5RGC

Crystal Structure of Kemp Eliminase HG3.7 with bound transition state analogue, 277K

Summary for 5RGC

• Entry DOI: 10.2210/pdb5rgc/pdb
• Group deposition: Crystal structures of HG-series of Kemp Eliminases at Room-temperature (G_1002148)
• Descriptor: Kemp Eliminase HG3, 6-NITROBENZOTRIAZOLE, SULFATE ION, ... (4 entities in total)
• Functional Keywords: hydrolase
• Biological source: Thermoascus aurantiacus
• Total number of polymer chains: 2
• Total formula weight: 69902.25

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

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: 32968058
DOI: 10.1038/s41467-020-18619-x

Experimental method

X-RAY DIFFRACTION (1.39 Å)