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dc.contributor.authorHernández, Heather Louise
dc.date.accessioned2014-03-04T02:27:15Z
dc.date.available2014-03-04T02:27:15Z
dc.date.issued2006-12
dc.identifier.otherhernandez_heather_l_200612_phd
dc.identifier.urihttp://purl.galileo.usg.edu/uga_etd/hernandez_heather_l_200612_phd
dc.identifier.urihttp://hdl.handle.net/10724/23645
dc.description.abstractA new class of Fe-S proteins, termed radical-SAM enzymes, catalyzes radical reactions in a 2+,+variety of biosynthetic processes. These [4Fe-4S] cluster-containing enzymes initiate radical enzymatic reactions via reductive cleavage of S-adenosyl-L-methionine (SAM) to yield methionine and an extremely reactive 5'-deoxyadenosyl radical. A growing number of radical-SAM enzymes have recently been discovered to contain a second Fe-S cluster of unknown function, although possible roles include acting as sacrificial S-donor or anchoring and possibly activating the substrate. The combination of analytical and spectroscopic studies, including EPR, Mössbauer, UV-visible absorption/circular dichroism/variable temperature magnetic circular dichroism, and resonance Raman, have been used to investigate the cofactor composition and properties of the two cluster containing radical-SAM enzymes Escherichia coli and Bacillus subtilis biotin synthase (BioB), human MOCS1A, and Thermotoga maritima MiaB. These enzymes are involved in crucial steps in the biosynthesis of biotin and molybdopterin, and in the thiomethylation of tRNA. E. coli and B. subtilis BioB are shown to house a radical-SAM [4Fe-4S] cluster and a [2Fe-2S] cluster in separate binding sites. The function and relevance of the [2Fe-2S] cluster, which has been suggested to be the S-donor to biotin, is addressed. In E. coli BioB, the most active form of the enzyme contains a 1:1 ratio of [2Fe-2S]/[4Fe-4S] clusters and the [2Fe-2S] cluster degrades during turnover. However, Mössbauer studies of E. coli BioB show that [2Fe-2S] cluster degradation is at least an order of magnitude faster than the initial rate of biotin formation and B. subtilis BioB exhibits comparable activity in samples depleted in the [2Fe-2S] cluster. Taken together, these results suggest that a [2Fe-2S] cluster degradation product may be the physiologically relevant S-donor. Both MOCS1A and T. maritima MiaB are characterized and shown to contain a radical-SAM [4Fe-4S] cluster as well as a second [4Fe-4S] cluster. In MOCS1A, the second cluster now appears to play a role in anchoring the 5’-GTP substrate. In T. maritima MiaB, the second cluster does not appear to interact with the tRNA substrate and may function as a S-donor in the thiomethylation reaction.
dc.languageeng
dc.publisheruga
dc.rightspublic
dc.subjectRadical-SAM superfamily
dc.subjectSAM
dc.subjectiron-sulfur cluster
dc.subjectradical generation
dc.subjectbiotin synthase
dc.subjectMOCS1A
dc.subjectMiaB
dc.subjectelectron paramagnetic resonance
dc.subjectresonance Raman
dc.subjectmagnetic circular dichroism
dc.subjectMý0 2ssbauer
dc.titleRadical-SAM enzymes with two iron-sulfur clusters
dc.title.alternativecofactor composition and spectroscopic studies of Escherichia coli and Bacillus subtilis biotin synthase, human MOCS1A, and Thermotoga maritima MIAB
dc.typeDissertation
dc.description.degreePhD
dc.description.departmentChemistry
dc.description.majorChemistry
dc.description.advisorMichael K. Johnson
dc.description.committeeMichael K. Johnson
dc.description.committeeMichael W. W. Adams
dc.description.committeeRobert A. Scott


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