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Evolution of a designed retro-aldolase leads to complete active site remodeling

Nature Chemical Biology volume 9, pages 494498 (2013) | Download Citation

Abstract

Evolutionary advances are often fueled by unanticipated innovation. Directed evolution of a computationally designed enzyme suggests that pronounced molecular changes can also drive the optimization of primitive protein active sites. The specific activity of an artificial retro-aldolase was boosted >4,400-fold by random mutagenesis and screening, affording catalytic efficiencies approaching those of natural enzymes. However, structural and mechanistic studies reveal that the engineered catalytic apparatus, consisting of a reactive lysine and an ordered water molecule, was unexpectedly abandoned in favor of a new lysine residue in a substrate-binding pocket created during the optimization process. Structures of the initial in silico design, a mechanistically promiscuous intermediate and one of the most evolved variants highlight the importance of loop mobility and supporting functional groups in the emergence of the new catalytic center. Such internal competition between alternative reactive sites may have characterized the early evolution of many natural enzymes.

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Acknowledgements

We thank T. Tomizaki, M. Müller, V. Olieric, G. Pompidor and A. Pauluhn at the Swiss Light Source for their outstanding support and all of the members of the Ban laboratory for suggestions and discussions. We are also grateful to E. Althoff (University of Washington) for providing the genes for RA95.0 and RA95.5 and sharing data before publication and to D. Gillingham and S. Tonazzi (ETH Zurich) for inhibitor synthesis and substrate resolution. The authors acknowledge support from the Swiss National Science Foundation (SNSF) (N.B. and D.H.), the National Center of Excellence in Research Structural Biology program of the SNSF (N.B.), the ETH Zurich (P.K., N.B. and D.H.), the Defense Advanced Research Projects Agency (D.B. and D.H.) and the Howard Hughes Medical Institute (D.B.). L.G. was funded by the Stipendienfonds der Schweizerischen Chemischen Industrie.

Author information

Author notes

    • Lars Giger
    •  & Sami Caner

    These authors contributed equally to this work.

Affiliations

  1. Laboratory of Organic Chemistry, ETH Zurich, Zurich, Switzerland.

    • Lars Giger
    • , Richard Obexer
    • , Peter Kast
    •  & Donald Hilvert
  2. Institute of Molecular Biology and Biophysics, ETH Zurich, Zurich, Switzerland.

    • Sami Caner
    •  & Nenad Ban
  3. Department of Biochemistry, University of Washington, Seattle, Washington, USA.

    • David Baker

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Contributions

D.H., N.B., D.B., P.K., L.G. and S.C. designed the experiments. L.G. and R.O. evolved and biochemically characterized the variants; S.C. crystallized the proteins and solved their structures. The manuscript and figures were prepared by L.G., S.C., P.K., N.B. and D.H.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Nenad Ban or Donald Hilvert.

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DOI

https://doi.org/10.1038/nchembio.1276

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