Natural products are biologically active metabolites produced by fungi, bacteria, and plants that have an extended application in pharmaceutical and chemical industries. Because of their chemical versatility, flavoenzymes are commonly involved in natural product biosynthetic pathways. This has given rise to the identification of flavoenzymes that are promising candidates for biomedical and biotechnical applications. This dissertation discusses the characterization of three flavoenzymes involved in natural product biosynthesis. The class B flavin-dependent monooxygenases S-monoooxygenase from Allium sativum (AsFMO) and N-hydroxylating monooxygenase from Streptomyces sp. XY332 (FzmM) were studied. Both enzymes perform heteroatom oxidation as part of allicin or fosfazinomycin biosynthesis respectively. AsFMO was predicted to oxidize S-allyl-L-cysteine (SAC) to alliin in allicin biosynthesis. Surprisingly, AsFMO exhibited negligible activity with SAC, and instead was highly active with allyl mercaptan and NADPH. This contradicted the initial proposal and suggested that AsFMO is involved in an alternative path producing allicin directly from allyl mercaptan. FzmM was identified to perform multiple N-oxidations which lead to the formation of a nitro group. FzmM performed a highly coupled and specific reaction with L-aspartate and NADPH to produce nitrosuccinate. Both AsFMO and FzmM followed a kinetic mechanism representative of class B flavin-dependent monooxygenases with a rapid pro-R stereospecific reduction and the formation of a C(4a)-hydroperoxyflavin intermediate during oxidation. In addition, the AsFMO structure was obtained and consisted of two domains for FAD and NADPH binding signature of class B monooxygenases. The biochemical and structural study of the Acinetobacter baumannii siderophore interacting protein (BauF) was also accomplished. This enzyme is essential in acinetobactin mediated iron assimilation and is important for virulence. The characterization of the binding and reduction of acinetobactin-ferric iron complex revealed that BauF is specific for this substrate and does not utilize NAD(P)H as an electron donor. The unique activity and structure of BauF can aid future drug design. / Doctor of Philosophy / Plants, fungi, and bacteria synthesize and excrete unique chemicals called secondary metabolites or natural products. These compounds are used for many applications including dyes, flavorings, fragrances, and medicine. To make natural products, organisms use enzymes to perform complex reactions. Studying the enzymes that are involved in natural product pathways is important for understanding how secondary metabolites are made. Additionally, these enzymes can be engineered to perform reactions relevant to biotechnical applications. Our lab specializes in the study of flavoenzymes which use flavin chemistry for catalysis. Flavin is a yellow coenzyme that contributes to wide array of reactions by performing 1 or 2 electron transfers. This dissertation discussed the characterization of three flavoenzymes. The first enzyme is a S- monooxygenase from Allium sativum (garlic) called AsFMO. Reported here is the kinetic and structural characterization of AsFMO. We demonstrated that AsFMO was cabable of performing an unexpected reaction with allyl mercaptan likely converting it into allicin, the main flavor ingredient of garlic. Secondly, we reported the kinetic characterization of a nitro- forming enzyme termed FzmM. Nitro- formation is a valuable process as nitro- compounds are used in industrial organic synthesis. It was shown that FzmM performs nitro- formation with high efficiency and is specific for the substrate L-aspartate. Lastly, this work described the characterization of the the siderophore-interacting protein from Acinetobacter baumannii, BauF, which was predicted to be involved in iron acqusition. A. baumannii is a serious human pathogen with multidrug resistance, and inhibiting iron acquisition has been shown to prevent its survival. The characterization of the enzymes involved in this pathway is essential for developing new treatments for A. baumannii infection. We report the structure and function of BauF confirming its role in A. baumannii iron uptake and providing information that will aid in future drug design.

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.

Flavin-dependent monooxygenases (FMOs) are a large family of enzymes found in microbes, plants, animals, and humans involved in defense pathways, xenobiotic metabolism, and natural product biosynthesis. One class of FMOs, Class B, carries out the oxidation of heteroatomic substrates, via hydroxylation, S-oxygenation, Baeyer-Villiger oxidation, and decarboxylation, using NAD(P)H as a coenzyme. In this dissertation, the characterization of several FMOs from bacteria and plants is described. The putrescine N-monooxygenase (NMO) FbsI from Acinetobacter baumannii is involved in the fimsbactin siderophore biosynthetic pathway, a virulence factor that allows acquisition of free iron from a human host by a pathogen. We show that putrescine is hydroxylated to form N-hydroxyputrescine and is favored over the aliphatic diamine cadaverine and amino acid L-ornithine. The three-dimensional structure of FbsI was solved and shown to have similarities to other NMOs, and characterization of active site mutants revealed residues essential for catalysis and cofactor specificity. The flavin-dependent S-monooxygenase TvMAS1 from the society garlic Tulbaghia violacea has been implicated in the production of marasmin, a natural product with human health benefits. We find that TvMAS1 has a broad substate scope among thiol and sulfide-containing compounds, particularly L-cysteine derivatives. Additionally, we show that S-allyl-L-cysteine is the preferred substrate and propose TvMAS1 to primarily have a physiological role in allicin, not marasmin biosynthesis. Lastly, we characterized the auxin-producing FMO YUC10 from Arabidopsis thaliana and showed the enzyme to only have steady-state activity with aromatic α-keto acids indole-3-pyruvic acid (IPA) and phenylpyruvic acid (PPA). We also propose that a C4a-peroxyflavin intermediate acts as a nucleophile to perform the oxidative decarboxylation on IPA and PPA. The work in this dissertation fills several knowledge gaps among bacterial and plant FMOs and with the established mechanisms aims to guide future drug discovery, green chemistry, and agricultural bioengineering efforts. / Doctor of Philosophy / The water-soluble vitamin riboflavin, also known as Vitamin B2, is an essential nutrient with numerous human health benefits and known for its characteristic yellow-orange color. The major role of riboflavin is in the synthesis of the coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are involved in biological processes such as energy metabolism and detoxification. Flavoenzymes are proteins found in all living organisms that use either FMN or FAD to facilitate otherwise unfavorable biochemical reactions in living systems. One class of flavoenzymes, known as flavin-dependent monooxygenases (FMOs), carries out oxidation-reduction reactions on an array of small molecules with the aid of molecular oxygen. In this dissertation, we present the characterization of three FMOs and propose their roles in nature. The pathogenic bacterium Acinetobacter baumannii can survive in human hosts using several virulence factors, one of which are siderophores – molecules that scavenge iron and transport it back to the organism. We determined that the enzyme FbsI hydroxylates the amine-containing substrate putrescine into N-hydroxyputrescine, a building block of the A. baumannii siderophore fimsbactin A. By solving the enzyme's three-dimensional structure and characterizing its reaction mechanism, we hope to guide future drug discovery studies of this target. We also describe the role of two FMOs from the society garlic Tulbaghia violacea (TvMAS1) and the thale cress Arabidopsis thaliana (YUC10). We show that TvMAS1 performs S-oxidation of S-allyl-L-cysteine to alliin with high efficiency, implicating a role in the production of allicin - a flavorful compound with health benefits. Finally, we show that YUC10 is involved in the production of auxin, a plant hormone that directs growth and development. A novel chemical mechanism for the YUCCAs is proposed, providing insight from in vitro experiments to guide in vivo findings. Our work with TvMAS1 and YUC10 will help guide future protein engineering and green chemistry efforts in the agricultural industry.