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dc.contributor.advisorLamb, Audrey L
dc.contributor.authorRonnebaum, Trey A
dc.date.accessioned2019-05-19T02:10:35Z
dc.date.available2019-05-19T02:10:35Z
dc.date.issued2018-12-31
dc.date.submitted2018
dc.identifier.otherhttp://dissertations.umi.com/ku:16176
dc.identifier.urihttp://hdl.handle.net/1808/28051
dc.description.abstractPathogenic bacteria are becoming increasingly resistant to antibiotics. In response to this alarming trend, the scientific community must determine therapeutic targets. Essential nutrients, such as iron, are necessary for pathogens to survive and become virulent within a host system. One mechanism used by pathogenic bacteria to acquire iron from its surrounding environment is to produce low-molecular weight compounds which have a high affinity towards ferric iron. These compounds are called siderophores, and studies have shown their production to be essential for growth and virulence of some pathogens in iron-limited environments, such as the human host. Siderophores are often biosynthesized by nonribosomal peptide synthetases (NRPSs), which rarely have human homologs, making them attractive targets for novel therapeutics. NRPSs are enzymes utilized by bacteria, fungi, and plants to generate bioactive peptides. These bioactive peptides are not only used as secondary metabolites (toxins, pigments, siderophores) but have also found their way into the clinic as antibiotics, anticancer drugs, and immunosuppressants. To elicit their unique bioactivity, these peptides are tailored, making the compound chemically unique. Natural product chemists, metabolic engineers, and researchers in biochemistry and biotechnology work to exploit NRPS biosynthesis to generate new compounds for clinical use. This dissertation describes mechanistic and structural analyses of the adenylation and tailoring domains of the NRPS biosystem responsible for the production of pyochelin, a siderophore produced by antibiotic resistant Pseudomonas aeruginosa. A large portion of this work aims to better understand adenylation and “stuffed” tailoring didomains which lack structural characterization and have limited mechanistic understanding yet are ubiquitous in NRPS bioassembly. Pyochelin biosynthesis employs an adenylation-epimerase stuffed didomain in PchE and an adenylation-methyltransferase stuffed didomain in PchF. Substrate and product analogs were synthesized and steady-state adenylation, epimerase, and methyltransferase assays, along with onium chalcogen effects of the methyltransferase reaction, were used to characterize the adenylation-tailoring stuffed domains in pyochelin bioassembly. Similarly, substrate analogs were generated and used in steady-state kinetic and crystallography experiments with the stand-alone tailoring, NADPH-dependent reductase, PchG, and homolog, Irp3, of yersiniabactin biosynthesis. Finally, a steady-state adenylation assay was developed for the stand-alone salicylate adenylation enzyme, PchD, and potential warhead inhibitors were synthesized and co-crystallized laying the groundwork for future inhibitor design.
dc.format.extent188 pages
dc.language.isoen
dc.publisherUniversity of Kansas
dc.rightsCopyright held by the author.
dc.subjectBiochemistry
dc.subjectChemistry
dc.subjectepimerase
dc.subjectmethyltransferase
dc.subjectnatural product biosynthesis
dc.subjectnonribosomal peptide synthesis
dc.subjectpyochelin
dc.subjectstuffed domains
dc.titleADENYLATION AND TAILORING ACTIVITIES IN THE NONRIBOSOMAL PEPTIDE SYNTHESIS OF THE SIDEROPHORE PYOCHELIN
dc.typeDissertation
dc.contributor.cmtememberDunn, Robert C
dc.contributor.cmtememberJackson, Timothy A
dc.contributor.cmtememberBlakemore, James D
dc.contributor.cmtememberHageman, Michael J
dc.thesis.degreeDisciplineChemistry
dc.thesis.degreeLevelPh.D.
dc.identifier.orcidhttps://orcid.org/0000-0002-9214-3564
dc.rights.accessrightsopenAccess


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