Purification and properties of phloroglucinol reductase from Eubacterium oxidoreducens G-41.
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 alpha 2 homodimer. The enzyme is specific for phloroglucinol and NADPH. The Km and Vmax are 600 microM and 640 mumol min-1 mg-1 (pH 7.2) for phloroglucinol, and 6.7 microM and 550 mumol min-1 mg-1 (pH 6.8) for NADPH; the Km and Vmax for the reverse direction are 290 microM and 140 mumol min-1 mg-1 (pH 7.2) for dihydrophloroglucinol, and 27 microM and 220 mumol min-1 mg-1 (pH 7.2) for NADP. Temperature and pH optima are 40 degrees 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.
Haddock
J D
JD
Department of Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg 24061.
Ferry
J G
JG
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002845:Chromatography; D004355:Drug Stability; D005051:Eubacterium / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008670:Metals / Q000032:analysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010696:Phloroglucinol / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity; D013696:Temperature; D014461:Ultracentrifugation
D000596:Amino Acids; D046911:Macromolecular Substances; D008670:Metals; D009249:NADP; D010696:Phloroglucinol; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C058829:phloroglucinol reductase