Mechanistic Studies of Flavin-Dependent Monooxygenases Involved in Bacterial Defense and Plant Metabolism

Abstract

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.