Date of Award
Doctor of Philosophy (PhD)
The rapid emergence of bacteria that are resistant to todayfs antibiotics makes them more and more ineffective. Consequently, the need for a novel class of antibacterial agents is rapidly increasing. The importance of this project is emphasized by the emergence of several pathogenic bacterial strains that are resistant to all currently available antibiotics on the market today. One way to approach this problem is to develop drugs that target essential bacterial biosynthetic pathways. Based on bacterial genetic information, the meso]diaminopimelate (mDAP)/lysine biosynthetic pathway offers several potential anti]bacterial targets that have not been yet explored. One of these, the dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) catalyzes the hydrolysis of N-succinyl-L,L-diaminopimelate to L,L-diaminopimelate and succinate. Since there are no similar pathways in mammals, inhibitors that target one or more of the enzymes in the mDAP/lysine biosynthetic pathway will likely exhibit selective toxicity against only bacteria.
It has been shown experimentally that deletion of the gene encoding for DapE is lethal to Helicobacter pylori and Mycobacterium smegmatis. Therefore, DapEs are crucial for bacterial cell growth and proliferation, making them potential targets for a new class of antibiotics. Rational design of inhibitors for DapE relies on understanding the active site structure and catalytic mechanism. Therefore, the DapE enzymes from Haemophilus influenzae and Neisseria meningitidis were characterized biochemically and crystallographically. In addition, residues situated within the active site were mutated and the mutated enzymes were characterized via kinetic and crystallographic methods in order to gain insight into substrate binding. In order to examine the role of the catalytic domain of DapE truncated enzymes from Vibrio cholera and H. influenzae were engineered by deleting their dimerization domains. And finally, a reliable and reproducible spectrophotometric assay for DapE enzymes that is conducive to high-throughput screening was developed.
Starus, Anna, "The DapE-Encoded N-Succinyl-L,L-Diaminopimelic Acid Desuccinylase (DapE) from Haemophilus Influenzae as a Prospective Target for the Development of Novel Antibiotics" (2014). Dissertations. 1306.
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