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Francisella tularensis IspD: A Target for Novel Antibiotics

Francisella tularensis IspD: A Target for Novel Antibiotics Arthur Tsang, MS George Mason University, 2012 Thesis Director: Dr. Robin Couch Francisella tularensis is the pathogenic bacteria responsible for causing Tularemia, a severe disease that has been explored as a biological weapon, leading to its classification as a Category A bioterrorist agent. To facilitate the development of novel therapeutics against F. tularensis, we investigated the metabolic enzyme IspD, which catalyzes an early intermediate step in the 2-C-methylerythritol-4-phosphate (MEP) pathway (or non-mevalonate pathway) responsible for isoprenoid biosynthesis in a variety of bacteria, including Francisella tularensis. Since the MEP pathway has no homologs in the human genome, it is an attractive target for antibiotic development. IspD was cloned, expressed, and purified. An assay which could be adapted to high throughput screening was used to determine the KM = 177.9 +/35.2 μM, Kcat=1.0 +/0.10 s, and Kcat/Km = 3.4×10 +/6.7×10 M s. FtIspD was found to exclusively prefer Mg for catalytic activity, and demonstrated the highest nucleotide specificity for CTP, although deoxy-CTP, ATP, GTP, TTP, and UTP resulted in some enzymatic activity. Site directed mutants were used to probe whether the enzyme could be regulated by phosphorylation at a conserved T141 site. Bioprospecting was also evaluated as an alternative to costly high throughput screening, and although a ligand for IspD was not discovered in a tested natural product molecular library, the bioprospecting method was validated by isolating fosmidomycin from its known target, IspC.