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

Nathalie Preiswerk, Tobias Beck, Jessica D. Schulz, Peter Milovník, Clemens Mayer, Justin B. Siegel, David Baker, Donald Hilvert*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

105 Citations (Scopus)


By combining targeted mutagenesis, computational refinement, and directed evolution, amodestly 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.

Original languageEnglish
Pages (from-to)8013-8018
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number22
Publication statusPublished - 3-Jun-2014
Externally publishedYes


  • Biocatalysis
  • Computational enzyme design
  • Diels-Alder reaction
  • Enzyme mechanism
  • Laboratory evolution

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