5rg5

Crystal Structure of Kemp Eliminase HG3.3b in unbound state, 277K

spinqualitylabelsall models 5rg5, resolution 1.62Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 5rg5 is a 2 chain structure with sequence from Theau. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Ligands: ACT, SO4 Activity: Endo-1,4-beta-xylanase, with EC number 3.2.1.8 Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Publication Abstract from PubMed 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 (kcat/KM 146 M(-1)s(-1)). 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 (kcat/KM 103,000 M(-1)s(-1)) 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. Ensemble-based enzyme design can recapitulate the effects of laboratory directed evolution in silico.,Broom A, Rakotoharisoa RV, Thompson MC, Zarifi N, Nguyen E, Mukhametzhanov N, Liu L, Fraser JS, Chica RA Nat Commun. 2020 Sep 23;11(1):4808. doi: 10.1038/s41467-020-18619-x. PMID:32968058 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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