Watching Pseudomonas mevalonii HMG-CoA Reductase in Action

HMG CoA Reductase catalyzed the interconversion of a thioester, HMG CoA, and mevalonic acid, a key step in the isoprenoid pathway, through a complex reaction mechanism involving three distinct chemical steps with two molecules of cofactor and large-scale rearrangements of the enzyme. Here, we investigate the second step, the formation of a thiohemiacetal from CoA and mevaldehyde, using time resolved crystallography and molecular dynamics (MD) simulations. After triggering the reaction by a pH jump from pH 6.7 to pH9, the formation of the carbon-sulfur bond can be observed in the two structures at 2.5 and 4 minutes. The structures obtained close to the activated complex of the reaction serve as the starting point for MD simulations of different possible protonation states of the catalytically active residues. Changes to the active site geometry, specifically the residues Ser 85, Glu 83 and His 381 that are important for catalysis of the reaction are discussed in detail. This work demonstrates the applicability of the combination of time resolved crystallography using a pH trigger with D simulations to obtain a detailed view of a complex reaction in an enzyme active site.