The white rot basidiomycete Gelatoporia subvermispora produces fatty aldehydes that enable fungal manganese peroxidases to degrade recalcitrant lignin structures.

The ability of some white rot basidiomycetes to remove lignin selectively from wood indicates that low molecular weight oxidants have a role in ligninolysis. These oxidants are likely free radicals generated by fungal peroxidases from compounds in the biodegrading wood. Past work supports a role for manganese peroxidases (MnPs) in the production of ligninolytic oxidants from fungal membrane lipids. However, the fatty acid alkylperoxyl radicals initially formed during this process are not reactive enough to attack the major structures in lignin. Here, we evaluate the hypothesis that the peroxidation of fatty aldehydes might provide a source of more reactive acylperoxyl radicals. We found that Gelatoporia subvermispora produced trans-2-nonenal, trans-2-octenal, and n-hexanal (a likely metabolite of trans-2,4-decadienal) during the incipient decay of aspen wood. Fungal fatty aldehydes supported the in vitro oxidation by MnPs of a nonphenolic lignin model dimer, and also of the monomeric model veratryl alcohol. Experiments with the latter compound showed that the reactions were partially inhibited by oxalate, the chelator that white rot fungi employ to detach Mn3+ from the MnP active site, but nevertheless proceeded at its physiological concentration of 1 mM. The addition of catalase was inhibitory, which suggests that the standard MnP catalytic cycle is involved in the oxidation of aldehydes. MnP oxidized trans-2-nonenal quantitatively to trans-2-nonenoic acid with the consumption of one O2 equivalent. The data suggest that when Mn3+ remains associated with MnP, it can oxidize aldehydes to their acyl radicals, and the latter subsequently add O2 to become ligninolytic acylperoxyl radicals.IMPORTANCEThe biodegradation of lignin by white rot fungi is essential for the natural recycling of plant biomass and has useful applications in lignocellulose bioprocessing. Although fungal peroxidases have a key role in ligninolysis, past work indicates that biodegradation is initiated by smaller, as yet unidentified oxidants that can infiltrate the substrate. Here, we present evidence that the peroxidase-catalyzed oxidation of naturally occurring fungal aldehydes may provide a source of ligninolytic free radical oxidants.

Fungal lignin peroxidase does not produce the veratryl alcohol cation radical as a diffusible ligninolytic oxidant.

Peroxidases are considered essential agents of lignin degradation by white-rot basidiomycetes. However, low-molecular-weight oxidants likely have a primary role in lignin breakdown because many of these fungi delignify wood before its porosity has sufficiently increased for enzymes to infiltrate. It has been proposed that lignin peroxidases (LPs, EC 1.11.1.14) fulfill this role by oxidizing the secreted fungal metabolite veratryl alcohol (VA) to its aryl cation radical (VA+•), releasing it to act as a one-electron lignin oxidant within woody plant cell walls. Here, we attached the fluorescent oxidant sensor BODIPY 581/591 throughout beads with a nominal porosity of 6 kDa and assessed whether peroxidase-generated aryl cation radical systems could oxidize the beads. As positive control, we used the 1,2,4,5-tetramethoxybenzene (TMB) cation radical, generated from TMB by horseradish peroxidase. This control oxidized the beads to depths that increased with the amount of oxidant supplied, ultimately resulting in completely oxidized beads. A reaction-diffusion computer model yielded oxidation profiles that were within the 95% confidence intervals for the data. By contrast, bead oxidation caused by VA and the LPA isozyme of Phanerochaete chrysosporium was confined to a shallow shell of LP-accessible volume at the bead surface, regardless of how much oxidant was supplied. This finding contrasted with the modeling results, which showed that if the LP/VA system were to release VA+•, it would oxidize the bead interiors. We conclude that LPA releases insignificant quantities of VA+• and that a different mechanism produces small ligninolytic oxidants during white rot.

An optical method for carbon dioxide isotopes and mole fractions in small gas samples: Tracing microbial respiration from soil, litter, and lignin.

RATIONALE: Carbon dioxide isotope (δ13 C value) measurements enable quantification of the sources of soil microbial respiration, thus informing ecosystem C dynamics. Tunable diode lasers (TDLs) can precisely measure CO2 isotopes at low cost and high throughput, but are seldom used for small samples (≤5 mL). We developed a TDL method for CO2 mole fraction ([CO2 ]) and δ13 C analysis of soil microcosms. METHODS: Peaks in infrared absorbance following constant volume sample injection to a carrier were used to independently measure [12 CO2 ] and [13 CO2 ] for subsequent calculation of δ13 C values. Using parallel soil incubations receiving differing C substrates, we partitioned respiration from three sources using mixing models: native soil organic matter (SOM), added litter, and synthetic lignin containing a 13 C label at Cß of the propyl side chain. RESULTS: Once-daily TDL calibration enabled accurate quantification of δ13 C values and [CO2 ] compared with isotope ratio mass spectrometry (IRMS), with long-term external precision of 0.17 and 0.31‰ for 5 and 1 mL samples, respectively, and linear response between 400 and 5000 µmol mol-1 CO2 . Production of CO2 from native soil C, added litter, and lignin Cß varied over four orders of magnitude. Multiple-pool first-order decay models fitted to data (R2  > 0.98) indicated substantially slower turnover for lignin Cß (17 years) than for the dominant pool of litter (1.3 years) and primed soil C (3.9 years). CONCLUSIONS: Our TDL method provides a flexible, precise, and high-throughput (60 samples h-1 ) alternative to IRMS for small samples. This enables the use of C isotopes in increasingly sophisticated experiments to test biogeochemical controversies, such as the fate of lignins in soil.

Basidiomycete DyPs: Genomic diversity, structural-functional aspects, reaction mechanism and environmental significance.

The first enzyme with dye-decolorizing peroxidase (DyP) activity was described in 1999 from an arthroconidial culture of the fungus Bjerkandera adusta. However, the first DyP sequence had been deposited three years before, as a peroxidase gene from a culture of an unidentified fungus of the family Polyporaceae (probably Irpex lacteus). Since the first description, fewer than ten basidiomycete DyPs have been purified and characterized, but a large number of sequences are available from genomes. DyPs share a general fold and heme location with chlorite dismutases and other DyP-type related proteins (such as Escherichia coli EfeB), forming the CDE superfamily. Taking into account the lack of an evolutionary relationship with the catalase-peroxidase superfamily, the observed heme pocket similarities must be considered as a convergent type of evolution to provide similar reactivity to the enzyme cofactor. Studies on the Auricularia auricula-judae DyP showed that high-turnover oxidation of anthraquinone type and other DyP substrates occurs via long-range electron transfer from an exposed tryptophan (Trp377, conserved in most basidiomycete DyPs), whose catalytic radical was identified in the H2O2-activated enzyme. The existence of accessory oxidation sites in DyP is suggested by the residual activity observed after site-directed mutagenesis of the above tryptophan. DyP degradation of substituted anthraquinone dyes (such as Reactive Blue 5) most probably proceeds via typical one-electron peroxidase oxidations and product breakdown without a DyP-catalyzed hydrolase reaction. Although various DyPs are able to break down phenolic lignin model dimers, and basidiomycete DyPs also present marginal activity on nonphenolic dimers, a significant contribution to lignin degradation is unlikely because of the low activity on high redox-potential substrates.

Evidence from Serpula lacrymans that 2,5-dimethoxyhydroquinone Is a lignocellulolytic agent of divergent brown rot basidiomycetes.

Basidiomycetes that cause brown rot of wood are essential biomass recyclers in coniferous forest ecosystems and a major cause of failure in wooden structures. Recent work indicates that distinct lineages of brown rot fungi have arisen independently from ligninolytic white rot ancestors via loss of lignocellulolytic enzymes. Brown rot thus proceeds without significant lignin removal, apparently beginning instead with oxidative attack on wood polymers by Fenton reagent produced when fungal hydroquinones or catechols reduce Fe(3+) in colonized wood. Since there is little evidence that white rot fungi produce these metabolites, one question is the extent to which independent lineages of brown rot fungi may have evolved different Fe(3+) reductants. Recently, the catechol variegatic acid was proposed to drive Fenton chemistry in Serpula lacrymans, a brown rot member of the Boletales (D. C. Eastwood et al., Science 333:762-765, 2011). We found no variegatic acid in wood undergoing decay by S. lacrymans. We found also that variegatic acid failed to reduce in vitro the Fe(3+) oxalate chelates that predominate in brown-rotting wood and that it did not drive Fenton chemistry in vitro under physiological conditions. Instead, the decaying wood contained physiologically significant levels of 2,5-dimethoxyhydroquinone, a reductant with a demonstrated biodegradative role when wood is attacked by certain brown rot fungi in two other divergent lineages, the Gloeophyllales and Polyporales. Our results suggest that the pathway for 2,5-dimethoxyhydroquinone biosynthesis may have been present in ancestral white rot basidiomycetes but do not rule out the possibility that it appeared multiple times via convergent evolution.

Genome, transcriptome, and secretome analysis of wood decay fungus Postia placenta supports unique mechanisms of lignocellulose conversion.

Brown-rot fungi such as Postia placenta are common inhabitants of forest ecosystems and are also largely responsible for the destructive decay of wooden structures. Rapid depolymerization of cellulose is a distinguishing feature of brown-rot, but the biochemical mechanisms and underlying genetics are poorly understood. Systematic examination of the P. placenta genome, transcriptome, and secretome revealed unique extracellular enzyme systems, including an unusual repertoire of extracellular glycoside hydrolases. Genes encoding exocellobiohydrolases and cellulose-binding domains, typical of cellulolytic microbes, are absent in this efficient cellulose-degrading fungus. When P. placenta was grown in medium containing cellulose as sole carbon source, transcripts corresponding to many hemicellulases and to a single putative beta-1-4 endoglucanase were expressed at high levels relative to glucose-grown cultures. These transcript profiles were confirmed by direct identification of peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Also up-regulated during growth on cellulose medium were putative iron reductases, quinone reductase, and structurally divergent oxidases potentially involved in extracellular generation of Fe(II) and H(2)O(2). These observations are consistent with a biodegradative role for Fenton chemistry in which Fe(II) and H(2)O(2) react to form hydroxyl radicals, highly reactive oxidants capable of depolymerizing cellulose. The P. placenta genome resources provide unparalleled opportunities for investigating such unusual mechanisms of cellulose conversion. More broadly, the genome offers insight into the diversification of lignocellulose degrading mechanisms in fungi. Comparisons with the closely related white-rot fungus Phanerochaete chrysosporium support an evolutionary shift from white-rot to brown-rot during which the capacity for efficient depolymerization of lignin was lost.

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.

An NADH:quinone oxidoreductase active during biodegradation by the brown-rot basidiomycete Gloeophyllum trabeum.

The brown-rot basidiomycete Gloeophyllum trabeum uses a quinone redox cycle to generate extracellular Fenton reagent, a key component of the biodegradative system expressed by this highly destructive wood decay fungus. The hitherto uncharacterized quinone reductase that drives this cycle is a potential target for inhibitors of wood decay. We have identified the major quinone reductase expressed by G. trabeum under conditions that elicit high levels of quinone redox cycling. The enzyme comprises two identical 22-kDa subunits, each with one molecule of flavin mononucleotide. It is specific for NADH as the reductant and uses the quinones produced by G. trabeum (2,5-dimethoxy-1,4-benzoquinone and 4,5-dimethoxy-1,2-benzoquinone) as electron acceptors. The affinity of the reductase for these quinones is so high that precise kinetic parameters were not obtainable, but it is clear that k(cat)/K(m) for the quinones is greater than 10(8) M(-1) s(-1). The reductase is encoded by a gene with substantial similarity to NAD(P)H:quinone reductase genes from other fungi. The G. trabeum quinone reductase may function in quinone detoxification, a role often proposed for these enzymes, but we hypothesize that the fungus has recruited it to drive extracellular oxyradical production.