Elucidating the HMG-COA Reductase Reaction Mechanism Using PH-Triggered Time-Resolved X-Ray Crystallography

Vatsal Purohit, Purdue University

Abstract

HMG-CoA reductase from Pseudomonas mevalonii (PmHMGR) catalyzes the oxidation of mevalonate and mevaldyl-CoA to form HMG-CoA using CoA-SH and two NAD+ cofactors. While the enzyme has been used extensively as a drug target in humans to treat hypercholesterolemia, its pathway has also been found to be critical for the survival of antibiotic resistant gram-positive bacteria. Structural studies using non-productive and slow-substrate binary complexes as well as biochemical studies using half and full reactions led to the proposal that the conversion of mevalonate to HMG-CoA occurs through the generation of two intermediates, mevaldehyde and mevaldyl-CoA (Shown in Fig 1.1). However, several intermediary changes along the PmHMGR reaction pathway remain unclear. By gathering information about the enzyme’s intermediate states via structural studies, we could identify potential allosteric sites that further the reaction mechanism. Using this knowledge, we could design enzyme inhibitors that act as novel antibacterials. The application of time-resolved crystallographic methods would provide structural information about transitory states in thePmHMGR reaction mechanism. The PmHMGR crystal has been shown to be suitable for time-resolved crystallographic measurements for the reaction steps resulting in mevaldyl-CoA formation. However, our structural investigations of the mevalonate, CoA and NAD+ complex that are expected to result in the formation of mevaldehyde (Fig 1.1) do not show any changes corresponding to a turnover in the crystal environment. To investigate the factors limiting enzymatic activity in the crystal, we investigated the effects of pH and specific ions in the crystallization environment. Kinetic studies indicated a strong PmHMHGR inhibition in the crystallization buffer that is dependent on the concentration of the crystallization precipitant ammonium sulfate. These studies also indicated an increase in enzyme turnover with increasing pH. Utilizing the ionic concentration and pH-dcrystalsependent properties of the enzyme in the crystallization environment, we have developed a reaction triggering approach using pH changes for PmHMGR crystals. We have demonstrated our application of this ‘pH-jump’ method by observing changes in PmHMGR crystals after reaction initiation. Changes in the density of mevalonate, CoA and NAD+have indicated mevaldehyde and mevaldyl-CoA formation. Additionally, the appearance of a unique NADH absorbance peak after the pH-change has also highlighted the initiation of the PmHMGR reaction and the occurrence of a hydride transfer step. Our analysis of the movements using time-resolved structures post reaction-initiation have also highlighted structural changes and inter-domain contacts in the small and flap domain that would allow cofactor exchange and product release. The pH-jump method can hence be utilized as a novel approach for triggering the PmHMGR reaction in crystals and further studying transitory states along its reaction pathway.

Degree

Ph.D.

Advisors

Stauffacher, Purdue University.

Subject Area

Genetics|Microbiology|Pharmaceutical sciences

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