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Kemp elimination catalysts by computational enzyme design

Nature volume 453, pages 190–195 (2008) Cite this article

Abstract

The design of new enzymes for reactions not catalysed by naturally occurring biocatalysts is a challenge for protein engineering and is a critical test of our understanding of enzyme catalysis. Here we describe the computational design of eight enzymes that use two different catalytic motifs to catalyse the Kemp elimination—a model reaction for proton transfer from carbon—with measured rate enhancements of up to 105 and multiple turnovers. Mutational analysis confirms that catalysis depends on the computationally designed active sites, and a high-resolution crystal structure suggests that the designs have close to atomic accuracy. Application of in vitro evolution to enhance the computational designs produced a >200-fold increase in kcat/Km (kcat/Km of 2,600 M-1s-1 and kcat/kuncat of >106). These results demonstrate the power of combining computational protein design with directed evolution for creating new enzymes, and we anticipate the creation of a wide range of useful new catalysts in the future.

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Figure 1: Reaction scheme and catalytic motifs used in design.
BERJAYAThe alternative text for this image may have been generated using AI.
Figure 2: Kinetic characterization of designed catalysts.
BERJAYAThe alternative text for this image may have been generated using AI.
Figure 3: Computational design models of the two most active catalysts.
BERJAYAThe alternative text for this image may have been generated using AI.
Figure 4: Comparison of the designed model of KE07 and the crystal structure.
BERJAYAThe alternative text for this image may have been generated using AI.

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Protein Data Bank

Data deposits

The crystal structure of KE07 has been deposited in the RCSB Protein Data Bank (http://www.rcsb.org) under the accession number 2rkx.

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Acknowledgements

We thank R. Stanfield and I. Wilson for providing D-2-deoxyribose-5-phosphate aldolase wild-type protein (PDB code 1jcl) and W. A. Greenberg and C.-H. Wong for providing the expression plasmid. We thank Rosetta@home participants for their contributions of computing power. This work was supported by a postdoctoral fellowship from the Swiss National Science Foundation to D.R., an Adams Fellowship (Israel Academy of Science) to O.K., research grants from the Minerva Foundation and the Fannie Sherr Estate to D.S.T., and NSF and NIH-CBI grants to K.N.H. We are also thankful for financial support from the Defense Advances Research Projects Agency (DARPA) and the Howard Hughes Medical Institute (HHMI) for this research.

Author Contributions D.R. performed computational design using carboxylate and the His–Asp motif, and purified and experimentally characterized designed catalysts; O.K. synthesized the substrate, performed in vitro evolution and experimentally characterized evolved variants; A.M.W. performed computational design on the His–Asp motif; L.J. performed initial computational design on the carboxylate motif; J.D. and K.N.H. computed idealized active sites using quantum mechanics; J.B. and J.L.G. expressed and purified designed catalysts; E.A.A. helped with enzyme design set-up; A.Z. wrote RosettaMatch and helped with computational set-up; O.D. and S.A. crystallized KE07; and D.R., A.M.W., D.B., K.N.H., O.K. and D.S.T. designed the experiment and wrote the manuscript.

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Author notes

  1. Daniela Röthlisberger, Olga Khersonsky and Andrew M. Wollacott: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry,,

    Daniela Röthlisberger, Andrew M. Wollacott, Lin Jiang, Eric A. Althoff, Alexandre Zanghellini & David Baker

  2. Biomolecular Structure and Design, and,,

    Lin Jiang, Alexandre Zanghellini & David Baker

  3. Howard Hughes Medical Institute, University of Washington, Seattle, Washington 98195, USA ,

    Jamie Betker, Jasmine L. Gallaher & David Baker

  4. Department of Biological Chemistry, and,

    Olga Khersonsky & Dan S. Tawfik

  5. Israel Structural Proteomics Center, Weizmann Institute of Science, Rehovot 76100, Israel ,

    Orly Dym & Shira Albeck

  6. Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, USA,

    Jason DeChancie & Kendall N. Houk

Authors
  1. Daniela Röthlisberger
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  2. Olga Khersonsky
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  13. Dan S. Tawfik
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Corresponding authors

Correspondence to Dan S. Tawfik or David Baker.

Supplementary information

Supplementary Information (download PDF )

The file contains Supplementary Discussion with Supplementary Figures 1-25 and Legends, Supplementary Tables 1-10 and additional references. (PDF 1656 kb)

Supplementary Data (download ZIP )

The file contains Supplementary Data 1/KE07.pdb with xyz coordinates of KE07 design model in pdb format; Supplementary Data 2/KE59.pdb with xyz coordinates of KE59 design model in pdb format and Supplementary Data 3/KE70.pdb with xyz coordinates of KE70 design model in pdb format. (ZIP 151 kb)

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Röthlisberger, D., Khersonsky, O., Wollacott, A. et al. Kemp elimination catalysts by computational enzyme design. Nature 453, 190–195 (2008). https://doi.org/10.1038/nature06879

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