Template:ABSTRACT PMID 18354394
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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 10<sup>5</sup> 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''<sub>cat</sub>/''K''<sub>m</sub> (''k''<sub>cat</sub>/''K''<sub>m</sub> of 2,600 M<sup>-1</sup>s<sup>-1</sup> and ''k''<sub>cat</sub>/''k''<sub>uncat</sub> of >10<sup>6</sup>). 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. | + | {{Abstract |
| + | |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<sup>5</sup> 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''<sub>cat</sub>/''K''<sub>m</sub> (''k''<sub>cat</sub>/''K''<sub>m</sub> of 2,600 M<sup>-1</sup>s<sup>-1</sup> and ''k''<sub>cat</sub>/''k''<sub>uncat</sub> of >10<sup>6</sup>). 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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Revision as of 10:23, 27 June 2008
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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From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
