N-hydroxylating monooxygenases (NMOs) are Class B flavin-dependent monooxygenases found only in fungi and bacteria. These enzymes catalyze the hydroxylation of nucleophilic primary amines, such as those found in histamine, L-ornithine, L-lysine, and small aliphatic diamines. The hydroxamate moiety produced by this reaction is key for the production of siderophores, small chelating compounds that allow survival in iron limiting conditions. NMOs involved in siderophore biosynthesis have been shown to be essential for pathogenesis in organisms such as Aspergillus fumigatus, Pseudomonas aeruginosa, and Mycobacterium tuberculosis. Therefore, NMOs are considered novel drug targets for the treatment associated with these diseases. Herein we present the characterization of TheA, an NMO from Thermocrispum agreste. The enzyme mechanism was studied using steady state kinetic measurements, thermostability, and stopped flow spectrophotometry assays. Using these techniques, the catalytic rates, substrate binding affinities, thermal stability, and coenzyme specificities were determined. Additionally, NADPH analogues were produced to use as tools to study FAD reduction in NMOs. An unspecific reduction reaction of NADP+ using NaB2H4 yielded [6-2H]-NADPH, [2-2H]-NADPH, and [4-2H]-NADPH. Compound identity was confirmed by mass spectrometry and unidimensional proton nuclear magnetic resonance (NMR). Results presented in this thesis lay the foundation for future studies of NMOs using NADPH analogues. In conjunction, these results will improve the general knowledge and understanding of flavoenzymes, ornithine monooxygenases, and their associated mechanisms. / Master of Science in Life Sciences / Due to the surge of more potent and prevalent microbial pathogens, there is a constant search for new and more specific drugs. One approach is to identify and inhibit enzymes that are key for growth of these pathogens. An example of such enzymes is a group called N-hydroxylating monooxygenases (NMOs) that are key for the production of siderophores, small chelating compounds that allow survival of some fungi and bacteria in iron limiting conditions. NMOs involved in siderophore biosynthesis have been shown to be essential for pathogenesis in organisms such as Aspergillus fumigatus, Pseudomonas aeruginosa, and Mycobacterium tuberculosis. Therefore, NMOs are considered novel drug targets for the treatment associated with these diseases. To develop specific inhibitors of NMOs that can be used as drugs to treat these infections, we first need to understand how these enzymes work. Herein, we characterized a novel NMO from Thermocrispum agrestre. Our results highlight the similarities and differences from previously characterized NMOs. Furthermore, we produced analogues of NADPH, a molecule needed for the mechanism of NMOs. The produced compounds will be used in future studies to understand the step-bystep mechanism of the enzyme. In conjunction, these results will improve the general knowledge and understanding of NMOs and their associated mechanisms and lay the foundation for future studies on the identification of drugs that can be used to treat these diseases.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/83785 |
| Date | 26 June 2018 |
| Creators | Mena Aguilar, Didier Philippe |
| Contributors | Biochemistry, Sobrado, Pablo, Xu, Bin, Slade, Daniel J. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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