Developing A Ph-Jump Chemical Triggering Method For Time-Resolved Diffraction In Bacterial Hmg-Coa Reductase

HMG-CoA reductase (HMGR) in Pseudomonas Mevalonii uses Mevalonate as a carbon source and converts it into 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) using the substrate Coenzyme A and cofactor NAD. The mevalonate pathway is critical for the survival of multiple gram-positive bacteria making HMGR a novel target for antibacterials. Previous studies propose that this enzyme's reaction mechanism involves the production of intermediates Mevaldehyde and Mevaldyl-CoA with structural evidence only for Mevaldyl-CoA. Using time-resolved crystallography we can observe structural changes along the reaction pathway and detect intermediates. By understanding the reaction mechanism in more detail, we can design inhibitors with higher specificity. Time-resolved techniques require a triggering system to achieve temporal control over the enzymatic reaction. To develop a pH-based trigger, we measured the effect of pH on the enzyme's activity in the crystallization environment. The enzyme's pH profile in crystallization conditions shows significantly reduced activity with increasing activity at alkaline pH. This is attributed to the presence of precipitant, ammonium sulfate. This was also observed outside the crystallization environment and with other ammonium salts. A strong correlation was observed between the dissociation constant of the ammonium salts and inhibition. Using this information, we developed a chemical triggering method in which we exchange the crystal constituents with an ammonium acetate buffer and introduce the ligands at crystallization pH with no enzymatic activity. To initiate the enzymatic reaction, these crystals are transferred to a higher pH for different time-periods before being flash-frozen. Structures generated from these crystals showed the formation of Mevaldyl-CoA and the release of HMG-CoA / NADH. They also captured movement in regions associated with catalytic site formation and product release. Currently, we are using this triggering system to collect data at faster timescales and at multiple time-points in individual crystals.