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dc.contributor.advisorChairperson, Graduate Committee: Jennifer DuBoisen
dc.contributor.authorCelis Luna, Arianna I.en
dc.contributor.otherBennett R. Streit, Garrett C. Moraski, Timothy D. Lash, Gudrun S. Lukat-Rodgers, Kenton R. Rodgers and Jennifer L. DuBois were co-authors of the article, 'Unusual peroxide-dependent, heme-transforming reaction catalyzed by hemQ' in the journal 'Biochemistry' which is contained within this thesis.en
dc.contributor.otherGeorge H. Gauss, Bennett R. Streit, Krista Shisler, Garrett C. Moraski, Kenton R. Rodgers, Gudrun S. Lukat-Rodgers, John W. Peters and Jennifer L. DuBois were co-authors of the article, 'A structure-based mechanism for oxidative decarboxylation reactions mediated by amino acids and heme propionates in coproheme decarboxylase (hemQ)' in the journal 'Journal of the American Chemical Society' which is contained within this thesis.en
dc.contributor.otherDissertation contains two articles of which Arianna I. Celis Luna is not the main author.en
dc.date.accessioned2018-12-05T19:30:05Z
dc.date.available2018-12-05T19:30:05Z
dc.date.issued2018en
dc.identifier.urihttps://scholarworks.montana.edu/xmlui/handle/1/14685
dc.description.abstractHeme b is one of nature's most ancient and versatile co-factors and is essential for aerobic life. As such, heme b is synthesized by almost every living organism and plays a major role in bacterial virulence. A pathway for heme b biosynthesis, which is unique to some of the most primitive gram-positive bacteria including many important pathogens, was recently discovered. This pathway, now known as the coproprophyrin-dependent (CPD) branch, ends in a step catalyzed by an unusual enzyme known alternately as coproheme decarboxylase (ChdC) or HemQ. This research aimed to understand ChdC function at the molecular, structural, and cellular levels. Using the ChdC enzyme from Staphylococcus aureus (SaChdC) and a variety of biochemical and analytical tools (conventional and stopped-flow UV-Vis spectroscopy, resonance Raman, HPLC, LC-MS, site-directed mutagenesis, EPR, and X-ray crystallography), the work presented here describes how the coproheme substrate is accommodated in the SaChdC active site and poised for reactivity. The cumulative results show that ChdC catalyzes the oxidative decarboxylation of coproheme III to generate heme b in a sequential and clock-wise fashion, generating harderoheme III in the process. This reaction is H 2 O 2-dependent and the mechanism involves the formation of the high-valent Fe(IV) intermediate (Compound I) and a tyrosine radical (Tyr °). The coproheme-bound ChdC structure revealed a helical-loop that is flexible and moves in towards the active site in the presence of substrate. This loop is hypothesized to act as an 'active site gate' which mediates substrate entry and product egress. Due to the cytotoxicity of heme and its porphyrin precursors, we proposed that the metabolite flux in this pathway is controlled by transient protein-protein interactions. Using the UV-Vis characteristics of porphyrins and phenotype characterization of the deltachdC knock-out strain of S. aureus complemented with ChdC point mutants, we present preliminary evidence for an interaction between ChdC the preceding enzyme of the pathway, CpfC. The same approaches also implicated potential interactions between ChdC and an unidentified heme-chaperone, which delivers heme to its final cellular destination. We propose that this chaperone is HemW. Experiments to test this hypothesis are outlined. This work elucidates yet different way that nature has equipped cells to perform radical chemistry in order to accomplish essential molecule transformations, such as that of decarboxylation and the simultaneous generation of CO 2, and emphasizes the importance of substrate/product post-catalysis cellular trafficking.en
dc.language.isoenen
dc.publisherMontana State University - Bozeman, College of Letters & Scienceen
dc.subject.lcshHeme.en
dc.subject.lcshBiosynthesis.en
dc.subject.lcshBacteria.en
dc.subject.lcshLyases.en
dc.subject.lcshCatalysts.en
dc.titleA molecular, structural, and cellular multiple-level study aimed at understanding the unique reaction catalyzed by the last enzyme in the heme-biosynthesis pathway of gram-positive bacteria, coproheme decarboxylase (CHDC)en
dc.typeDissertationen
dc.rights.holderCopyright 2018 by Arianna I. Celis Lunaen
thesis.degree.committeemembersMembers, Graduate Committee: Jennifer DuBois (chairperson); Brian Bothner; Valorie Copie; Joan B. Broderick; John W. Peters.en
thesis.degree.departmentChemistry & Biochemistry.en
thesis.degree.genreDissertationen
thesis.degree.namePhDen
thesis.format.extentfirstpage1en
thesis.format.extentlastpage288en
mus.data.thumbpage250en


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