| Non-mammalian isoprenoid biosynthesis pathway, the 2 C-methyl-D-erythritol phosphate (MEP) pathway, produces isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), which are the 5--carbon building blocks of >30,000 isoprenoids. The MEP pathway has been shown to be essential in pathogenic organisms including Plasmodium falciparum and Mycobacterium tuberculosis. Due to the absence of the MEP pathway enzymes in humans, it has emerged as a potential target for the generation of a new class of antibiotics.;The fifth enzyme of the pathway, the cyclodiphosphate synthase IspF catalyzes the conversion of 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate (CDPME2P) to the cyclic diphosphate 2C-methyl- D-erythritol 2,4-cyclodiphosphate (MEcDP) with concomitant release of cytidine 5'-diphosphate (CMP). Crystallographic and biochemical studies have shown that IspF is active as a homotrimer with the active sites at the interfaces of the monomeric subunits, and requires Zn2+ and Mg2+ for catalysis.;In this dissertation, we examined the regulation of isoprenoid biosynthesis at the fifth step (IspF) of the MEP pathway. We also designed and synthesized IspF modulators toward the ultimate goal of developing potential inhibitors of this remarkable enzyme. First, we tested the hypothesis that downstream products of the MEP pathway (farnesyl diphosphate (FDP), geranyl diphosphate (GDP), IDP and DMADP) modulate IspF activity through a feedback mechanism. We found that 2C-methyl-D-erythritol 4-phosphate (MEP) activates and sustains the activity of IspF, and that only this IspF-MEP complex is sensitive to inhibition by the downstream isoprenoid diphosphate FDP. Further investigation shows that this MEP-induced activation/stabilization of IspF is driven by the 2C-methyl-D-erythritol (ME) scaffold itself. Second, we designed, synthesized and evaluated IspF substrate analogs as potential modulators of IspF activity. We highlight the synthetic challenges to accessing stable, bisphosphonate (BP)-containing substrate analogs, and subsequently show that BP-containing substrate analogs exhibit different effects on IspF and the IspF-MEP complex compared to their diphosphate counterparts. |
