{"@context":"http://iiif.io/api/presentation/2/context.json","@id":"https://repo.library.stonybrook.edu/cantaloupe/iiif/2/manifest.json","@type":"sc:Manifest","label":"Inhibitors of 3&beta-Hydroxysteroid? Dehydrogenase and Metabolic? and \nEnzymatic Characterization of the Cholesterol? ? Metabolizing Intracellular \nGrowth? Operon? of? Mycobacterium tuberculosis","metadata":[{"label":"dc.description.sponsorship","value":"This work is sponsored by the Stony Brook University Graduate School in compliance with the requirements for completion of degree."},{"label":"dc.format","value":"Monograph"},{"label":"dc.format.medium","value":"Electronic Resource"},{"label":"dc.identifier.uri","value":"http://hdl.handle.net/1951/59889"},{"label":"dc.language.iso","value":"en_US"},{"label":"dc.publisher","value":"The Graduate School, Stony Brook University: Stony Brook, NY."},{"label":"dcterms.abstract","value":"New drugs with novel mechanisms of action are required to meet the \nsevere threat to human health posed by the emergence of multidrug and extensively drug \nresistant strains of Mycobacterium tuberculosis (M. tuberculosis). The cholesterol \nmetabolism pathway in M. tuberculosis is a potential source of energy as well as secondary \nmetabolite production that is important for survival of M. tuberculosis in the host \nmacrophage. Oxidation and isomerization of 3\u0392-hydroxysterols to 4-en-3-ones by M. \ntuberculosis 3\u0392-hydroxysteroid dehydrogenase (3\u0392-HSD) is required for sterol \nmetabolism, and inhibitors of 3\u0392-HSD are important for targeting the cholesterol \nmetabolic pathway. In this work, we evaluated a series of azasteroids for inhibition of \n3\u0392-HSD. Our structure-activity studies indicate that the 6-aza version of cholesterol \nis the best and tightest binding competitive inhibitor (Ki = 100 nM) of the steroid substrate \nand are consistent with cholesterol being the preferred substrate of M. tuberculosis \n3\u0392-HSD. The intracellular growth (igr) operon is required for in vitro growth using \ncholesterol as a sole carbon source. The function of igr operon and its role in cholesterol \nmetabolism is yet to be established. Here we describe the biosynthetic preparation of \nisotopically labeled 13C- [1,7,15,22,26]-cholesterol and employ it as a tool to investigate \nthe cholesterol-derived metabolite profile of the M. tuberculosis H37Rv \u0394igr mutant \nstrain by high resolution LC/MS. Culture supernatants from the \u0394igr mutant accumulate \na cholesterol-derived metabolite not observed in H37Rv wild type or complemented strains. \nMultidimensional NMR and mass spectral analysis revealed the structure of this \ncholesterol-derived catabolite to be a late stage metabolic product: methyl \n1\u0392-(2'-propanoate)-3a\u0391-H-4\u0391(3'-propanoic \nacid)-7a\u0391-methylhexahydro-5-indanone. The computationally annotated functions of the \nsix genes of the igr operon are a lipid transfer protein (ltp2/Rv3540c), two (R)-specific \nenoyl-CoA hydratases (Rv3541c and Rv3542c), two acyl-CoA dehydrogenases (fadE29/Rv3543c and \nfadE28/Rv3544c), and a cytochrome P450 (cyp125/Rv3545c). Heterologous expression in E. coli \ndemonstrated that FadE28 forms a heteromeric complex with FadE29, and that likewise, Rv3542c \nforms a heteromeric complex with Rv3541c. Biophysical characterization of each complex \nestablished they form novel \u03912\u03922 heterotetramers. Using synthetic substrates \nanalogous to the metabolite identified in M. tuberculosis H37Rv \u0394igr mutant strain, we \nverified the catalytic activity of the purified, recombinant FadE28-FadE29 and \nRv3541c-Rv3542c protein complexes to be dehydrogenation and hydration of the \n2'-propanoate-CoA side chain. We conclude the igr operon is required for degradation of \nthe 2'-propanoate side chain fragment during metabolism of cholesterol by M. \ntuberculosis."},{"label":"dcterms.available","value":"2015-04-24T14:47:34Z"},{"label":"dcterms.contributor","value":"Sch? ? rer, Orlando"},{"label":"dcterms.creator","value":"Thomas, Suzanne T."},{"label":"dcterms.dateAccepted","value":"2015-04-24T14:47:34Z"},{"label":"dcterms.dateSubmitted","value":"2013-05-22T17:35:42Z"},{"label":"dcterms.description","value":"Department of \nChemistry"},{"label":"dcterms.extent","value":"151 pg."},{"label":"dcterms.format","value":"Monograph"},{"label":"dcterms.identifier","value":"http://hdl.handle.net/11401/71437"},{"label":"dcterms.issued","value":"2012-08-01"},{"label":"dcterms.language","value":"en_US"},{"label":"dcterms.provenance","value":"Made available in DSpace on 2013-05-22T17:35:42Z (GMT). No. of bitstreams: 1\nThomas_grad.sunysb_0771E_11070.pdf: 16908050 bytes, checksum: 3a89c1162e8eb93be99bdb58435ae84c (MD5)\n Previous issue date: 1"},{"label":"dcterms.publisher","value":"The Graduate School, Stony Brook University: Stony Brook, NY."},{"label":"dcterms.subject","value":"Chemistry"},{"label":"dcterms.title","value":"Inhibitors of 3&beta-Hydroxysteroid? Dehydrogenase and Metabolic? and \nEnzymatic Characterization of the Cholesterol? ? Metabolizing Intracellular \nGrowth? Operon? of? Mycobacterium tuberculosis"},{"label":"dcterms.type","value":"Dissertation"},{"label":"dc.type","value":"Dissertation"}],"description":"This manifest was generated dynamically","viewingDirection":"left-to-right","sequences":[{"@type":"sc:Sequence","canvases":[{"@id":"https://repo.library.stonybrook.edu/cantaloupe/iiif/2/canvas/page-1.json","@type":"sc:Canvas","label":"Page 1","height":1650,"width":1275,"images":[{"@type":"oa:Annotation","motivation":"sc:painting","resource":{"@id":"https://repo.library.stonybrook.edu/cantaloupe/iiif/2/85%2F76%2F56%2F85765645237170696565788278276971165770/full/full/0/default.jpg","@type":"dctypes:Image","format":"image/jpeg","height":1650,"width":1275,"service":{"@context":"http://iiif.io/api/image/2/context.json","@id":"https://repo.library.stonybrook.edu/cantaloupe/iiif/2/85%2F76%2F56%2F85765645237170696565788278276971165770","profile":"http://iiif.io/api/image/2/level2.json"}},"on":"https://repo.library.stonybrook.edu/cantaloupe/iiif/2/canvas/page-1.json"}]}]}]}