Purification and Characterization of a 1,2,4-Trihydroxybenzene 1,2-Dioxygenase from the Basidiomycete

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. 1802 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%o of the optimal rate. THB dioxygenase catalyzes a key step in the degradation pathway of vanillate, an intermediate in lignin degradation. Maleylacetate, the product ofTHB cleavage, is reduced to j0-ketoadipate by an NADPH-requiring enzyme present in partially purified extracts. The white rot basidiomycete Phanerochaete chrysosporium degrades the plant cell wall polymer lignin (11, 15, 17, 24, 28