Purification and characterization of an extracellular H 2O 2-requiring diarylpropane oxygenase from the white rot basidiomycete, Phanerochaete chrysosporium

Abstract

An H 2O 2-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 M r 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 β-ether dinier, 1-(4′-ethoxy-3′-methoxyphenyl)glycerol-β-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.