title: Purification and characterization of a 1,2,4-trihydroxybenzene 1,2-dioxygenase from the basidiomycete Phanerochaete chrysosporium.
abstract: 1,2,4-Trihydroxybenzene (THB) is an intermediate in the Phanerochaete chrysosporium degradation of vanillate and aromatic pollutants. A P. chrysosporium intracellular enzyme able to oxidatively cleave the aromatic ring of THB was purified by ammonium sulfate precipitation, hydrophobic and ion-exchange chromatographies, and native gel electrophoresis. The native protein has a molecular mass of 90 kDa and a subunit mass of 45 kDa. The enzyme catalyzes an intradiol cleavage of the substrate aromatic ring to produce maleylacetate. 18O2 incorporation studies demonstrate that molecular oxygen is a cosubstrate in the reaction. The enzyme exhibits high substrate specificity for THB; however, catechol cleavage occurs at approximately 20% of the optimal rate. THB dioxygenase catalyzes a key step in the degradation pathway of vanillate, an intermediate in lignin degradation. Maleylacetate, the product of THB cleavage, is reduced to beta-ketoadipate by an NADPH-requiring enzyme present in partially purified extracts.
authors.affiliation: Department of Chemistry, Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Portland 97291-1000.
authors.forename: S
authors.initials: S
authors.lastname: Rieble
authors.forename: D K
authors.initials: DK
authors.lastname: Joshi
authors.forename: M H
authors.initials: MH
authors.lastname: Gold
mesh_terms: D001487:Basidiomycota / Q000201:enzymology*; D049308:Dioxygenases; D008401:Gas Chromatography-Mass Spectrometry; D008031:Lignin / Q000378:metabolism; D008298:Maleates / Q000378:metabolism; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity; D014641:Vanillic Acid / Q000378:metabolism
chemical_list: D008298:Maleates; C011905:maleoylacetic acid; D008031:Lignin; D010105:Oxygenases; D049308:Dioxygenases; C041894:hydroxyquinol 1,2-dioxygenase; D014641:Vanillic Acid