Evolution of an enzyme conformational ensemble guides design of an efficient biocatalyst
| Autor: | Michael C. Thompson, Mukhametzhanov N, Erin Nguyen, Lin Liu, Aron Broom, R.V. Rakotoharisoa, James S. Fraser, Roberto A. Chica, Zarifi N |
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| Rok vydání: | 2020 |
| Předmět: |
chemistry.chemical_classification
0303 health sciences biology Stereochemistry Chemistry Protein design Active site Crystallographic data 010402 general chemistry Directed evolution 01 natural sciences 0104 chemical sciences 03 medical and health sciences Enzyme Biocatalysis biology.protein Enzyme kinetics Conformational ensembles 030304 developmental biology |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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