The long term objective of this research plan is to gain an understanding of the structural basis for isoprenoid biosynthesis in bacteria and parasites, which will serve as a foundation for the design of new classes of antibacterial and antimalarial agents. In particular, a key aim of this proposal is to understand the structural and mechanistic features governing the biosynthesis of the isoprenoid precursors isopentenyl disphosphate (IPP) and the isomeric compound, dimethylallyl diphosphate (DMAPP) catalyzed by enzymes comprising the mevalonate-independent pathway. In addition, the structural and functional analysis of a recently described and novel IPP isomerase that differs mechanistically and structurally from previously described IPP isomerases will be undertaken. This type 2 IPP isomerase, unlike the well-studied type 1 enzymes, depends on flavin mononucleotide and NADPH for activity. Notably, this type 2 class of IPP isomerase is found in several Gram-positive bacteria such as Staphylococcus aureus suggesting that it will serve as an effective target for new anti-staphylococcal drugs. Finally, the structure of the bacterial form of the enzyme farnesyl diphosphate synthase obtained from Escherichia coli will be determined. IPP and DMAPP are the fundamental building blocks of isoprenoids in all organisms and are essential metabolites for the construction of numerous higher order terpenoids. Recent independent studies demonstrated the existence of a novel, mevalonate-independent pathway for IPP synthesis known as the 1-deoxy-D-xylulose 5-phosphate / 2-C-methyl-D-erythritol 4-phosphate (DXP/MEP) pathway. This latter mevalonate-independent pathway utilizes pyruvate and glyceraldehyde 3-phosphate as starting materials for production of IPP. The DXPIMEP pathway occurs in a variety of eubacteria that includes several pathogenic species such as Mycobacterium tuberculosis, in algae, in the plastids of plant cells and in the apicoplast of Plasmodium falciparum the parasite that causes malaria. Given the essential nature of the DXP/MEP pathway in these organisms and the absence of this pathway in mammals, the enzymes comprising the DXP/MEP pathway represent targets for the generation of selective antibacterial and antimalarial agents. In order to better understand the mechanistic features of this pathway and the subsequent formation of a critical diphosphate intermediate farnesyl diphosphate (FPP), we have begun the structural elucidation of the enzymes comprising the DXP/MEP pathway and the formation of DMAPP and FPP from IPP isomerase and FPP synthase, respectively, using protein x-ray crystallography.
