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Precision is essential for efficient catalysis in an evolved Kemp eliminase

Nature volume 503, pages 418–421 (2013)Cite this article

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Abstract

Linus Pauling established the conceptual framework for understanding and mimicking enzymes more than six decades ago1. The notion that enzymes selectively stabilize the rate-limiting transition state of the catalysed reaction relative to the bound ground state reduces the problem of design to one of molecular recognition. Nevertheless, past attempts to capitalize on this idea, for example by using transition state analogues to elicit antibodies with catalytic activities2, have generally failed to deliver true enzymatic rates. The advent of computational design approaches, combined with directed evolution, has provided an opportunity to revisit this problem. Starting from a computationally designed catalyst for the Kemp elimination3—a well-studied model system for proton transfer from carbon—we show that an artificial enzyme can be evolved that accelerates an elementary chemical reaction 6 × 108-fold, approaching the exceptional efficiency of highly optimized natural enzymes such as triosephosphate isomerase. A 1.09 Å resolution crystal structure of the evolved enzyme indicates that familiar catalytic strategies such as shape complementarity and precisely placed catalytic groups can be successfully harnessed to afford such high rate accelerations, making us optimistic about the prospects of designing more sophisticated catalysts.

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Figure 1: Kemp elimination.
Figure 2: Directed evolution of Kemp eliminase HG3.
Figure 3: Crystal structure of HG3.17 complexed with 6-nitrobenzotriazole.
Figure 4: Catalytic improvement of HG3.

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Accession codes

Accessions

Protein Data Bank

Data deposits

The crystal structure of HG3.17 was deposited in the RCSB Protein Data Bank (PDB) under the accession number 4BS0.

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Acknowledgements

The authors are grateful to A. Aires-Trapote and C. Mayer for experimental assistance. We also thank C. Stutz and B. Blattmann for help in protein crystallization, and the beamline staff at the Swiss Light Source for support during data collection. This work was supported by the Swiss National Science Foundation (SNSF), the National Center of Excellence in Research (NCCR) Structural Biology program of the SNSF, the ETH Zurich, and the Defense Advanced Research Projects Agency (DARPA). Fellowships from the Fonds des Verbandes der chemischen Industrie (to R.B.), the Stipendienfonds der Schweizer Chemischen Industrie (to H.K.), the Studienstiftung des deutschen Volkes (to R.B. and H.K.), and the National Security Science and Engineering Faculty Fellowship (to S.L.M.) are gratefully acknowledged.

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Author notes

  1. Rebecca Blomberg & Heidi K. Privett

    Present address: Present addresses: Corporate R&D Division, Firmenich SA, 1211 Geneva, Switzerland (R.B.); Protabit, Pasadena, California 91101, USA (H.K.P.).,

Authors and Affiliations

  1. Laboratory of Organic Chemistry, ETH Zurich, 8093 Zurich, Switzerland,

    Rebecca Blomberg, Hajo Kries, Daniel M. Pinkas & Donald Hilvert

  2. Department of Biochemistry, University of Zurich, 8057 Zurich, Switzerland,

    Peer R. E. Mittl & Markus G. Grütter

  3. Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, 91125, California, USA

    Heidi K. Privett

  4. Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, 91125, California, USA

    Stephen L. Mayo

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Contributions

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

Corresponding author

Correspondence to Donald Hilvert.

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The authors declare no competing financial interests.

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Blomberg, R., Kries, H., Pinkas, D. et al. Precision is essential for efficient catalysis in an evolved Kemp eliminase. Nature 503, 418–421 (2013). https://doi.org/10.1038/nature12623

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