Mechanistic analysis of enzymes often requires a broad range of specific conditions, but X-ray crystallography is limited by the conditions of crystallization. Cryo-EM can facilitate structural comparison at multiple solution conditions (e.g., pH, ligands, or temperature), which will be addressed with ketol-acid reductoisomerase (KARI) from archaea Sulfolobus solfataricus, which is a dodecamer in solution, displays optimal activity at pH 7-8, and is bi-specific to coenzymes NADH and NADPH. While crystals were obtainable only at pH 8.5, cryo-EM structures were solved at pH 7.5 and 8.5 for KARI:2Mg2+ to address the pH effect.
In addition, cryo-EM structures of two KARI complexes, with NADH+inhibitor and NADPH+inhibitor at pH 7.5, were solved to address the structural basis of cofactor bi-specificity. Most importantly, structures of both the Mg2+-form (KARI:2Mg2+) and its ternary complex (KARI:2Mg2+:NADH:inhibitor) were solved at six temperatures from 4-70° C, leading to dissection of the induced-fit mechanism into ligand-induced and temperature-induced effects, and capturing of temperature-resolved intermediates of the temperature-induced conformational change.
(1) Use of Cryo-EM to Uncover Structural Bases of pH Effect and Cofactor Bi-specificity of Ketol-acid Reductoisomerase. Chen CY, Chang YC, Lin BL, Lin KF, Huang CH, Hsieh DL, Ko TP, Tsai MD. J. Am. Chem. Soc. 141, 6136-6140 (2019). doi: 10.1021/jacs.9b01354
(2) Temperature-resolved Cryo-EM Uncovers Structural Bases of Temperature-Dependent Enzyme Functions. Chen CY, Chang YC, Lin BL, Huang CH, Tsai MD. J. Am. Chem. Soc. 141, 19983-19987 (2019). doi: 10.1021/jacs.9b10687.