Characterization of the pyrogallol-phloroglucinol isomerase of Eubacterium oxidoreducens.
Cell extracts of Eubacterium oxidoreducens, in the presence of dimethyl sulfoxide, catalyzed the conversion of pyrogallol to phloroglucinol with methyl sulfide as a product. The isomerization reaction also proceeded when 1,2,3,5-benzenetetrol was present rather than dimethyl sulfoxide. An assay to quantitate this activity was developed. The assay followed the disappearance of 1,2,4-benzenetriol as determined colorimetrically after incubation with sodium molybdate at neutral pH. The products of this reaction were resorcinol and 2,6-dihydroxyquinone. The enzyme(s) catalyzing this reaction was purified fivefold from cells grown on gallate plus H2. The purification procedure involved treatment with 40% acetone, precipitation with ammonium sulfate, DEAE-cellulose chromatography, concentration by ultrafiltration (molecular weight cutoff, greater than 100,000), and hydroxylapatite chromatography. This preparation had a specific activity of 14.7 mumol/min per mg of protein and a pH optimum of about 7.3. It was strongly inhibited by p-chloromercuribenzoate. The mechanism of the reaction involved oxidation of the pyrogallol followed by introduction of water. The benzenetetrol intermediate was then reduced and dehydrated to phloroglucinol.
Krumholz
L R
LR
Department of Animal Sciences, University of Illinois, Urbana 61801.
Bryant
M P
MP
D003124:Colorimetry; D004121:Dimethyl Sulfoxide / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005051:Eubacterium / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D006873:Hydroquinones / Q000378:metabolism; D019751:Intramolecular Transferases; D007535:Isomerases / Q000378:metabolism*; D007700:Kinetics; D010696:Phloroglucinol / Q000378:metabolism; D011748:Pyrogallol / Q000378:metabolism; D013997:Time Factors
D006873:Hydroquinones; D011748:Pyrogallol; C013038:hydroxyhydroquinone; D010696:Phloroglucinol; D007535:Isomerases; D019751:Intramolecular Transferases; C056152:pyrogallol-phloroglucinol isomerase; D004121:Dimethyl Sulfoxide