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Precision is essential for efficient catalysis in an evolved Kemp eliminase

Blomberg, Rebecca and Kries, Hajo and Pinkas, Daniel M. and Mittl, Peer R. E. and Grütter, Markus G. and Privett, Heidi K. and Mayo, Stephen L. and Hilvert, Donald (2013) Precision is essential for efficient catalysis in an evolved Kemp eliminase. Nature, 503 (7476). pp. 418-421. ISSN 0028-0836. doi:10.1038/nature12623.

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Linus Pauling established the conceptual framework for understanding and mimicking enzymes more than six decades ago. 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 activities, 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 elimination—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 × 10^8-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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Mayo, Stephen L.0000-0002-9785-5018
Additional Information:© 2013 Macmillan Publishers Limited. Received 7 June; accepted 30 August 2013; Published online 16 October 2013. 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. Author 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. The crystal structure of HG3.17 was deposited in the RCSB Protein Data Bank (PDB) under the accession number 4BS0.
Funding AgencyGrant Number
Swiss National Science Foundation (SNSF)UNSPECIFIED
Defense Advanced Research Projects Agency (DARPA)UNSPECIFIED
Fonds des Verbandes der chemischen IndustrieUNSPECIFIED
Stipendienfonds der Schweizer Chemischen IndustrieUNSPECIFIED
Studienstiftung des deutschen VolkesUNSPECIFIED
National Security Science and Engineering Faculty FellowshipUNSPECIFIED
Subject Keywords:X-ray crystallography; Protein design; Enzyme mechanisms
Issue or Number:7476
Record Number:CaltechAUTHORS:20131220-135748515
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:43121
Deposited By: Jason Perez
Deposited On:20 Dec 2013 23:03
Last Modified:10 Nov 2021 16:33

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