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Barry Goodell - One of the best experts on this subject based on the ideXlab platform.
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oxidation states of iron and manganese in lignocellulose altered by the brown rot fungus Gloeophyllum trabeum measured in situ using x ray absorption near edge spectroscopy xanes
2021Co-Authors: Samuel L Zelinka, Grant T. Kirker, Amy B. Bishell, Jody Jellison, Joseph E Jakes, Charles R Boardman, Barry Lai, George E Sterbinsky, Barry GoodellAbstract:Abstract Brown rot fungi utilize iron as part of a chelator-mediated Fenton (CMF) reaction during wood biodegradation. Research suggests these fungi reduce Fe3+ to promote oxygen radical generation resulting in depolymerization of the wood cell wall. High levels of Mn are also found in wood decayed by brown rot fungi. However, little is known about the oxidation states of Fe and Mn during the decay process. X-ray absorption near edge spectroscopy (XANES) can be used to examine metal oxidation states and coordination chemistry. XANES experiments were conducted on wood decayed by Gloeophyllum trabeum over 2–8 weeks with results showing that Mn2+ and Fe3+ predominated for metal oxidation states. However, Fe2+ was present at sites of greater fungal growth In certain cases, the μXANES measurements showed that the fraction of Fe2+ in the wood samples was as high as 50%. Localized areas of reduced iron corresponded with areas of greater fungal hyphal mass which is in agreement with how brown rot fungi decay wood via the CMF reaction. The limited change in oxidation state of Mn observed in wood with active fungal activity suggests that the role of manganese in CMF biodegradation chemistry should be further explored.
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Transcriptome analysis of the brown rot fungus Gloeophyllum trabeum during lignocellulose degradation.
2020Co-Authors: Kiwamu Umezawa, Barry Goodell, Mai Niikura, Yuka Kojima, Makoto YoshidaAbstract:Brown rot fungi have great potential in biorefinery wood conversion systems because they are the primary wood decomposers in coniferous forests and have an efficient lignocellulose degrading system. Their initial wood degradation mechanism is thought to consist of an oxidative radical-based system that acts sequentially with an enzymatic saccharification system, but the complete molecular mechanism of this system has not yet been elucidated. Some studies have shown that wood degradation mechanisms of brown rot fungi have diversity in their substrate selectivity. Gloeophyllum trabeum, one of the most studied brown rot species, has broad substrate selectivity and even can degrade some grasses. However, the basis for this broad substrate specificity is poorly understood. In this study, we performed RNA-seq analyses on G. trabeum grown on media containing glucose, cellulose, or Japanese cedar (Cryptomeria japonica) as the sole carbon source. Comparison to the gene expression on glucose, 1,129 genes were upregulated on cellulose and 1,516 genes were upregulated on cedar. Carbohydrate Active enZyme (CAZyme) genes upregulated on cellulose and cedar media by G. trabeum included glycoside hyrolase family 12 (GH12), GH131, carbohydrate esterase family 1 (CE1), auxiliary activities family 3 subfamily 1 (AA3_1), AA3_2, AA3_4 and AA9, which is a newly reported expression pattern for brown rot fungi. The upregulation of both terpene synthase and cytochrome P450 genes on cedar media suggests the potential importance of these gene products in the production of secondary metabolites associated with the chelator-mediated Fenton reaction. These results provide new insights into the inherent wood degradation mechanism of G. trabeum and the diversity of brown rot mechanisms.
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a lytic polysaccharide monooxygenase with broad xyloglucan specificity from the brown rot fungus Gloeophyllum trabeum and its action on cellulose xyloglucan complexes
2016Co-Authors: Yuka Kojima, Barry Goodell, Jody Jellison, Gry Alfredsen, Dejan Petrovic, Aniko Varnai, Takuya Ishida, Naoki Sunagawa, Kiyohiko Igarashi, Bjorge WesterengAbstract:Fungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases (LPMOs) to degrade plant polysaccharides. Brown-rot fungi, such as Gloeophyllum trabeum , tend to have few LPMOs and information on these enzymes is scarce. The genome of G. trabeum encodes four AA9 LPMOs, whose coding sequences were amplified from cDNA. Due to alternative splicing, two variants of Gt LPMO9A seem to be produced, a single domain variant, Gt LPMO9A-1, and a longer variant, Gt LPMO9A-2, which contains a C-terminal domain comprising approximately 55 residues without a predicted function. We have overexpressed the phylogenetically distinct Gt LPMO9A-2 in Pichia pastoris and investigated its properties. Standard analyses, using HPAEC-PAD and MS, showed that Gt LPMO9A-2 is active on cellulose, carboxymethylcellulose and xyloglucan. Importantly, compared to other known xyloglucan-active LPMOs, Gt LPMO9A-2 has broad specificity, cleaving at any position along the β-glucan backbone of xyloglucan, regardless of substitutions. Using dynamic viscosity measurements to compare the hemicellulolytic action of Gt LPMO9A-2 to that of a well-characterized hemicellulolytic LPMO, Nc LPMO9C from Neurospora crassa , revealed that Gt LPMO9A-2 is more efficient in depolymerizing xyloglucan. These measurments also revealed minor activity on glucomannan that could not be detected by the analysis of soluble products by HPAEC-PAD and MS and that was lower than the activity of Nc LPMO9C. Experiments with co-polymeric substrates showed an inhibitory effect of hemicellulose-coating on cellulolytic LPMO activity and did not reveal additional activities of Gt LPMO9A-2. These results provide insight into the LPMO-potential of G. trabeum and provide a novel sensitive method, measurement of dynamic viscosity, for monitoring LPMO activity. Importance Currently, there are only a few methods available to analyze end-products of lytic polysaccharide monooxygenase (LPMO) activity, the most common ones being liquid chromatography and mass spectrometry. Here we present an alternative and sensitive method based on measurement of dynamic viscosity, for real-time continuous monitoring of LPMO activity in the presence of water-soluble hemicelluloses such as xyloglucan. We have used both this novel and existing analytical methods to characterize a xyloglucan-active LPMO from a brown rot fungus. This enzyme, Gt LPMO9A-2, differs from previously characterized LPMOs, in having broad substrate specificity, enabling almost random cleavage of the xyloglucan backbone. Gt LPMO9A-2 acts preferentially on free xyloglucan, suggesting a preference for xyloglucan chains that tether cellulose fibres together. The xyloglucan-degrading potential of Gt LPMO9A-2 suggests a role in decreasing wood strength at the initial stage of brown-rot, through degradation of the primary cell wall.
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a lytic polysaccharide monooxygenase with broad xyloglucan specificity from the brown rot fungus Gloeophyllum trabeum and its action on cellulose xyloglucan complexes
2016Co-Authors: Yuka Kojima, Barry Goodell, Jody Jellison, Gry Alfredsen, Dejan Petrovic, Aniko Varnai, Takuya Ishida, Naoki Sunagawa, Kiyohiko Igarashi, Bjorge WesterengAbstract:ABSTRACT Fungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases (LPMOs) to degrade plant polysaccharides. Brown-rot fungi, such as Gloeophyllum trabeum, tend to have few LPMOs, and information on these enzymes is scarce. The genome of G. trabeum encodes four auxiliary activity 9 (AA9) LPMOs (GtLPMO9s), whose coding sequences were amplified from cDNA. Due to alternative splicing, two variants of GtLPMO9A seem to be produced, a single-domain variant, GtLPMO9A-1, and a longer variant, GtLPMO9A-2, which contains a C-terminal domain comprising approximately 55 residues without a predicted function. We have overexpressed the phylogenetically distinct GtLPMO9A-2 in Pichia pastoris and investigated its properties. Standard analyses using high-performance anion-exchange chromatography–pulsed amperometric detection (HPAEC-PAD) and mass spectrometry (MS) showed that GtLPMO9A-2 is active on cellulose, carboxymethyl cellulose, and xyloglucan. Importantly, compared to other known xyloglucan-active LPMOs, GtLPMO9A-2 has broad specificity, cleaving at any position along the β-glucan backbone of xyloglucan, regardless of substitutions. Using dynamic viscosity measurements to compare the hemicellulolytic action of GtLPMO9A-2 to that of a well-characterized hemicellulolytic LPMO, NcLPMO9C from Neurospora crassa revealed that GtLPMO9A-2 is more efficient in depolymerizing xyloglucan. These measurements also revealed minor activity on glucomannan that could not be detected by the analysis of soluble products by HPAEC-PAD and MS and that was lower than the activity of NcLPMO9C. Experiments with copolymeric substrates showed an inhibitory effect of hemicellulose coating on cellulolytic LPMO activity and did not reveal additional activities of GtLPMO9A-2. These results provide insight into the LPMO potential of G. trabeum and provide a novel sensitive method, a measurement of dynamic viscosity, for monitoring LPMO activity. IMPORTANCE Currently, there are only a few methods available to analyze end products of lytic polysaccharide monooxygenase (LPMO) activity, the most common ones being liquid chromatography and mass spectrometry. Here, we present an alternative and sensitive method based on measurement of dynamic viscosity for real-time continuous monitoring of LPMO activity in the presence of water-soluble hemicelluloses, such as xyloglucan. We have used both these novel and existing analytical methods to characterize a xyloglucan-active LPMO from a brown-rot fungus. This enzyme, GtLPMO9A-2, differs from previously characterized LPMOs in having broad substrate specificity, enabling almost random cleavage of the xyloglucan backbone. GtLPMO9A-2 acts preferentially on free xyloglucan, suggesting a preference for xyloglucan chains that tether cellulose fibers together. The xyloglucan-degrading potential of GtLPMO9A-2 suggests a role in decreasing wood strength at the initial stage of brown rot through degradation of the primary cell wall.
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Investigating oxalate biosynthesis in the wood-decaying fungus Gloeophyllum trabeum using 13C metabolic flux analysis
2015Co-Authors: Liangpeng Zhuang, Xueyang Feng, Makoto Yoshida, Barry GoodellAbstract:Oxalate synthesis was rigorously investigated in a wood-decaying fungus, Gloeophyllum trabeum, using 13C metabolic flux analysis, a method not previously explored in this type of system. Peroxisomal glyoxylate dehydrogenase and cytosolic oxaloacetate acetylhydrolase were found to contribute to the majority of oxalate synthesized under low and high nitrogen conditions, respectively.
Jody Jellison - One of the best experts on this subject based on the ideXlab platform.
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oxidation states of iron and manganese in lignocellulose altered by the brown rot fungus Gloeophyllum trabeum measured in situ using x ray absorption near edge spectroscopy xanes
2021Co-Authors: Samuel L Zelinka, Grant T. Kirker, Amy B. Bishell, Jody Jellison, Joseph E Jakes, Charles R Boardman, Barry Lai, George E Sterbinsky, Barry GoodellAbstract:Abstract Brown rot fungi utilize iron as part of a chelator-mediated Fenton (CMF) reaction during wood biodegradation. Research suggests these fungi reduce Fe3+ to promote oxygen radical generation resulting in depolymerization of the wood cell wall. High levels of Mn are also found in wood decayed by brown rot fungi. However, little is known about the oxidation states of Fe and Mn during the decay process. X-ray absorption near edge spectroscopy (XANES) can be used to examine metal oxidation states and coordination chemistry. XANES experiments were conducted on wood decayed by Gloeophyllum trabeum over 2–8 weeks with results showing that Mn2+ and Fe3+ predominated for metal oxidation states. However, Fe2+ was present at sites of greater fungal growth In certain cases, the μXANES measurements showed that the fraction of Fe2+ in the wood samples was as high as 50%. Localized areas of reduced iron corresponded with areas of greater fungal hyphal mass which is in agreement with how brown rot fungi decay wood via the CMF reaction. The limited change in oxidation state of Mn observed in wood with active fungal activity suggests that the role of manganese in CMF biodegradation chemistry should be further explored.
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a lytic polysaccharide monooxygenase with broad xyloglucan specificity from the brown rot fungus Gloeophyllum trabeum and its action on cellulose xyloglucan complexes
2016Co-Authors: Yuka Kojima, Barry Goodell, Jody Jellison, Gry Alfredsen, Dejan Petrovic, Aniko Varnai, Takuya Ishida, Naoki Sunagawa, Kiyohiko Igarashi, Bjorge WesterengAbstract:Fungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases (LPMOs) to degrade plant polysaccharides. Brown-rot fungi, such as Gloeophyllum trabeum , tend to have few LPMOs and information on these enzymes is scarce. The genome of G. trabeum encodes four AA9 LPMOs, whose coding sequences were amplified from cDNA. Due to alternative splicing, two variants of Gt LPMO9A seem to be produced, a single domain variant, Gt LPMO9A-1, and a longer variant, Gt LPMO9A-2, which contains a C-terminal domain comprising approximately 55 residues without a predicted function. We have overexpressed the phylogenetically distinct Gt LPMO9A-2 in Pichia pastoris and investigated its properties. Standard analyses, using HPAEC-PAD and MS, showed that Gt LPMO9A-2 is active on cellulose, carboxymethylcellulose and xyloglucan. Importantly, compared to other known xyloglucan-active LPMOs, Gt LPMO9A-2 has broad specificity, cleaving at any position along the β-glucan backbone of xyloglucan, regardless of substitutions. Using dynamic viscosity measurements to compare the hemicellulolytic action of Gt LPMO9A-2 to that of a well-characterized hemicellulolytic LPMO, Nc LPMO9C from Neurospora crassa , revealed that Gt LPMO9A-2 is more efficient in depolymerizing xyloglucan. These measurments also revealed minor activity on glucomannan that could not be detected by the analysis of soluble products by HPAEC-PAD and MS and that was lower than the activity of Nc LPMO9C. Experiments with co-polymeric substrates showed an inhibitory effect of hemicellulose-coating on cellulolytic LPMO activity and did not reveal additional activities of Gt LPMO9A-2. These results provide insight into the LPMO-potential of G. trabeum and provide a novel sensitive method, measurement of dynamic viscosity, for monitoring LPMO activity. Importance Currently, there are only a few methods available to analyze end-products of lytic polysaccharide monooxygenase (LPMO) activity, the most common ones being liquid chromatography and mass spectrometry. Here we present an alternative and sensitive method based on measurement of dynamic viscosity, for real-time continuous monitoring of LPMO activity in the presence of water-soluble hemicelluloses such as xyloglucan. We have used both this novel and existing analytical methods to characterize a xyloglucan-active LPMO from a brown rot fungus. This enzyme, Gt LPMO9A-2, differs from previously characterized LPMOs, in having broad substrate specificity, enabling almost random cleavage of the xyloglucan backbone. Gt LPMO9A-2 acts preferentially on free xyloglucan, suggesting a preference for xyloglucan chains that tether cellulose fibres together. The xyloglucan-degrading potential of Gt LPMO9A-2 suggests a role in decreasing wood strength at the initial stage of brown-rot, through degradation of the primary cell wall.
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a lytic polysaccharide monooxygenase with broad xyloglucan specificity from the brown rot fungus Gloeophyllum trabeum and its action on cellulose xyloglucan complexes
2016Co-Authors: Yuka Kojima, Barry Goodell, Jody Jellison, Gry Alfredsen, Dejan Petrovic, Aniko Varnai, Takuya Ishida, Naoki Sunagawa, Kiyohiko Igarashi, Bjorge WesterengAbstract:ABSTRACT Fungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases (LPMOs) to degrade plant polysaccharides. Brown-rot fungi, such as Gloeophyllum trabeum, tend to have few LPMOs, and information on these enzymes is scarce. The genome of G. trabeum encodes four auxiliary activity 9 (AA9) LPMOs (GtLPMO9s), whose coding sequences were amplified from cDNA. Due to alternative splicing, two variants of GtLPMO9A seem to be produced, a single-domain variant, GtLPMO9A-1, and a longer variant, GtLPMO9A-2, which contains a C-terminal domain comprising approximately 55 residues without a predicted function. We have overexpressed the phylogenetically distinct GtLPMO9A-2 in Pichia pastoris and investigated its properties. Standard analyses using high-performance anion-exchange chromatography–pulsed amperometric detection (HPAEC-PAD) and mass spectrometry (MS) showed that GtLPMO9A-2 is active on cellulose, carboxymethyl cellulose, and xyloglucan. Importantly, compared to other known xyloglucan-active LPMOs, GtLPMO9A-2 has broad specificity, cleaving at any position along the β-glucan backbone of xyloglucan, regardless of substitutions. Using dynamic viscosity measurements to compare the hemicellulolytic action of GtLPMO9A-2 to that of a well-characterized hemicellulolytic LPMO, NcLPMO9C from Neurospora crassa revealed that GtLPMO9A-2 is more efficient in depolymerizing xyloglucan. These measurements also revealed minor activity on glucomannan that could not be detected by the analysis of soluble products by HPAEC-PAD and MS and that was lower than the activity of NcLPMO9C. Experiments with copolymeric substrates showed an inhibitory effect of hemicellulose coating on cellulolytic LPMO activity and did not reveal additional activities of GtLPMO9A-2. These results provide insight into the LPMO potential of G. trabeum and provide a novel sensitive method, a measurement of dynamic viscosity, for monitoring LPMO activity. IMPORTANCE Currently, there are only a few methods available to analyze end products of lytic polysaccharide monooxygenase (LPMO) activity, the most common ones being liquid chromatography and mass spectrometry. Here, we present an alternative and sensitive method based on measurement of dynamic viscosity for real-time continuous monitoring of LPMO activity in the presence of water-soluble hemicelluloses, such as xyloglucan. We have used both these novel and existing analytical methods to characterize a xyloglucan-active LPMO from a brown-rot fungus. This enzyme, GtLPMO9A-2, differs from previously characterized LPMOs in having broad substrate specificity, enabling almost random cleavage of the xyloglucan backbone. GtLPMO9A-2 acts preferentially on free xyloglucan, suggesting a preference for xyloglucan chains that tether cellulose fibers together. The xyloglucan-degrading potential of GtLPMO9A-2 suggests a role in decreasing wood strength at the initial stage of brown rot through degradation of the primary cell wall.
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induction and catalytic properties of an intracellular nadh dependent 1 4 benzoquinone reductase from the brown rot basidiomycete Gloeophyllum trabeum
2004Co-Authors: Jody JellisonAbstract:Abstract The expression of intracellular NADH-dependent 1,4-benzoquinone reductase in the brown-rot fungus Gloeophyllum trabeum was increased in the presence of 2,6-dimethoxy-1,4-benzoquinone (2,6-DMBQ) and, to a lesser extent, vanillic acid. Expression was time-dependent and influenced by nitrogen levels. An intracellular NADH-dependent 1,4-benzoquinone reductase was purified from G. trabeum . The subunit molecular mass was 22 kDa . Flavin mononucleotide was the coenzyme. The pI of the enzyme was 4.2. The quinone reductase catalyzed the reduction of multiple 1,4-quinones and exhibited Ping–Pong kinetics. For enzyme-catalyzed 2,6-DMBQ reduction, the apparent K M was 6.8 μM and the k cat 1.0×10 3 s −1 ; the pH optimum was between 5.5 and 7; the activation energy of the reaction was estimated at 43.7 kJ mol −1 . The one-to-one stoichiometric ratio of NADH oxidation versus 2,6-DMBQ reduction suggested a two-electron transfer mechanism for the enzyme. Dicumarol and Cibacron blue were competitive inhibitors with K i values of 0.5 and 0.2 μM , respectively.
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Characterization of a transplasma membrane redox system of the brown rot fungus Gloeophyllum trabeum
2004Co-Authors: Jody JellisonAbstract:The transplasma membrane redox system of the brown rot fungus Gloeophyllum trabeum was characterized with ferricyanide reduction kinetics. Nitrogen deficiency did not statistically affect the ferricyanide reduction rate, which depended on initial ferricyanide concentration and initial mycelial mass. The reduction rate increased with pH above pH 5.0, and was statistically lower in HEPES buffer (pH 8.0) than in potassium phosphate buffer (pH 8.0). Carbonyl cyanide m-chloromethoxyphenyl hydrazone, 2,4-dinitrophenol and sodium azide were efficient inhibitors of the transplasma membrane redox system. The quinone reducing activity of extracellular, membrane-bound, and intracellular quinone reductases of G. trabeum was tested, extracellular quinone reducing activity was not observed under the examined culture conditions. Quinone reduction by mycelium and the intracellular enzyme showed different kinetic constants. The fungus produced constitutive intracellular benzoquinone reductases, which are NAD(P)H-dependent.
Frank A Fekete - One of the best experts on this subject based on the ideXlab platform.
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Oxidation of 2-keto-4-thiomethylbutyric acid (KTBA) by iron-binding compounds produced by the wood-decaying fungus Gloeophyllum trabeum
2002Co-Authors: Vikas Chandhoke, Barry Goodell, Jody Jellison, Frank A FeketeAbstract:The ability of iron-binding compounds isolated from the brown-rot fungus Gloeophyllum trabeum to carry out one-electron oxidation reactions was established using a model substrate, 2-keto-4-thiomethylbutyric acid (KTBA). The oxidation reaction was monitored by measuring the amount of ethylene produced from the substrate by gas chromatography. The extent of the reaction was found to be influenced by the concentration of the chelators, and by iron and manganese.
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the isolation and immunolocalization of iron binding compounds produced by Gloeophyllum trabeum
1991Co-Authors: Jody Jellison, Barry Goodell, Vikas Chandhoke, Frank A FeketeAbstract:Low molecular weight iron-binding compounds are produced by the brown-rot fungus Gloeophyllum trabeum. These chelators may function in scavenging transition metals for fungal metabolism and extracellular enzyme production. Because of the low molecular mass of the chelate-metal complex (below 1000 Da), and the oxidizing potential of the bound transition metals, certain chelating compounds could also play a role in the early stages of cellulose depolymerization by brown-rot fungi. High-affinity iron-binding compounds were isolated and partially purified from both liquid cultures of the brown-rot Gloeophyllum trabeum and from infected wood. Chelating compounds purified by thin-layer chromatography were used to prepare specific antibodies. These antibodies were shown to detect the chelator in infected wood and liquid fungal cultures by enzyme-linked immunosorbent assay and could be used in immunotransmission electron microscopy to visualize the high-affinity iron-binding compounds in situ. Elucidating the physiological roles of fungal chelate-metal complexes and determining their function in lignocellulose depolymerization will help us to better understand the mechanism of wood biodegradation.
Kenneth E. Hammel - One of the best experts on this subject based on the ideXlab platform.
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processive endoglucanase active in crystalline cellulose hydrolysis by the brown rot basidiomycete Gloeophyllum trabeum
2005Co-Authors: Roni Cohen, Melissa R Suzuki, Kenneth E. HammelAbstract: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 β-glucosidase, it is now clear that this brown rot fungus produces enzymes capable of yielding assimilable glucose from crystalline cellulose.
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differential stress induced regulation of two quinone reductases in the brown rot basidiomycete Gloeophyllum trabeum
2004Co-Authors: Roni Cohen, Melissa R Suzuki, Kenneth E. HammelAbstract: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.
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an nadh quinone oxidoreductase active during biodegradation by the brown rot basidiomycete Gloeophyllum trabeum
2002Co-Authors: Kenneth A Jensen, Zachary C Ryan, Amber Vanden Wymelenberg, Daniel Cullen, Kenneth E. HammelAbstract: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.
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pathways for extracellular fenton chemistry in the brown rot basidiomycete Gloeophyllum trabeum
2001Co-Authors: Kenneth A Jensen, Carl J Houtman, Zachary C Ryan, Kenneth E. HammelAbstract:The brown rot fungus Gloeophyllum trabeum uses an extracellular hydroquinone-quinone redox cycle to reduce Fe(3+) and produce H(2)O(2). These reactions generate extracellular Fenton reagent, which enables G. trabeum to degrade a wide variety of organic compounds. We found that G. trabeum secreted two quinones, 2,5-dimethoxy-1,4-benzoquinone (2,5-DMBQ) and 4,5-dimethoxy-1,2-benzoquinone (4,5-DMBQ), that underwent iron-dependent redox cycling. Experiments that monitored the iron- and quinone-dependent cleavage of polyethylene glycol by G. trabeum showed that 2,5-DMBQ was more effective than 4,5-DMBQ in supporting extracellular Fenton chemistry. Two factors contributed to this result. First, G. trabeum reduced 2,5-DMBQ to 2,5-dimethoxyhydroquinone (2,5-DMHQ) much more rapidly than it reduced 4,5-DMBQ to 4,5-dimethoxycatechol (4,5-DMC). Second, although both hydroquinones reduced ferric oxalate complexes, the predominant form of Fe(3+) in G. trabeum cultures, the 2,5-DMHQ-dependent reaction reduced O(2) more rapidly than the 4,5-DMC-dependent reaction. Nevertheless, both hydroquinones probably contribute to the extracellular Fenton chemistry of G. trabeum, because 2,5-DMHQ by itself is an efficient reductant of 4,5-DMBQ.
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biodegradative mechanism of the brown rot basidiomycete Gloeophyllum trabeum evidence for an extracellular hydroquinone driven fenton reaction
1999Co-Authors: Zohar Kerem, Kenneth A Jensen, Kenneth E. HammelAbstract:We have identified key components of the extracellular oxidative system that the brown rot fungus Gloeophyllum trabeum uses to degrade a recalcitrant polymer, polyethylene glycol, via hydrogen abstraction reactions. G. trabeum produced an extracellular metabolite, 2,5-dimethoxy-1,4-benzoquinone, and reduced it to 2,5-dimethoxyhydroquinone. In the presence of 2,5-dimethoxy-1,4-benzoquinone, the fungus also reduced extracellular Fe3+ to Fe2+ and produced extracellular H2O2. Fe3+ reduction and H2O2 formation both resulted from a direct, non-enzymatic reaction between 2,5-dimethoxyhydroquinone and Fe3+. polyethylene glycol depolymerization by G. trabeum required both 2,5-dimethoxy-1,4-benzoquinone and Fe3+ and was completely inhibited by catalase. These results provide evidence that G. trabeum uses a hydroquinone-driven Fenton reaction to cleave polyethylene glycol. We propose that similar reactions account for the ability of G. trabeum to attack lignocellulose.
Wei Wang - One of the best experts on this subject based on the ideXlab platform.
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antifungal ability and decay resistance of fokienia hodginsii heartwood extract and its inhibitory effect on Gloeophyllum trabeum
2020Co-Authors: Xuefeng Zhuang, Wei Wang, Hongmei Yuan, Jinguo LinAbstract:The inhibitory ability of Fokienia hodginsii heartwood (FHH) extracts on Trametes versicolor (TV) and Gloeophyllum trabeum (GT) as well as the toxic effect of its heartwood extracts on GT were studied. The growth inhibition ability of the samples was analyzed using the growth rate method. The results showed that in the experiment of inhibiting TV, extracts using hot water had little effect, acetone extracts had the best inhibiting effect, and the lowest value of acetone EC50 was 0.409 g/L. The parameter EC50 is the concentration of the corresponding agent that inhibits the growth of 50% fungi. In the antifungal experiment of GT, methanol extract had the best inhibition effect, and the lowest EC50 value was 0.283 g/L. The antifungal effect of five solvent extracts of FHH was good when the concentration was 10% (w/w), and at this time, the mass loss rate of the test pieces was below 11%, all of them were Class I, indicating a strong antifungal level. After observing the samples of GT with SEM, it was found that the structure of methanol extract treatment was more complete and the antiseptic effect was better than that of the hot water extract treatment.
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lignin degradation by a novel peptide gt factor from brown rot fungus Gloeophyllum trabeum
2006Co-Authors: Wei Wang, Feng Huang, Pei Ji GaoAbstract:Gt factor, a pure component isolated from Gloeophyllum trabeum, was used to decompose lignin materials. The radical intermediates, degradation products and structural changes of treated materials were analyzed by infrared spectrum, NMR and electron paramagnetic resonance, etc. The results indicate that Gt factor makes an oxidative attack on lignin via HO., which might be the initial step in lignocellulose degradation by brown rot fungus.
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function and mechanism of a low molecular weight peptide produced by Gloeophyllum trabeum in biodegradation of cellulose
2003Co-Authors: Wei Wang, Pei Ji GaoAbstract:Abstract A special low-molecular-weight peptide named Gt factor, was isolated and purified via HPLC from the culture extract of the brown-rot fungus Gloeophyllum trabeum. It had high-affinity Fe3+-chelating ability and could reduce Fe3+ to Fe2+. In the presence of O2, it could produce hydroxyl radicals HO . The effects of Gt factor on cellulose degradation suggested that Gt factor could disrupt inter- and intra- hydrogen bonds in cellulose chains by a HO -involved mechanism. This resulted in depolymerization of cellulose chains, which produced more reducing and non-reducing ends, thus making cellulose accessible for further degradation. This pathway was quite different from the hydrolytic processes driven by cellulases, and Gt factor might play an important role in the early stage of cellulose depolymerization by brown-rot fungi.
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a possible role of a low molecular weight peptide from Gloeophyllum trabeum in cellulose degradation
2002Co-Authors: Wei Wang, Pei Ji GaoAbstract:A low-molecular-weight peptide(named Gt factor) was first isolated and purified from the extracellular culture of a brown-rot fungi Gloeophyllum trabeum. It could produce hydroxyl radical HO. in presence of O2 and Fe3+, might destroy hydrogen bonds of cellulose by HO.-involved oxidative mechanism, which was quite different from hydrolysis mechanism of cellulase in filamentous fungi. Gt factor might attack crystalline region of cellulose, bring about more reducing ends and non-reducing ends exposed, thus making cellulose accessible to further degradation.