N-Hydroxylating monooxygenases (NMOs) are flavin dependent enzymes that primarily catalyze the hydroxylation of L-ornithine or L-lysine. This is the first, committed step to siderophore biosynthesis. Pathogenic microbes including Aspergillus fumigatus and Mycobacterium tuberculosis secrete these low molecular weight compounds in order to uptake FeIII from their hosts for their metabolic needs when establishing infection. Therefore, members of this family of enzymes represent novel drug targets for the development of antibiotics. Here, we present the detailed functional and structural analysis of the L-ornithine monooxygenase SidA from Aspergillus fumigatus and the L-lysine monooxygenases MbsG from Mycobacterium smegmatis and NbtG from Nocardia farcinica. The detailed chemical mechanism for flavin oxidation in SidA was elucidated for formation of the C4a-hydroperoxyflavin, deprotonation of L-ornithine, and for the chemical steps of hydrogen peroxide elimination and water elimination. This was performed through a combination of kinetic isotope effect, pH, and density functional theory studies. Also, important residues involved in substrate binding and catalysis were characterized using site-directed mutagenesis for both SidA and NbtG. These include residues involved in coenzyme selectivity, substrate binding, and residues important in C4a-hydroperoxyflavin stabilization and flavin oxidation. The kinetic mechanisms of the L-lysine monooxygenases MbsG and NbtG were characterized which show unique differences with SidA. These include differences in coenzyme selectivity, and C4a-hydroperoxyflavin stabilization. Lastly, the three-dimensional structure of NbtG was solved using X-ray crystallography which is the first structure of a lysine monooxygenase. The structure shows the NADPH-binding domain is rotated ~30° relative to the FAD-binding domain which occludes NADP+ binding in NbtG. Unlike SidA, NbtG does not stabilize a C4a-hydroperoxyflavin and this occlusion observed in the structure might explain this difference. This highlights both the structural and mechanistic diversities among NMOs and the data presented here provides valuable information for the future development of specific inhibitors of NMOs. / Ph. D.

Natural products are secondary metabolites produced by plants and microorganisms that often possess medicinal properties and are implicated in organismal defense. Drawbacks to utilizing natural products in the pharmaceutical industry are difficulties with isolation from biological sources and low yields that can lack stereospecificity from synthetic sources. It is paramount to solve these issues and to develop novel natural products to combat the growing antimicrobial resistance crisis, which was responsible for ~5 million deaths in 2019 alone. One approach is utilizing enzymes to synthesize existing natural products to improve the yields and stereospecificity issue. This dissertation is focused on the biochemical characterization of three enzymes-ZvFMO, OxaD, and CreE-that are implicated in the detoxification of natural products used for organismal defense or participate in the biosynthesis of novel natural products. Each of these enzymes belong to the flavin-dependent monooxygenase (FMO) family, which catalyze the oxygenation of a substrate, generating an oxidized product. ZvFMO, from the insect food crop pest, Zonocerus variegatus, was determined to catalyze a highly uncoupled oxygenation reaction of the nitrogen or sulfur atom of various substrates. OxaD, from Penicillium oxalicum F30, catalyzes novel sequential oxidation reactions of the indole nitrogen of roquefortine C. CreE, from Streptomyces cremeus, also catalyzes sequential nitrogen oxidation reactions to convert L-aspartate to nitrosuccinate en route to biosynthesis of cremeomycin. For each enzyme, the steady-state kinetics have been determined using an oxygen consumption assay and the rapid-reaction kinetics were measured using anaerobic time-resolved spectroscopy. All three enzymes feature a fast flavin reduction step and a slow flavin dehydration step. The oxygenation chemistry of each enzyme was found to proceed through a highly reactive oxygenating species, the C4a-hydroperoxyflavin. Site-directed mutagenesis efforts led to the identification of key active site residues involved in flavin motion and substrate binding, revealing important information about the active site architecture for enzyme engineering applications and drug discovery efforts. / Doctor of Philosophy / Natural products are compounds that are produced by many plants, fungi, and bacteria that have potent medicinal properties and can be used to defend the organism against pests. Unfortunately, using these compounds widely in the pharmaceutical industry is difficult because it is hard to isolate the compound of interest from the organism that produces it and attempts to produce it chemically can result in low yields. Additionally, the overuse of the current natural products, which are most of the antibiotics on the market today, has led to an extreme increase in the resistance of bacteria, fungi, and parasites to the natural product-based drug. Therefore, it is essential that a method is developed to produce novel natural products at high yields to combat the antimicrobial resistance crisis. One method is by using enzymes to generate the natural products of interest. Enzymes are biological catalysts that speed up reactions by ensuring that less energy is required to transition from a reactant to a product and are highly efficient. This dissertation focuses on the characterization of three enzymes that could aid in our understanding of natural product chemistry. All three enzymes insert an oxygen atom on a nitrogen of their respective reactant. The first enzyme ZvFMO, is from an insect and its reactivity causes the insect to become resistant to the natural product-based plant defense mechanism, demonstrating that ZvFMO is a great candidate for inhibitor design. OxaD is the second enzyme and is involved in producing natural products that have antimicrobial and anticancer properties. The last enzyme, CreE, is involved in generating the natural product, cremeomycin, which possesses potent antimicrobial and anticancer properties as well. The reactions of OxaD and CreE positions these enzymes as candidates to produce novel natural products and other efforts to expand their reactivity. The rates of each reaction step have been determined in this work. Key amino acids that contribute to the reaction chemistry and the uptake of the reactant have been identified, laying a solid foundation for drug discovery efforts.