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
Submitter: niayesh zarifi
Submission Date: May 5, 2020, 6:22 a.m.
|Number of data points
|Kemp Eliminase HG3.17 ; Kemp Eliminase HG4 ; Kemp Eliminase HG3.14 ; Kemp Eliminase HG3.7 ; Kemp Eliminase HG3.3b ; Kemp Eliminase HG3
|Experimental Assay: Steady-state kinetics ; Derived Quantity: SD of Steady-state kinetics
|Catalytic efficiency measured in kcat/Km for all the kemp eliminases of the HG series
|Kemp Eliminase HG3