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Nature. 2008 May 8;453(7192):190-5. doi: 10.1038/nature06879. Epub 2008 Mar 19.

Kemp elimination catalysts by computational enzyme design.

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  • 1Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA.

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 10(5) 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(-1)s(-1) and k(cat)/k(uncat) of >10(6)). 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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PMID:
18354394
[PubMed - indexed for MEDLINE]
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