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Purification and properties of phloroglucinol reductase from Eubacterium oxidoreducens G-41

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      Phloroglucinol reductase was purified 90-fold to homogeneity from the anaerobic rumen organism Eubacterium oxidoreducens strain G-41. The enzyme is stable in the presence of air and is found in the soluble fraction after ultracentrifugation of cell extract. Ion-exchange, hydrophobic interaction, and affinity chromatography were used to purify the enzyme. The native Mr is 78,000, and the subunit Mr is 33,000 indicating an α2 homodimer. The enzyme is specific for phloroglucinol and NADPH. The Km and Vmax are 600 µM and 640 µmol min−1 mg−1 (pH 7.2) for phloroglucinol, and 6.7 µM and 550 µmol min−1 mg−1 (pH 6.8) for NADPH; the Km and Vmax for the reverse direction are 290 µM and 140 µmol min−1 mg−1 (pH 7.2) for dihydrophloroglucinol, and 27 µM and 220 µmol min−1 mg−1 (pH 7.2) for NADP. Temperature and pH optima are 40 ° C and 7.8 in the forward direction. The pure enzyme is colorless in solution and flavins are absent. Analysis for cobalt, manganese, molybdenum, vanadium, tungsten, selenium, copper, nickel, iron, and zinc indicated that these metals are not components of the phloroglucinol reductase. Cupric chloride, n-ethylmaleimide, and p-chloromercuribenzoate are potent inhibitors of enzyme activity. The properties of phloroglucinol reductase indicate that it functions in the pathway of anaerobic degradation of trihydroxybenzenes by catalyzing reduction of the aromatic nucleus prior to ring fission.

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