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

Aron Broom,Nurzhan Mukhametzhanov,Lin Liu,Rojo V Rakotoharisoa,Erin Nguyen,James S Fraser,Roberto A Chica,Niayesh Zarifi,Michael C Thompson

doi:10.1038/s41467-020-18619-x

Aron Broom, Nurzhan Mukhametzhanov + Show 7 more

Open Access

https://doi.org/10.1038/s41467-020-18619-x

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Abstract

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.

Highlights

The creation of artificial enzymes is a key objective of computational protein design
Perhaps the most successful example of the improvement of a de novo enzyme by directed evolution has been the engineering of HG3.17, the most active Kemp eliminase reported to date[11]
Unlike previous examples where the active sites of de novo enzymes were completely remodeled during evolution[25,26], or where the binding pose of the substrate or transition-state analogue was significantly altered[13,17], we observed only subtle changes to the active site geometry or 6NTbinding pose in the HG-series of Kemp eliminases

Summary

Introduction

The creation of artificial enzymes is a key objective of computational protein design. 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. We observe that during evolution, catalytic residues were increasingly rigidified through improved packing, the active site became better pre-organized to favor productive binding of the substrate, and the active-site entrance was widened to facilitate substrate entry and product release Based on these observations, we generate a variant that contains all mutations necessary to establish these structural features, which are found at positions within or close to the active site. We demonstrate that HG4 can be successfully designed using a crystallographically derived ensemble of backbone templates approximating conformational flexibility, but not with the single template used to design HG3, offering insights for improving enzyme design methodologies

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