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Access Type

WSU Access

Date of Award

January 2019

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Biochemistry and Molecular Biology

First Advisor

David R. Evans

Abstract

Sepsis affects 1.7 million people in the United States every year and nearly 270,000 people die as a result. Sepsis is characterized by systemic inflammation from an infection leading to organ dysfunction and death. Multi-drug resistance in bacteria is increasing globally, and Pseudomonas aeruginosa and Staphylococcus aureus are notorious for their multi-drug resistance and pose a serious need for the development of new antibiotics. The levels of pyrimidines in blood are too low to sustain the growth of bacteria, so they must rely on pyrimidine biosynthesis. Previous studies have shown that a defect in several pyrimidine biosynthetic enzymes resulted in 1000-fold decrease in the titer of bacteria growing in blood. Aspartate transcarbamoylase (ATCase) catalyzes the first committed step of the de novo pyrimidine pathway, making it an attractive drug target in pyrimidine biosynthesis. The following dissertation utilizes biochemical and biophysical tools to understand the regulatory and structural properties of ATCase in P. aeruginosa and S. aureus as well as to identify potential sites for ATCase inhibition.

In this dissertation, I demonstrate that pDHO is a requirement for P. aeruginosa ATCase because ATCase does not self-associate to form a stable trimer and that pDHO facilitates ATCase trimer formation. The basis for why pDHO was a requirement for ATCase activity was not explored previously. Additionally, I explore the role of N- and C-terminal extensions of P. aeruginosa ATCase and demonstrate that these extensions are not involved in the ATCase-pDHO complex formation or nucleotide inhibition by ATP. This led me to conclude that the binding site of ATP is not located in the 11-residue extension of ATCase as previously suggested. Furthermore, I demonstrate that ATP inhibition of ATCase is not an allosteric mechanism, rather it is a consequence of the destabilization of the ATCase-pDHO complex. Lastly, I solved the crystal structure of P. aeruginosa delN ATCase-pDHO complex with a molecule of TEW bound in the active site of delN ATCase and demonstrated that TEW is a potent inhibitor of P. aeruginosa ATCase.

In my dissertation, I demonstrate that S. aureus ATCase is a stable catalytic trimer. S. aureus ATCase functions independently and does not associate with DHOase. I solved the crystal structure of S. aureus ATCase bound to an active site inhibitor, N-phosphonacetyl-L-aspartate (PALA), and validated that S. aureus ATCase undergoes domain closure upon PALA binding as observed in other ATCases. Using this crystal structure of S. aureus ATCase, I identified two sites for inhibition, including the interdomain cleft and the trimer interface. I screened top-ranking FDA-approved compounds against S. aureus ATCase in an in vitro assay after virtual screening. Although none of the compounds had a significant inhibitory effect on S. aureus ATCase, nitazoxanide showed dose-dependent destabilization of ATCase. This suggests that nitazoxanide does not show inhibitory activity probably due to its poor binding to ATCase.

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