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|Title:||Metabolism of Gallic Acid by Eubacterium Oxidoreducens|
|Author(s):||Krumholz, Lee Richard|
|Department / Program:||Animal Science|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||Gallate was decarboxylated by cell extracts of Eubacterium oxidoreducens with pyrogallol as the only detectable product. A phloro-glucinol reductase catalyzed the conversion of phloroglucinol to dihydrophloroglucinol, using NADPH as the source of electrons. Extracts of cells grown on gallate and formate contained formate dehydrogenase (EC 220.127.116.11) and hydrogenase (EC 18.104.22.168), which were both NADP-linked. These results suggest that the oxidation of formate or H$\sb2$ may be indirectly linked to the reduction of phloroglucinol. A dihydrophloroglucinolase was present, which hydrolyzed dihydrophloroglucinol to 3-hydroxy-5-oxohexanoate. This six-carbon ring cleavage product presumably can be broken down by a series of reactions similar to $\beta$-oxidation. These reactions cleaved the six carbon acid to 3-hydroxybutyryl-CoA yielding acetate and butyrate as end products. A number of key enzymes involved in $\beta$-oxidation and substrate level phosphorylation were demonstrated in cell extracts.
Cell extracts, in the presence of dimethylsulfoxide (DMSO), catalyzed the conversion of pyrogallol to phloroglucinol with dimethylsulfide as a product. The isomerization reaction would also proceed if 1,2,3,5-benzenetetrol was present rather than DMSO. To quantitate this activity, an assay was developed that followed the disappearance of 1,2,4-benzenetriol colorimetrically after incubation with sodium molybdate at neutral pH. The products of this reaction are resorcinol and 1,6-dihydroxyquinone. The enzyme(s) catalyzing this reaction was purified 5-fold from cells grown on gallate. The purification procedure involved fractionation with 40% acetone, precipitation with ammonium sulfate, DEAE-cellulose chromatography, concentration by ultrafiltration (molecular weight retention above 100,000), and hydroxylapatite chromatography. This preparation had a specific activity of 14.7 umol/min/mg protein and a pH optimum about 7.5. Activity was strongly inhibited by p-chloromercuribenzoate. The mechanism of the reaction involves an oxidation of the substrate followed by introduction of water. The benzenetetrol intermediate is then reduced and dehydrated to form the product.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1988.
|Date Available in IDEALS:||2014-12-16|