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

dc.contributor.authorLyons, Noah Scotten
dc.contributor.committeechairSobrado, Pabloen
dc.contributor.committeememberLemkul, Justin Alanen
dc.contributor.committeememberSun, Weien
dc.contributor.committeememberAllen, Kylie Dawnen
dc.contributor.departmentBiochemistryen
dc.date.accessioned2025-03-13T08:00:19Zen
dc.date.available2025-03-13T08:00:19Zen
dc.date.issued2025-03-12en
dc.description.abstractFlavin-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.en
dc.description.abstractgeneralThe 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.en
dc.description.degreeDoctor of Philosophyen
dc.format.mediumETDen
dc.identifier.othervt_gsexam:42640en
dc.identifier.urihttps://hdl.handle.net/10919/124847en
dc.language.isoenen
dc.publisherVirginia Techen
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subjectFlavin-dependent monooxygenasesen
dc.subjectenzyme mechanismen
dc.subjectsiderophoresen
dc.subjectN-hydroxylatingen
dc.subjectAcinetobacter baumanniien
dc.subjectS-oxygenatingen
dc.subjectmarasminen
dc.subjectTulbaghia violaceaen
dc.subjectYUCCAen
dc.subjectauxinen
dc.subjectArabidopsis thalianaen
dc.titleMechanistic Studies of Flavin-Dependent Monooxygenases Involved in Bacterial Defense and Plant Metabolismen
dc.typeDissertationen
thesis.degree.disciplineBiochemistryen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.leveldoctoralen
thesis.degree.nameDoctor of Philosophyen

Files

Original bundle
Now showing 1 - 1 of 1
Name:
Lyons_NS_D_2025.pdf
Size:
8.35 MB
Format:
Adobe Portable Document Format