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Proc Natl Acad Sci U S A. 2014 Jun 3;111(22):8013-8. doi: 10.1073/pnas.1401073111. Epub 2014 May 20.

Impact of scaffold rigidity on the design and evolution of an artificial Diels-Alderase.

Author information

1
Laboratory of Organic Chemistry, Eidgenössische Technische Hochschule Zürich, 8093 Zurich, Switzerland;
2
Department of Biochemistry, University of Washington, Seattle, WA 98195; andDepartments of Biochemistry and Molecular Medicine andChemistry, andGenome Center, University of California, Davis, CA 95616.
3
Department of Biochemistry, University of Washington, Seattle, WA 98195; and.
4
Laboratory of Organic Chemistry, Eidgenössische Technische Hochschule Zürich, 8093 Zurich, Switzerland; hilvert@org.chem.ethz.ch.

Abstract

By combining targeted mutagenesis, computational refinement, and directed evolution, a modestly active, computationally designed Diels-Alderase was converted into the most proficient biocatalyst for [4+2] cycloadditions known. The high stereoselectivity and minimal product inhibition of the evolved enzyme enabled preparative scale synthesis of a single product diastereomer. X-ray crystallography of the enzyme-product complex shows that the molecular changes introduced over the course of optimization, including addition of a lid structure, gradually reshaped the pocket for more effective substrate preorganization and transition state stabilization. The good overall agreement between the experimental structure and the original design model with respect to the orientations of both the bound product and the catalytic side chains contrasts with other computationally designed enzymes. Because design accuracy appears to correlate with scaffold rigidity, improved control over backbone conformation will likely be the key to future efforts to design more efficient enzymes for diverse chemical reactions.

KEYWORDS:

Diels–Alder reaction; biocatalysis; computational enzyme design; enzyme mechanism; laboratory evolution

PMID:
24847076
PMCID:
PMC4050586
DOI:
10.1073/pnas.1401073111
[Indexed for MEDLINE]
Free PMC Article
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