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

Substrate independent ATPase activity may complicate high throughput screening

Inorganic phosphate release, [Pi], is often measured in an enzymatic reaction in a high throughput setting. Based on the published mechanism, we designed a protocol for our screening for inhibitors of SAICAR synthetase (PurC), and we found a gradual increase in [Pi] in positive control samples over the course of the day. Further investigation indicated that hydrolysis of ATP catalyzed by PurC, rather than substrate-related phosphate release, was responsible for a partial contribution to the signals in the control samples. Thus substrate-independent ATPase activity may complicate high throughput screening

Structure and dynamics of the liver receptor homolog 1–PGC1α complex

Peroxisome proliferator-activated gamma coactivator 1-α (PGC1α) regulates energy metabolism by directly interacting with transcription factors to modulate gene expression. Among the PGC1α binding partners is liver receptor homolog 1 (LRH-1; NR5A2), an orphan nuclear hormone receptor that controls lipid and glucose homeostasis. Although PGC1α is known to bind and activate LRH-1, mechanisms through which PGC1α changes LRH-1 conformation to drive transcription are unknown. Here, we used biochemical and structural methods to interrogate the LRH-1–PGC1α complex. Purified, full-length LRH-1, as well as isolated ligand binding domain, bound to PGC1α with higher affinity than to the coactivator, nuclear receptor coactivator-2 (Tif2), in coregulator peptide recruitment assays. We present the first crystal structure of the LRH-1–PGC1α complex, which depicts several hydrophobic contacts and a strong charge clamp at the interface between these partners. In molecular dynamics simulations, PGC1α induced correlated atomic motion throughout the entire LRH-1 activation function surface, which was dependent on charge-clamp formation. In contrast, Tif2 induced weaker signaling at the activation function surface than PGC1α but promoted allosteric signaling from the helix 6/β-sheet region of LRH-1 to the activation function surface. These studies are the first to probe mechanisms underlying the LRH-1–PGC1α interaction and may illuminate strategies for selective therapeutic targeting of PGC1α-dependent LRH-1 signaling pathways