6U1Z

Crystal Structure of Ketosteroid Isomerase from Pseudomonas Putida (pKSI) at 280 K

Summary for 6U1Z

• Entry DOI: 10.2210/pdb6u1z/pdb
• Related: 6TZD
• Descriptor: Steroid Delta-isomerase, MAGNESIUM ION, CHLORIDE ION, ... (4 entities in total)
• Functional Keywords: isomerase
• Biological source: Pseudomonas putida
• Total number of polymer chains: 2
• Total formula weight: 29192.21

Authors

Yabukarski, F.,Herschlag, D. (deposition date: 2019-08-18, release date: 2020-08-19, Last modification date: 2023-10-11)

Primary citation

Yabukarski, F.,Biel, J.T.,Pinney, M.M.,Doukov, T.,Powers, A.S.,Fraser, J.S.,Herschlag, D.
Assessment of enzyme active site positioning and tests of catalytic mechanisms through X-ray-derived conformational ensembles.
Proc.Natl.Acad.Sci.USA, 117:33204-33215, 2020

PubMed Abstract: How enzymes achieve their enormous rate enhancements remains a central question in biology, and our understanding to date has impacted drug development, influenced enzyme design, and deepened our appreciation of evolutionary processes. While enzymes position catalytic and reactant groups in active sites, physics requires that atoms undergo constant motion. Numerous proposals have invoked positioning or motions as central for enzyme function, but a scarcity of experimental data has limited our understanding of positioning and motion, their relative importance, and their changes through the enzyme's reaction cycle. To examine positioning and motions and test catalytic proposals, we collected "room temperature" X-ray crystallography data for ketosteroid isomerase (KSI), and we obtained conformational ensembles for this and a homologous KSI from multiple PDB crystal structures. Ensemble analyses indicated limited change through KSI's reaction cycle. Active site positioning was on the 1- to 1.5-Å scale, and was not exceptional compared to noncatalytic groups. The KSI ensembles provided evidence against catalytic proposals invoking oxyanion hole geometric discrimination between the ground state and transition state or highly precise general base positioning. Instead, increasing or decreasing positioning of KSI's general base reduced catalysis, suggesting optimized Ångstrom-scale conformational heterogeneity that allows KSI to efficiently catalyze multiple reaction steps. Ensemble analyses of surrounding groups for WT and mutant KSIs provided insights into the forces and interactions that allow and limit active-site motions. Most generally, this ensemble perspective extends traditional structure-function relationships, providing the basis for a new era of "ensemble-function" interrogation of enzymes.

PubMed: 33376217
DOI: 10.1073/pnas.2011350117

Experimental method

X-RAY DIFFRACTION (1.5005 Å)