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dc.contributor.authorSuits, Michael Douglas Leo, 1978-en
dc.date2007-04-27 11:34:50.272
dc.date.accessioned2007-07-05T15:06:47Z
dc.date.available2007-07-05T15:06:47Z
dc.date.issued2007-07-05T15:06:47Z
dc.identifier.urihttp://hdl.handle.net/1974/429
dc.descriptionThesis (Ph.D, Biochemistry) -- Queen's University, 2007-04-27 11:34:50.272en
dc.description.abstractFor pathogenic microorganisms, heme uptake and degradation is a critical mechanism for iron acquisition that enables multiplication and survival within hosts they invade. While the bacterial proteins involved in heme transport had been identified at the initiation of our investigation, the fate of heme once it reached the cytoplasm was largely uncharacterized. Here we report the first crystal structures of two members of the heme utilization operon from the human pathogen Escherichia coli O157:H7. These are the heme oxygenase ChuS in its apo and heme-complexed forms, and the apo form of heme binding protein ChuX. Surprisingly, despite minimal sequence similarity between the N- and C-terminal halves, the structure of ChuS is a structural repeat. Furthermore, the ChuS monomer forms a topology that is similar to the homodimeric structure of ChuX. Based on spectral analysis and carbon monoxide measurement by gas chromatography, we demonstrated that ChuS is a heme oxygenase, the first to be identified in any E. coli strain. We also show that ChuS coordinates heme in a unique fashion relative to other heme oxygenases, potentially contributing to its enhanced activity. As ChuS and ChuX share structural homology, we extended the structural insight gained in our analysis of ChuS to purport a hypothesis of heme binding for ChuX. Furthermore, we demonstrated that ChuX may serve to modulate cytoplasmic stores of heme by binding heme and transferring it to other hemoproteins such as ChuS. Based on sequence and structural comparisons, we designed a number of site-directed mutations in ChuS and ChuX to probe heme binding sites and mechanisms in each. ChuS and ChuX mutants were analyzed through reconstitution experiments with heme and functional analyses, including enzyme catalysis by ChuS and mutants, and in culture development during heme challenge experiments by ChuX and mutants. Taken together, our results suggested that ChuX acts upstream of ChuS, and regulates heme uptake through ChuX-mediated heme binding and release. ChuS can degrade heme as a potential iron source or antioxidant, thereby contributing directly to E. coli O157:H7 pathogenesis. Functional implications that may be revealed from sequence and structure based information will be addressed as they pertained to our evaluation of ChuS and ChuX.en
dc.format.extent6886078 bytes
dc.format.mimetypeapplication/pdf
dc.language.isoengen
dc.relation.ispartofseriesCanadian Thesesen
dc.rightsThis publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.en
dc.subjectX-Ray Crystallographyen
dc.subjectChuSen
dc.subjectChuXen
dc.subjectEscherichia coli O157:H7en
dc.subjectHeme Utilization Operonen
dc.subjectStructureen
dc.subjectHeme Oxygenaseen
dc.subjectHemeen
dc.titleStructural determination and functional annotation of ChuS and ChuX, two members of the heme utilization operon in pathogenic Escherichia coli O157:H7en
dc.typethesisen
dc.description.degreePhDen
dc.contributor.supervisorJia, Zongchaoen
dc.contributor.departmentBiochemistryen
dc.degree.grantorQueen's University at Kingstonen


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