Optimization of the Turnover in Artificial Enzymes via Directed Evolution Results in the Coupling of Protein Dynamics to Chemistry

Joseph W. Schafer, Ioanna Zoi, Dimitri Antoniou, Steven D. Schwartz

Research output: Contribution to journalArticlepeer-review

19 Scopus citations


The design of artificial enzymes is an emerging field of research. Although progress has been made, the catalytic proficiency of many designed enzymes is low compared to natural enzymes. Nevertheless, recently Hilvert et al. (Nat. Chem. 2017, 9, 50-56) created a series of five artificial retro-aldolase enzymes via directed evolution, with the final variant exhibiting a rate comparable to the naturally occurring enzyme fructose 1,6 bisphosphate aldolase. We present a study of this system in atomistic detail that elucidates the effects of mutational changes on the chemical step. Transition path sampling is used to create ensembles of reactive trajectories, and committor analysis is used to identify the stochastic separatrix of each ensemble. The application of committor distribution analysis to constrained trajectories allows the identification of changes in important protein motions coupled to reaction across the generated series of the artificial retro-aldolases. We observed two different reaction mechanisms and analyzed the role of the residues participating in the reaction coordinate of each enzyme. However, only in the most evolved variant we identified a fast motion that promotes catalysis, suggesting that this rate promoting vibration was introduced during directed evolution. This study provides further evidence that protein dynamics must be taken into account in designing efficient artificial enzymes.

Original languageEnglish (US)
Pages (from-to)10431-10439
Number of pages9
JournalJournal of the American Chemical Society
Issue number26
StatePublished - Jul 3 2019

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry


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