Fungal hydroquinones contribute to brown rot of wood.

The fungi that cause brown rot of wood initiate lignocellulose breakdown with an extracellular Fenton system in which Fe(2+) and H(2)O(2) react to produce hydroxyl radicals (.OH), which then oxidize and cleave the wood holocellulose. One such fungus, Gloeophyllum trabeum, drives Fenton chemistry on defined media by reducing Fe(3+) and O(2) with two extracellular hydroquinones, 2,5-dimethoxyhydroquinone (2,5-DMHQ) and 4,5-dimethoxycatechol (4,5-DMC). However, it has never been shown that the hydroquinones contribute to brown rot of wood. We grew G. trabeum on spruce blocks and found that 2,5-DMHQ and 4,5-DMC were each present in the aqueous phase at concentrations near 20 microM after 1 week. We determined rate constants for the reactions of 2,5-DMHQ and 4,5-DMC with the Fe(3+)-oxalate complexes that predominate in wood undergoing brown rot, finding them to be 43 l mol(-1) s(-1) and 65 l mol(-1) s(-1) respectively. Using these values, we estimated that the average amount of hydroquinone-driven .OH production during the first week of decay was 11.5 micromol g(-1) dry weight of wood. Viscometry of the degraded wood holocellulose coupled with computer modelling showed that a number of the same general magnitude, 41.2 micromol oxidations per gram, was required to account for the depolymerization that occurred in the first week. Moreover, the decrease in holocellulose viscosity was correlated with the measured concentrations of hydroquinones. Therefore, hydroquinone-driven Fenton chemistry is one component of the biodegradative arsenal that G. trabeum expresses on wood.

Processive endoglucanase active in crystalline cellulose hydrolysis by the brown rot basidiomycete Gloeophyllum trabeum.

Brown rot basidiomycetes have long been thought to lack the processive cellulases that release soluble sugars from crystalline cellulose. On the other hand, these fungi remove all of the cellulose, both crystalline and amorphous, from wood when they degrade it. To resolve this discrepancy, we grew Gloeophyllum trabeum on microcrystalline cellulose (Avicel) and purified the major glycosylhydrolases it produced. The most abundant extracellular enzymes in these cultures were a 42-kDa endoglucanase (Cel5A), a 39-kDa xylanase (Xyn10A), and a 28-kDa endoglucanase (Cel12A). Cel5A had significant Avicelase activity--4.5 nmol glucose equivalents released/min/mg protein. It is a processive endoglucanase, because it hydrolyzed Avicel to cellobiose as the major product while introducing only a small proportion of reducing sugars into the remaining, insoluble substrate. Therefore, since G. trabeum is already known to produce a beta-glucosidase, it is now clear that this brown rot fungus produces enzymes capable of yielding assimilable glucose from crystalline cellulose.

Differential stress-induced regulation of two quinone reductases in the brown rot basidiomycete Gloeophyllum trabeum.

Quinone reductases (QRDs) have two important functions in the basidiomycete Gloeophyllum trabeum, which causes brown rot of wood. First, a QRD is required to generate biodegradative hydroxyl radicals via redox cycling between two G. trabeum extracellular metabolites, 2,5-dimethoxyhydroquinone (2,5-DMHQ) and 2,5-dimethoxy-1,4-benzoquinone (2,5-DMBQ). Second, because 2,5-DMBQ is cytotoxic and 2,5-DMHQ is not, a QRD is needed to maintain the intracellular pool of these metabolites in the reduced form. Given their importance in G. trabeum metabolism, QRDs could prove useful targets for new wood preservatives. We have identified two G. trabeum genes, each existing in two closely related, perhaps allelic variants, that encode QRDs in the flavodoxin family. Past work with QRD1 and heterologous expression of QRD2 in this study confirmed that both genes encode NADH-dependent, flavin-containing QRDs. Real-time reverse transcription PCR analyses of liquid- and wood-grown cultures showed that qrd1 expression was maximal during secondary metabolism, coincided with the production of 2,5-DMBQ, and was moderately up-regulated by chemical stressors such as quinones. By contrast, qrd2 expression was maximal during fungal growth when 2,5-DMBQ levels were low, yet was markedly up-regulated by chemical stress or heat shock. The total QRD activity in lysates of G. trabeum mycelium was significantly enhanced by induction beforehand with a cytotoxic quinone. The promoter of qrd2 contains likely antioxidant, xenobiotic, and heat shock elements, absent in qrd1, that probably explain the greater response of qrd2 transcription to stress. We conclude from these results that QRD1 is the enzyme G. trabeum routinely uses to detoxify quinones during incipient wood decay and that it could also drive the biodegradative quinone redox cycle. However, QRD2 assumes a more important role when the mycelium is stressed.