Purification and characterization of an extracellular H2O2-requiring diarylpropane oxygenase from the white rot basidiomycete, Phanerochaete chrysosporium.
An H2O2-requiring oxygenase found in the extracellular medium of ligninolytic cultures of the white rot fungus Phanerochaete chrysosporium was purified by DEAE-Sepharose ion-exchange chromatography and gel filtration on Sephadex G-100. Sodium dodecyl sulfate (SDS)-disc gel electrophoresis indicated that the purified protein was homogeneous. The Mr of the enzyme as determined by gel filtration and SDS-polyacrylamide gel electrophoresis was 41,000. The absorption spectrum of the enzyme indicated the presence of a heme prosthetic group. The absorption maximum of the native enzyme (407 nm) shifted to 435 nm in the reduced enzyme and to 420 nm in the reduced-CO complex. The pyridine hemochrome absorption spectrum indicated that the enzyme contained one molecule of heme as iron protoporphyrin IX. Both CN- and N-3 bound readily to the native enzyme, indicating an available coordination site and that the heme iron was high spin. The purified enzyme generated ethylene from 2-keto-4-thiomethyl butyric acid, and oxidized a variety of lignin model compounds, including the diarylpropane, 1-(3'4'-diethoxyphenyl)1,3-dihydroxy-2-(4"-methoxyphenyl)propane (I); a beta-ether dimer, 1-(4'-ethoxy-3'-methoxyphenyl)glycerol-beta-guaiacyl ether (V); an olefin, 1-(4'-ethoxy-3'-methoxyphenyl)-1,2 propene (III); and a diol, 1-(4'-ethoxy-3'-methoxyphenyl)-1,2-propane diol (IV). The products found were equivalent to the metabolic products previously isolated from intact ligninolytic cultures.
D001487:Basidiomycota / Q000201:enzymology*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000302:isolation & purification / Q000378:metabolism*; D010544:Peroxidases; D013053:Spectrophotometry; D013379:Substrate Specificity
D006861:Hydrogen Peroxide; D010544:Peroxidases; C042858:lignin peroxidase; D010105:Oxygenases