ISOPRENOID BIOSYNTHESIS: NON-MEVALONATE PATHWAY STUDIES

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The medical community is faced with increasing numbers of bacterial infections that are resistant to many of the currently used antibiotics. Identifying new molecular targets in bacteria is an approach that can lead to the development of antibiotics that may help to combat resistance. The non-mevalonate pathway (NMP) to isoprenoids which has recently been identified in some bacteria, higher plants, and algae through biosynthetic studies may prove to be such a target. Based on database searches of recently deposited whole genome DNA sequences, a number of pathogenic organisms have been identified that harbor genes with high homology to the first two genes identified for the NMP, suggesting that these organisms might be susceptible to antibiotics targeted to this pathway. Organisms implicated as causative agents of tuberculosis (Mycobacterium tuberculosis), some ulcers (Helicobacter pylori), sexually transmitted diseases (Chlamydia trachomatis), and malaria (Plasmodium falciparum) are included in this list. The long-term objectives of this research are to thoroughly understand the mechanisms of the enzymes of the NMP and to determine the feasibility of inhibiting these enzymes as a means to develop new antibacterial agents. The first specific aim of this project will be to fully characterize 1- deoxyxylulose-5-phosphate reductoisomerase (DXR), the second enzyme in the pathway and one which has been shown to be inhibited by the known antibacterial agent, fosmidomycin. This aim will be accomplished by 1) Purifying and obtaining kinetic data for the recombinant enzymes from the cyanobacterium Synechocystis sp. PCC6803 and Helicobacter pylori. 2) Examining the stereochemistry of the reduction step by using DXP specifically deuterated at C3 3) Preparing and characterizing eleven site-directed mutants of DXR 4) Synthesizing alternate substrates and proposed inhibitors and assaying for their effects on and 5) Testing in vitro inhibitors for antibacterial activity. The second aim will be to examine 1-deoxyxylulose-5-phosphate synthase from Synechocystis sp., the first enzyme involved in the pathway. This will be accomplished by 1) Cloning, overproducing, purifying, and characterizing the recombinant DXP synthase, 2) Testing alternate substrates with DXP synthase 3) Testing known inhibitors of TPP dependent enzymes with DXP, and 4) Designing and testing bisubstrate analogs to build the foundation for the later development of bisubstrate inhibitors.