Investigating the mechanism of DXP synthase catalysis

Isoprenoids are an important class of natural products that originate from two common precursors, isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP). In mammals, these compounds are synthesized by the mevalonate pathway; however, most bacteria and apicomplexan parasites utilize the novel methylerythritol phosphate (MEP) pathway for the production of these essential precursors. The first enzyme of the pathway, DXP synthase, catalyzes a thiamine diphosphate (ThDP)-dependent condensation reaction between pyruvate and D-glyceraldehyde-3-phosphate (D-GAP) to generate 1-deoxy-D-xylulose-5-phosphate (DXP). As DXP also feeds into thiamine and pyridoxal biosyntheses, DXP synthase holds potential as a target in central metabolism for new anti-infective agents. While achieving selectivity over other ThDP-dependent enzymes is a challenge, increasing evidence indicates that DXP synthase is unique in its class and can thus be selectively targeted. The active site of DXP synthase adopts a unique domain arrangement and is predicted to have twice the volume of other ThDP enzyme active sites. Additionally, DXP synthase catalyzes a unique kinetic mechanism, whereby productive catalysis must proceed through formation of a ternary complex. This research has focused efforts toward further understanding the unique aspects of DXP synthase catalysis with the long-term goal of using this insight in inhibitor design. Chapter 2 will describe kinetic and circular dichroism (CD) studies aimed at identifying active site residues with roles in LThDP formation and decarboxylation, steps representing potential targets of DXP synthase inhibitors. Our findings suggest that three active site histidines impact these and subsequent steps of DXP synthase catalysis. Chapter 3 will outline investigation into the behaviors of several small molecule pyruvate analogues that have been widely used as mechanistic probes in ThDP enzymology. We suggest these mimics behave distinctly in the DXP synthase system, which will have implications for their future use. Furthermore, we explore the binding determinants of a selective DXP synthase bisubstrate inhibitor, butyl acetylphosphonate (BAP) and other alkyl acetylphosphonates. Finally, Chapter 4 will describe studies aimed at understanding unique aspects of aromatic nitroso substrate binding and DXP synthase-catalyzed C-N bond formation. These studies will guide the design of future aromatic acetylphosphonate inhibitors of DXP synthase