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Glucose oxidase, an important source of hydrogen peroxide in lignin-degrading cultures of Phanerochaete chrysosporium, was purified to electrophoretic homogeneity by a combination of ion-exchange and molecular sieve chromatography. The enzyme is a flavoprotein with an apparent native molecular weight of 180,000 and a denatured molecular weight of 80,000. This enzyme does not appear to be a glycoprotein. It gives optimal activity with D-glucose, which is stoichiometrically oxidized to D-gluconate. The enzyme has a relatively broad pH optimum of 4 to 5. It is inhibited by Ag+ (10 mM) and o-phthalate (100 mM), but not by Cu2+, NaF, or KCN (each 10 mM).

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Phanerochaete chrysosporium produces two classes of extracellular heme proteins, designated lignin peroxidases and manganese peroxidases, that play a key role in lignin degradation. In this study we isolated and characterized a lignin peroxidase-negative mutant (lip mutant) that showed 16% of the ligninolytic activity (14C-labeled synthetic lignin -* '4CO2) exhibited by the wild type. The lip mutant did not produce detectable levels of lignin peroxidase, whereas the wild type, under identical conditions, produced 96 U of lignin peroxidase per liter. Both the wild type and the mutant produced comparable levels of manganese peroxidase and glucose oxidase, a key H202-generating secondary metabolic enzyme in P. chrysosporium. Fast protein liquid chromatographic analysis of the concentrated extracellular fluid of the lip mutant confirmed that it produced only heme proteins with manganese peroxidase activity but no detectable lignin peroxidase activity, whereas both lignin peroxidase and manganese peroxidase activities were produced by the wild type. The lip mutant appears to be a regulatory mutant that is defective in the production of all the lignin peroxidases.Phanerochaete chrysosporium, a white-rot basidiomycete, has been extensively studied as a model for fungal lignin degradation (25). Two classes of extracellular heme protein peroxidases, designated lignin peroxidases and manganese peroxidases, and an H202-generating system have been identified to date as the major components of the lignin-degrading enzyme system of this organism (25). Lignin peroxidases are glycosylated heme proteins that catalyze H202-dependent oxidation of a variety of phenolic and nonphenolic lignin model compounds, and that catalyze the oxidative cleavage of P-0-4 linkages (the most abundant linkage in lignin polymers), Cot-Co linkages, and other linkages in lignin and lignin substructure model compounds (25). The number of lignin peroxidase isozymes produced by P. chrysosporium is reported to vary from 2 to 15, based on the strain, culture conditions, and separation efficiency (24,30,41). All the lignin peroxidase isozymes oxidize veratryl alcohol to veratraldehyde but exhibit considerable differences in specific activities (9, 24). Manganese peroxidases constitute a second group of extracellular heme proteins that catalyze the H202-dependent oxidation of Mn(II) to Mn(III), which, in turn, oxidizes various phenolic substrates (25,44). Also, the manganese peroxidases have been reported to show properties of both an oxidase and a peroxidase (32). Both lignin peroxidases and manganese peroxidases require H202 for activity (15,40). Several enzymes, including glucose oxidase and glyoxal oxidase, have been reported to contribute to H202 production in lignin-degrading cultures of P. chrysosporium (8,21,22,23,25). Both lignin peroxidases and manganese peroxidases and H202-generating enzymes are produced during secondary metabolism, in response to nitrogen starvation, whereas cultures grown under nitrogenrich conditions produce no detectable pero...

We reported previously that mitochondrial tyrosyl-tRNA synthetase, which is encoded by the nuclear gene cyt-18 in Neurospora crassa, functions in splicing several group I introns in N. crassa mitochondria (R. A. Akins and A. M. Lambowitz, Cell 50:331-345, 1987 Group I introns include nuclear rRNA introns of Tetrahymena spp. and Physarum polycephalum, most fungal mitochondrial DNA introns, some chloroplast introns, and introns in T-even bacteriophages (5). All group I introns appear to use the same splicing mechanism first elucidated for the Tetrahymena thermophila nuclear rRNA intron by T. R. Cech and co-workers. This mechanism involves two sequential transesterification reactions: addition of guanosine to the 5' end of the intron coupled to cleavage at the 5' splice site and exon ligation coupled to cleavage at the 3' splice site (5). Some group I introns, including the Tetrahymena nuclear rRNA intron, some mitochondrial DNA introns in Neurospora crassa and Saccharomyces cerevisiae, and introns in T-even bacteriophage, are efficiently self splicing in vitro (5). Self-splicing indicates that both the structural information and catalytic activities required for splicing are contained in the structure of the intron RNAs. The basic outline of the catalytically active structure of group I introns has been elucidated by phylogenetic comparisons, analysis of in vivo and in vitro mutants, and direct RNA structure analysis (5). All a core secondary structure that includes base pairing between short sequence elements P3[5'], P, Q, R, P3[3'], and S. Group I introns also contain an internal guide sequence that base pairs with flanking sequences in the 5' exon to position the 5' splice site for splicing and may also play a role in positioning the 3' splice site (4, 5). The internal region of the intron, including the core structure, possesses the catalytic activities that cleave at the 5' splice site and add guanosine to the 5' end of the intron (35). In those group I introns that are self-splicing in vitro, the catalytically active structure must be favored relative to alternative structures of deproteinized precursor RNAs. Although some group I introns are self-splicing in vitro, genetic analysis has shown that most, if not all, group I introns are dependent upon proteins for splicing in vivo. The most likely hypothesis is that these proteins function to fold the RNA into the catalytically active conformation. We showed previously that N. crassa cob intron 1, which is efficiently self-splicing in vitro, is dependent on the protein encoded by the cyt-18 gene for splicing in vivo (6,12). The N. crassa mitochondrial large rRNA intron has been shown to require proteins for splicing both in vivo and in vitro (13).The proteins required for splicing group I introns can be divided into three classes: (i) maturases, a family of structurally related proteins encoded within some, but not all, group I introns (26, 36); (ii) intron-specific proteins encoded by nuclear genes, e.g., the CBP2 and MRS1 proteins of S. cerevisiae, whic...