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dc.contributor.advisorLamb, Audrey L.
dc.contributor.authorOlucha, José
dc.date.accessioned2012-10-28T17:26:55Z
dc.date.available2012-10-28T17:26:55Z
dc.date.issued2012-05-31
dc.date.submitted2012
dc.identifier.otherhttp://dissertations.umi.com/ku:12019
dc.identifier.urihttp://hdl.handle.net/1808/10330
dc.description.abstractTo survive and establish infections in host tissues, pathogens must compete with the host organism for iron. One strategy for iron acquisition is to excrete iron-chelators, called siderophores, with very high affinity to ferric iron. Studies have shown siderophores to be associated with growth and virulence of some pathogens. Inhibition of siderophore production is therefore considered an attractive target for the development of novel antimicrobial agents. This dissertation describes biochemical investigations of two enzymes involved in siderophore production in Pseudomonas aeruginosa, an opportunistic pathogen. PchB catalyzes two pericyclic reactions in a single active site: 1.) an isochorismate-pyruvate lyase reaction (breakdown of isochorismate to salicylate and pyruvate) and 2.) a chorismate mutase reaction (rearrangement of chorismate to prephanate). There is an ongoing debate in the field over the relative contributions of Near Attack Conformations (NAC) and Transition State Stabilization (TSS) to the molecular mechanism of pericyclic reactions. Steady-state kinetics of a K42H-PchB mutant with a pH-dependent charge on position 42 on the active site loop, previously shown to be important for catalysis reveals that lyase and mutase activities require the positive charge at that position for efficient catalysis. Covalent and non-covalent chemical-rescue experiments on mutants deficient of the positive charge at position 42 suggest that the positive charge at the 42 position must be organized within the active site for efficient catalysis. Finally, quantum mechanical/molecular mechanical experiments on wild type and K42H PchB models look at the mechanism of catalysis of the lyase activity in more detail. PvdA is an accessory enzyme to the siderophore pyoverdin biosynthetic pathway of Pseudomonas aeruginosa. PvdA is an N-hydroxylating ornithine hydroxylase: it catalyzes the FAD dependent addition of oxygen to the N5-amine of the ornithine using NADPH as electron donor and molecular oxygen. Here we present two structures of PvdA with FAD in oxidized (1.9 Å resolution) and reduced (3.03 Å resolution) flavin forms. These are the first two structures of an N-hydroxylating Class B flavoprotein monooxygenase and the first structures of a Class B flavoprotein monooxygenase to contain redox center, electron donor and product/substrate in the active site bound simultaneously.
dc.format.extent215 pages
dc.language.isoen
dc.publisherUniversity of Kansas
dc.rightsThis item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
dc.subjectBiochemistry
dc.subjectBiophysics
dc.subjectBioinformatics
dc.subjectIsochorismate-pyruvate lyase
dc.subjectOrnithine hydroxylase
dc.subjectPseudomonas aeruginosa
dc.subjectPyochelin
dc.subjectPyoverdin
dc.titleStructural, Functional and Computational Characterization of Pseudomonas aeruginosa Siderophore Biosynthetic Pathway Accessory Proteins PchB and PvdA
dc.typeDissertation
dc.contributor.cmtememberRichter, Mark
dc.contributor.cmtememberHefty, P. Scott
dc.contributor.cmtememberDe Guzman, Roberto N.
dc.contributor.cmtememberAzuma, Yoshiaki
dc.contributor.cmtememberPrisinzano, Thomas E.
dc.thesis.degreeDisciplineMolecular Biosciences
dc.thesis.degreeLevelPh.D.
kusw.oastatusna
kusw.oapolicyThis item does not meet KU Open Access policy criteria.
dc.rights.accessrightsopenAccess


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