Abstract:
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-1, and
Kcat/Km = 3.4×105 +/- 6.7×104 M-1 s-1. FtIspD was found to exclusively prefer Mg2+ 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.
