Structural and Functional Studies of Bacillus anthracis Enzymes in de novo Purine Synthesis

The de novo purine biosynthesis is an essential life sustaining process in many organisms, including bacterial pathogens. My work investigates the structural and functional properties of enzymes from this pathway in Bacillus anthracis (Ba) towards the identification of antimicrobials. The first enzyme I studied was PurK, which carboxylates aminoimidazole ribonucleotide (AIR), with bicarbonate in the presence of ATP, to N5-carboxyaminoimidazole ribonucleotide (N5-CAIR). PurK is unique to prokaryotes and lower eukaryotes. Divergence at this step in the pathway makes it an appealing target for antimicrobial development. I used x-ray crystallography to solve the structure of BaPurK. My work also produced several ligand-bound BaPurK structures; of which included bicarbonate in the active site, a first for any PurK structure. Based on these structures, a reaction mechanism was proposed. The second enzyme I focused on was PurC. BaPurC catalyzes the conversion of carboxyaminoimidazole ribonucleotide (CAIR) and aspartate (L-Asp) to succinoaminoimidazolecarboxamide ribonucleotide (SAICAR), with the use of ATP. Studies focused on the large difference in the purification yield between Bacillus anthracis and Streptococcus pneumoniae PurC. Although their amino acid sequences are very similar the recombinant protein yields differed by more than 10-fold. Results from biophysical studies with CD and fluorescence spectroscopies, and molecular modeling suggest that the variances in exposed hydrophobic surfaces are the cause of the difference in purification yield. BaPurC was targeted in high throughput screening, using malachite green, a common phosphate detection assay, in effort to find antimicrobials that inhibit this enzyme from Bacillus anthracis. A set of hit molecules was obtained from the screening of activity inhibition. While analyzing the data an unusual trend appeared in the controls of the assay, further investigation of this event reveals that PurC exhibits substrate-independent ATPase activity, which provides insights into the reaction mechanism