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dc.contributor.advisorLamb, Audrey L.
dc.contributor.authorLuo, Qianyi
dc.date.accessioned2010-06-09T04:19:35Z
dc.date.available2010-06-09T04:19:35Z
dc.date.issued2009-04-29
dc.date.submitted2009
dc.identifier.otherhttp://dissertations.umi.com/ku:10351
dc.identifier.urihttp://hdl.handle.net/1808/6305
dc.description.abstractIron is an essential element for most pathogenic bacteria. To survive and establish infections in host tissues, these pathogens must compete with the host organism for iron. One strategy is to excrete iron-chelator siderophores with very high affinity to ferric iron in the low iron environment of the host. The phenolate type siderophore, such as pyochelin in Pseudomonas aeruginosa, uses salicylate derived from chorismate as a precursor. Studies have shown that the salicylate activation by adenylation for incorporation to siderophores is associated with the growth and virulence of some pathogens. The inhibition of salicylate biosynthesis, and hence, siderophore production is considered an attractive target for the development of novel antimicrobial agents. In P. aeruginosa, salicylate is derived from chorismate via isochorismate by two enzymes: isochorismate synthase (PchA) and isochorismate-pyruvate lyase (IPL, PchB). PchB eliminates the enolpyruvyl side chain from isochorismate through a biologically unusual pericyclic reaction mechanism. PchB can also perform an adventitious pericyclic reaction that rearranges chorismate to prephenate possibly due to the homology to the E. coli chorismate mutase (CM). The primary contribution to lower the activation energy for enzymatic pericyclic reactions is controversial and may be arise from electrostatic stabilization of the transition state, or conformational stabilization of the reactive substrate. Structural and mutational studies on a key residue, lysine 42, of the active site loop suggest that rate enhancement of the two pericyclic reactions (IPL and CM) performed by PchB results from both the transition state stabilization and the reactive substrate conformation, but the relative contributions are different for each reaction. A mutation with less active site loop mobility, A43P indicates that the loop dynamics is related to catalysis. The I87T structure reveals a larger disordered region compared to the wild type structure, suggesting that conformational mobility may play a role in catalysis. PchA is an isochorismate synthase (ICS) in P. aeruginosa that removes the C4 hydroxyl group and adds a hydroxyl group to C2-chorismate. PchA is homologous to salicylate synthases from Yersinia spp. and Mycobacterium tuberculosis that convert chorismate to salicylate without requirement of an additional lyase such as PchB in P. aeruginosa. A sequence comparison between PchA with salicylate synthases of known structure suggests that two conserved residues are directly involved in the general acid and base chemistry in PchA: K221 as the general base and E269 as the general acid. Replacement of K221 and E269 with alanine respectively led to catalytically inactive enzymes, suggesting that K221 and E269 are critical for ICS catalysis. Preliminary pH dependence data for PchA supports the general acid and base mechanism of PchA catalysis. Two nonconserved residues A375 and D310 were also examined. Replacement of A375 by threonine, the corresponding residue in salicylate synthase, resulted in only residual ICS activity, indicating that A375 is not associated with the IPL-deficiency in PchA. The D310E mutant leads to two additional activities, IPL and CM. The additional activities in three reactions may due to the preferential orientation of substrates in the active site.
dc.format.extent148 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.subjectChemistry
dc.subjectBiochemistry
dc.subjectChorismate mutase pchb
dc.subjectIsochorismate synthase pcha
dc.subjectIsochorismate-pyruvate lyase pchb
dc.subjectSalicylate biosynthesis
dc.titleMechanistic and Structural Studies of Salicylate Biosynthesis in Pseudomonas aeruginosa
dc.typeDissertation
dc.contributor.cmtememberDe Guzman, Roberto N.
dc.contributor.cmtememberKuczera, Krzysztof
dc.contributor.cmtememberRichter, Mark
dc.contributor.cmtememberScott, Emily E.
dc.contributor.cmtememberPicking, William
dc.thesis.degreeDisciplineBiochemistry & Molecular Biology
dc.thesis.degreeLevelPh.D.
kusw.oastatusna
kusw.oapolicyThis item does not meet KU Open Access policy criteria.
dc.rights.accessrightsopenAccess


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