single-molecule paleoenzymology probes the chemistry of resurrected enzymes. Nat Struct Mol Biol 18, 592–596 (2011). https://doi.org/10.1038/nsmb.2020

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