N-hydroxylating monooxygenases (NMO) are members the class B flavoprotein monooxygenases. They catalyze the N-hydroxylation of lysine and ornithine and play and essential role in the biosynthesis of hydroxamate containing siderophores. Siderophores are high affinity iron-chelators composed of catechol and hydroxamate functional groups that are synthesized and secreted by several microorganisms and plants. It has been showed that many NMOs are essential for virulence in many opportunistic pathogens such as Aspergillus fumigatus and Pseudomonas aeruginosa. The focus of my research is on the N-hydroxylating enzymes: Siderophore A (SidA) from Aspergillus fumigatus and Amycolatoposis alba monooxygenase (AMO).
One of my projects is focusing on identifying inhibitors of SidA that will ultimately block the siderophore biosynthesis in A. fumigatus. Out of 973 compounds screened using an activity high-throughput assays two compounds were identified. These were, wortmannin a steroid metabolite and ebselen a benzoselenazole as SidA inhibitors with IC50 values of 369 µM and 11 µM respectively. A second part of this works investigates the hydroxamate formation of the siderophore albachelin in Amycolatoposis alba with the purpose of better understanding this class of enzymes and their catalytic mechanism. The enzyme was purified and characterized in its holo (FAD-bound) and apo (unbound) forms. Pre-steady and steady state kinetics shows that the two forms have different coenzyme preference; apo-AMO prefers NADH while holo-AMO has a higher affinity to NADPH. / Master of Science in Life Sciences
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/85845 |
| Date | 23 May 2017 |
| Creators | Bufkin, Kendra Bernice |
| Contributors | Biochemistry, Sobrado, Pablo, Mackey, Zachary B., White, Robert H. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Thesis |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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