Ensemble-based enzyme design can recapitulate the effects of laboratory directed evolution in silico

Nat Commun. 2020 Sep 23;11(1):4808. doi: 10.1038/s41467-020-18619-x.


The creation of artificial enzymes is a key objective of computational protein design. Although de novo enzymes have been successfully designed, these exhibit low catalytic efficiencies, requiring directed evolution to improve activity. Here, we use room-temperature X-ray crystallography to study changes in the conformational ensemble during evolution of the designed Kemp eliminase HG3 (kcat/KM 146 M-1s-1). We observe that catalytic residues are increasingly rigidified, the active site becomes better pre-organized, and its entrance is widened. Based on these observations, we engineer HG4, an efficient biocatalyst (kcat/KM 103,000 M-1s-1) containing key first and second-shell mutations found during evolution. HG4 structures reveal that its active site is pre-organized and rigidified for efficient catalysis. Our results show how directed evolution circumvents challenges inherent to enzyme design by shifting conformational ensembles to favor catalytically-productive sub-states, and suggest improvements to the design methodology that incorporate ensemble modeling of crystallographic data.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Catalysis
  • Catalytic Domain
  • Computer Simulation*
  • Crystallography, X-Ray
  • Directed Molecular Evolution / methods*
  • Enzyme Stability
  • Enzymes / chemistry*
  • Enzymes / genetics
  • Enzymes / metabolism
  • Evolution, Chemical*
  • Kinetics
  • Lyases / chemistry*
  • Lyases / genetics
  • Lyases / metabolism
  • Molecular Dynamics Simulation
  • Mutation
  • Protein Conformation
  • Protein Engineering


  • Enzymes
  • Lyases