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dc.contributor.authorLatimer, Ryan
dc.contributor.otherQueen's University (Kingston, Ont.). Theses (Queen's University (Kingston, Ont.))en
dc.date2010-07-22 16:39:52.059en
dc.date.accessioned2010-07-22T20:59:08Z
dc.date.available2010-07-22T20:59:08Z
dc.date.issued2010-07-22T20:59:08Z
dc.identifier.urihttp://hdl.handle.net/1974/5936
dc.descriptionThesis (Master, Chemistry) -- Queen's University, 2010-07-22 16:39:52.059en
dc.description.abstractThe flavin-dependent halogenases are the most prevalent class of halogenase responsible for the regio- and stereoselective incorporation of halogens into natural products. These enzymes require FAD, O2, NADH, and a flavin reductase to perform catalysis. The majority of these enzymes are observed to catalyze the halogenation of aromatic substrates. CmlS is a unique member of the flavin-dependent halogenase family in that it dichlorinates an alkyl group prior to incorporation onto the antibiotic chloramphenicol. CmlS from the chloramphenicol producing strain Streptomyces venezuelae was cloned and heterologously expressed in Escherichia coli in high yield. The X-ray crystal structure of CmlS was solved to 2.2 Å resolution, yielding key insights into a potential mechanism of catalysis. As well, a covalent bond between D277 and the FAD 8α methyl group was discovered. This is a novel post-translational modification that is likely conserved among a large subset of flavin dependent halogenases and raises interesting questions about the catalytic mechanism of flavin dependent halogenation. Another key enzyme involved in the incorporation of the dichloroacetyl moiety into chloramphenicol is CmlK, an acyl-CoA synthetase. Only through the tandem action of CmlS and CmlK enzymes will chloramphenicol be formed with its dichloroacetyl group. A variety of activity assays were performed with CmlS and CmlK with the hope of discovering their substrate specificities. However, in vitro reconstitution of the activity of CmlS or CmlK was not successful. Future studies are discussed which will hopefully lead to delineating the roles of CmlS and CmlK in the biosynthesis of the dichloroacetyl group, and ultimately a detailed mechanistic description of halogenation by CmlS.en
dc.languageenen
dc.language.isoenen
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.subjectHalogenaseen
dc.subjectChloramphenicolen
dc.titleStudies on the Chloramphenicol Halogenase CmlSen
dc.typeThesisen
dc.description.degreeMasteren
dc.contributor.supervisorZechel, L. Daviden
dc.contributor.departmentChemistryen


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