Article

Nature 453, 190-195 (8 May 2008) | doi:10.1038/nature06879; Received 25 October 2007; Accepted 3 March 2008; Published online 19 March 2008

Kemp elimination catalysts by computational enzyme design

Daniela Röthlisberger1,7, Olga Khersonsky4,7, Andrew M. Wollacott1,7, Lin Jiang1,2, Jason DeChancie6, Jamie Betker3, Jasmine L. Gallaher3, Eric A. Althoff1, Alexandre Zanghellini1,2, Orly Dym5, Shira Albeck5, Kendall N. Houk6, Dan S. Tawfik4 & David Baker1,2,3

  1. Department of Biochemistry,
  2. Biomolecular Structure and Design, and,
  3. Howard Hughes Medical Institute, University of Washington, Seattle, Washington 98195, USA
  4. Department of Biological Chemistry, and,
  5. Israel Structural Proteomics Center, Weizmann Institute of Science, Rehovot 76100, Israel
  6. Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, USA
  7. These authors contributed equally to this work.

Correspondence to: Dan S. Tawfik4David Baker1,2,3 Correspondence and requests for materials should be addressed to D.B. (Email: dabaker@u.washington.edu) or D.S.T. (Email: dan.tawfik@weizmann.ac.il).

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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 k cat/K m (k cat/K m of 2,600 M-1s-1 and k cat/k uncat 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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