Purification and Properties of Phloroglucinol Reductase from

Phloroglucinol reductase was purified 90-fold to homogeneity from the anaerobic rumen organism Eubacterium oxidoreducem strain G-41. The enzyme is stable in the presence of air and is found in the soluble fraction after ultracentrifugation of cell extract. Ionexchange, hydrophobic interaction, and affinity chromatography were used to purify the enzyme. The native M, is 78,000, and the subunit M, is 33,000 indicating an a2 homodimer. The enzyme is specific for phloroglucinol and NADPH. The K,,, and Vare 600 p~ and 640 pmol min-' mg" (pH 7.2) for phloroglucinol, and 6.7 p~ and 550 pmol min" mg" (pH 6.8) for NADPH; the K, and V, for the reverse direction are 290 p~ and 140 pmol min" mg" (pH 7.2) for dihydrophloroglucinol, and 27 p~ and 220 pmol min" mg" (pH 7.2) for NADP. Temperature and pH optima are 40 O 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, nethylmaleimide, 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.