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Evolution of an enzyme conformational ensemble guides design of an efficient biocatalyst

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

Posted 20 Mar 2020
bioRxiv DOI: 10.1101/2020.03.19.999235 (published 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 used room-temperature X-ray crystallography to study changes in the conformational ensemble during evolution of the designed Kemp eliminase HG3 (kcat/KM 160 M−1s−1). We observed that catalytic residues were increasingly rigidified, the active site became better pre-organized, and its entrance was widened. Based on these observations, we engineered HG4, an efficient biocatalyst (kcat/KM 120,000 M−1s−1) containing active-site mutations found during evolution but not distal ones. HG4 structures revealed that its active site was 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. 

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