5RG9
Crystal Structure of Kemp Eliminase HG4 in unbound state, 277K
Summary for 5RG9
| Entry DOI | 10.2210/pdb5rg9/pdb |
| Group deposition | Crystal structures of HG-series of Kemp Eliminases at Room-temperature (G_1002148) |
| Descriptor | Kemp Eliminase HG4, ACETATE ION, SULFATE ION, ... (4 entities in total) |
| Functional Keywords | hydrolase |
| Biological source | Thermoascus aurantiacus |
| Total number of polymer chains | 2 |
| Total formula weight | 69067.42 |
| Authors | Broom, A.,Rakotoharisoa, R.V.,Thompson, M.C.,Fraser, J.S.,Chica, R.A. (deposition date: 2020-03-19, release date: 2020-07-22, Last modification date: 2024-10-23) |
| Primary citation | Broom, A.,Rakotoharisoa, R.V.,Thompson, M.C.,Zarifi, N.,Nguyen, E.,Mukhametzhanov, N.,Liu, L.,Fraser, J.S.,Chica, R.A. Ensemble-based enzyme design can recapitulate the effects of laboratory directed evolution in silico. Nat Commun, 11:4808-4808, 2020 Cited by PubMed 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 (k/K 146 Ms). 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 (k/K 103,000 Ms) 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. PubMed: 32968058DOI: 10.1038/s41467-020-18619-x PDB entries with the same primary citation |
| Experimental method | X-RAY DIFFRACTION (1.47 Å) |
Structure validation
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