8USK

Crystal Structure of Kemp Eliminase HG185 in unbound state, 280 K

Summary for 8USK

• Entry DOI: 10.2210/pdb8usk/pdb
• Descriptor: Kemp eliminase (2 entities in total)
• Functional Keywords: de novo enzyme design; computational protein design; kemp eliminases; biocatalysis; directed evolution; x-ray crystallography, hydrolase
• Biological source: Thermoascus aurantiacus
• Total number of polymer chains: 2
• Total formula weight: 68695.09

Authors

Seifinoferest, B. (deposition date: 2023-10-27, release date: 2023-12-06, Last modification date: 2024-10-16)

Primary citation

Rakotoharisoa, R.V.,Seifinoferest, B.,Zarifi, N.,Miller, J.D.M.,Rodriguez, J.M.,Thompson, M.C.,Chica, R.A.
Design of Efficient Artificial Enzymes Using Crystallographically Enhanced Conformational Sampling.
J.Am.Chem.Soc., 146:10001-10013, 2024

PubMed Abstract: The ability to create efficient artificial enzymes for any chemical reaction is of great interest. Here, we describe a computational design method for increasing the catalytic efficiency of de novo enzymes by several orders of magnitude without relying on directed evolution and high-throughput screening. Using structural ensembles generated from dynamics-based refinement against X-ray diffraction data collected from crystals of Kemp eliminases HG3 (/ 125 M s) and KE70 (/ 57 M s), we design from each enzyme ≤10 sequences predicted to catalyze this reaction more efficiently. The most active designs display / values improved by 100-250-fold, comparable to mutants obtained after screening thousands of variants in multiple rounds of directed evolution. Crystal structures show excellent agreement with computational models, with catalytic contacts present as designed and transition-state root-mean-square deviations of ≤0.65 Å. Our work shows how ensemble-based design can generate efficient artificial enzymes by exploiting the true conformational ensemble to design improved active sites.

PubMed: 38532610
DOI: 10.1021/jacs.4c00677

Experimental method

X-RAY DIFFRACTION (1.6 Å)