Targeting a Unique Enzyme Mechanism in Non-Mammalian Isoprenoid Biosynthesis

Johns Hopkins University, Baltimore MD

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Abstract

DESCRIPTION (provided by applicant): Although antibiotics exist for M. tuberculosis and the malaria parasite, Plasmodium falciparum, the widespread use of anti-microbial agents has, over time, provided a selective growth advantage for more virulent drug-resistant pathogenic strains that are increasingly difficult to treat. To ensure the availability of effective antibiotics in the treatment of life threatening infections, novel antibacterial targets need to be investigated. Isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) are essential isoprenoid precursors found in all living organisms. Biogenesis of isoprenoids is carried out via the methylerythritol phosphate (MEP) pathway in most human pathogens, including Mycobacterium tuberculosis and the malaria parasite Plasmodium falciparum. The unique MEP pathway to produce isoprenoids is essential in these pathogens and absent in mammals, and therefore represents a potential drug target for the development of new anti-infective agents. 1-Deoxy-D-xylulose 5-phosphate (DXP) synthase catalyzes the first of seven enzymatic steps in the MEP pathway. This thiamin diphosphate (ThDP)-dependent enzyme catalyzes the formation of DXP from D-glyceraldehyde 3-phosphate (GAP) and pyruvate. DXP synthase is a potential drug target, yet there are few reports describing inhibitors of this enzyme. This proposal describes detailed mechanistic studies of E. coli and M. tuberculosis DXP synthase using tryptophan fluorescence to study substrate binding and steady state kinetics. On the basis of our preliminary results suggesting a novel mechanism in this ThDP-dependent enzyme class, we hypothesize that DXP synthase can be selectively inhibited by targeting a conformation that is unique to this enzyme, namely the conformation comprising the catalytically competent ternary complex. The rationale for the proposed studies is that developing strategies for selective inhibition of this enzyme will provide new tools for investigating this essential pathway in pathogens and will lead to new anti-infective agents.