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At least 631 records · Page 35Linked to original sources
PubMed2001

Investigation of the role of 3-hydroxyanthranilic acid in the degradation of lignin by white-rot fungus Pycnoporus cinnabarinus.

An aminophenol, 3-hydroxyanthranilic acid (3-HAA), has been proposed to play important roles in lignin degradation. Production of 3-HAA in Pycnoporus cinnabarinus was completely inhibited by a combination of tryptophan and S-(2-aminophenyl)-L-cysteine S,S-dioxide (APCD) while the fungus grew well and produced high amounts of laccase. The biosynthesis of 3-HAA is mainly through the metabolism of tryptophan in the kynurenine pathway. A minor pathway for 3-HAA synthesis is through the hydroxylation of anthranilic acid during the biosynthesis of tryptophan in the shikimic acid pathway. Through UV irradiation of wild-type P. cinnabarinus (WT-Pc) spores, a 3-HAA-less mutant was produced. Both WT-Pc, under the inhibitory culture condition, and the 3-HAA-less mutant were found to degrade lignin in unbleached kraft pulp as efficiently as the WT-Pc, which unambiguously demonstrated that 3-HAA does not play an important role in the fungal degradation of lignin.

Journal Article↗
PubMed2005

Screening of basidiomycetes and xylariaceous fungi for lignin peroxidase and laccase gene-specific sequences.

Lignin peroxidase and laccase gene-specific PCR primers were used to screen 38 diverse basidiomycetes and xylariaceous fungi. Lignin peroxidase gene-specific sequences were obtained for basidiomycetes only and were highly divergent. Possession of laccase genes was relatively widespread among basidiomycetes, and is shown for the first time in Xylariaceae. All sequences were highly conserved with no variation resulting in changes to predicted amino acid sequence. Those basidiomycetes shown to possess lignin peroxidase and laccase genes also produced the enzyme in vitro. Conversely none of the xylariaceous fungi shown to possess laccase genes were able to do so, whilst others decolorized Poly R yet yielded no PCR amplicons.

Amino Acid Sequence↗
PubMed1992

Structure of the active site of lignin peroxidase isozyme H2: native enzyme, compound III, and reduced form.

The wood-degrading fungus Phanerochaete chrysosporium secretes a number of extracellular enzymes called lignin peroxidases which are involved in the degradation of both lignin and a number of persistent environmental pollutants. Lignin peroxidase isozyme H2, a glycosylated protein of approximately 40 kDa, contains a single heme. X-ray absorption spectroscopy (XAS) has been used to probe the local environment of the iron in the active site of resting enzyme, reduced enzyme, and compound III. For the native and reduced forms, respectively, the average Fe-pyrrole nitrogen distances are 2.055 and 2.02 A (+/- 0.015 A); the Fe-proximal nitrogen distance is 1.93 and 1.91 A (+/- 0.02 A) while the Fe-distal ligand distance is 2.17 and 2.10 A (+/- 0.03 A). Although the results are not as well-defined, the active-site structure of compound III is largely 2.02 +/- 0.015 A for the average Fe-pyrrole nitrogen distance, 1.90 +/- 0.02 for the Fe-proximal nitrogen, and 1.74 +/- 0.03 A for the Fe-distal ligand distance. The heme iron-pyrrole nitrogen distance is more expanded in ligninase H2 than in other peroxidases. The possible significance of this is discussed in relation to other heme proteins.

Basidiomycota↗
PubMed1998

Irreversible oxidation of ferricytochrome c by lignin peroxidase.

Lignin peroxidase (LiP) from Phanerochaete chrysosporium catalyzes irreversible oxidative damage to ferricytochrome c (Cc3+) in the presence of H2O2 and 3,4-dimethoxybenzyl (veratryl) alcohol (VA). Atomic absorption analysis and UV/vis spectroscopy indicate that the oxidation of Cc3+ is accompanied by a loss of heme iron from the protein and probably oxidation of the porphyrin ring. At H2O2 concentrations of 7.5 microM or higher, this oxidation of Cc3+ by LiP is strictly dependent on the presence of VA. The latter is not oxidized to veratraldehyde at a significant rate in the presence of either ferrocytochrome c (Cc2+) or Cc3+, indicating it is not stimulating the reactions by specifically reducing LiP compound II. LiP is inactivated rapidly in 100 microM H2O2, and the presence of 500 microM VA protects LiP from this inactivation. Neither 20 microM Cc3+ nor 20 microM VA alone can protect LiP from inactivation; however, 20 microM each of VA and Cc3+ together protect LiP fully. This and other results strongly suggest that VA is acting as a protein-bound redox mediator in the oxidation of Cc3+. SDS-PAGE analysis of the Cc3+ oxidation products demonstrates the formation of some covalently linked dimer of Cc3+ in addition to the oxidized Cc3+ monomer. Amino acid analysis of the dimeric and monomeric products indicates the presence of oxidized Met and Tyr residues. This suggests that Tyr residues on the surface of the protein are oxidized to Tyr radicals during LiP oxidation and that some of these radicals subsequently undergo intermolecular radical coupling, resulting in dimerization of some of the Cc3+ molecules. However, most of the Cc3+ molecules appear to be irreversibly oxidized without dimerization. These results demonstrate that Cc3+ can serve as a useful polymeric model of the lignin substrate in studying the enzymatic mechanism of lignin oxidation and the role of VA in the reaction.

Basidiomycota↗
PubMed1998

Autocatalytic formation of a hydroxy group at C beta of trp171 in lignin peroxidase.

In the high-resolution crystal structures of two lignin peroxidase isozymes from the white rot fungus Phanerochaete chrysosporium a significant electron density at single bond distance from the C beta of Trp171 was observed and interpreted as a hydroxy group. To further clarify the nature of this feature, we carried out tryptic digestion of the enzyme and isolated the Trp171 containing peptide. Under ambient conditions, this peptide shows an absorbance spectrum typical of tryptophan. At elevated temperature, however, the formation of an unusual absorbance spectrum with lambda max = 333 nm can be followed that is identical to that of N-acetyl-alpha, beta-didehydrotryptophanamide, resulting upon water elimination from beta-hydroxy tryptophan. The Trp171 containing tryptic peptide isolated from the recombinant and refolded lignin peroxidase produced from Escherichia coli does not contain the characteristic 333 nm absorbance band at any temperature. However, treatment with 3 equiv of H2O2 leads to complete hydroxylation of Trp171. Reducing substrates compete with this process, e.g., in the presence of 0.5 mM veratryl alcohol, about 7 equiv of H2O2 is necessary for complete modification. We conclude that the hydroxylation at the C beta of Trp171 is an autocatalytic reaction which occurs readily under conditions of natural turnover, e.g., in the ligninolytic cultures of P. chrysosporium, which are known to contain an oxidase-based H2O2-generating system. No dependence on dioxygen was found for this oxidative process. Chemical modification of fungal lignin peroxidase with the tryptophan-specific agent N-bromo succinimide leads to a drastically reduced activity with respect to the substrate veratryl alcohol. This suggests that Trp171 is involved in catalysis and that electron transfer from this surface residue to the oxidized heme cofactor is possible under steady-state conditions.

Basidiomycota↗
PubMed2001

Overexpression of a heterologous sam gene encoding S-adenosylmethionine synthetase in flax (Linum usitatissimum) cells: Consequences on methylation of lignin precursors and pectins.

The Arabidopsis thaliana sam1 gene encoding S-adenosylmethionine synthetase (EC 2.5.1.6) was transferred to flax (Linum usitatissimum) cells via Agrobacterium tumefaciens. This enzyme catalyses the conversion of methionine to S-adenosylmethionine (SAM), the major methyl group donor in living cells. The aim of this work was to study the consequences of an increased SAM-synthetase (SAM-S) activity in transgenic cell lines on both the production of mono- and dimethoxylated lignin monomers and the degree of methylesterification of pectins. Hypocotyls were cocultivated with Agrobacterium tumefaciens strain GV3101 (pGV2260) harbouring the pO35SSAM binary vector carrying the sam1 gene under the control of the 35S promoter and the nptII gene for selection of putative transformed cells. Most of the transgenic cell lines exhibited a significant (up to 3.2-fold) increase in SAM-S activity compared to the controls. The results showed that for the cell lines analysed this transformation had no effect on caffeic acid O-methyltransferase (COMT, EC 2.1.1.68) in vitro activity, degree of methoxylation of lignin precursors or lignin deposition, pectin methyltransferase (PMT, EC 2.1.1) in vitro activity, but led to an increase of pectin methylesterification in friable and fast-growing transgenic cell lines.

Journal Article↗
PubMed1999

Veratryl alcohol-mediated oxidation of isoeugenyl acetate by lignin peroxidase.

The mechanism of the veratryl alcohol (VA)-mediated oxidation of isoeugenyl acetate (IEA) by lignin peroxidase, and the subsequent spontaneous Calpha-Cbeta cleavage of IEA to vanillyl acetate were studied. IEA oxidation only occurred in the presence of VA. It probably did not bind to lignin peroxidase as evidenced by an unaffected Km for VA in the presence of IEA, and by the fact that a 10-fold molar excess of the unreactive IEA counterpart, eugenyl acetate, did not affect the IEA oxidation rate. IEA was very efficient in recycling VA. Up to 34 mol of IEA were oxidized per mol VA. Formation of the predominant VA oxidation product, veratraldehyde, was postponed until IEA was almost completely oxidized. Together these findings suggest that IEA was oxidized by VA.+ rather than directly by lignin peroxidase. Thus, VA functioned as a redox mediator during IEA oxidation which is remarkable considering the high calculated ionization potential of 8.81 eV. Regardless of the presence of O2, approximately 2 mol of IEA were consumed per mol H2O2, which indicated that IEA was enzymatically oxidized by one electron to the putative radical cation (IEA.+). After formation of IEA.+, a series of O2-dependent chemical reactions were responsible for Calpha-Cbeta cleavage to the major oxidation product vanillyl acetate, as evidenced by the observation that an N2 atmosphere did not inhibit IEA oxidation, but almost completely inhibited vanillyl acetate formation. GC-MS analyses revealed that under an air atmosphere 1-(4'-acetoxy-3'-methoxyphenyl)-2-propanone, 1-(4'-acetoxy-3'-methoxyphenyl)-1-hydroxy-2-propanone, and 1-(4'-acetoxy-3'-methoxyphenyl)-2-hydroxy-1-propanone were also formed. Formation of the latter two was diminished under an N2 atmosphere.

Benzyl Alcohols↗
PubMed1987

Molecular cloning of complementary DNA encoding the lignin-forming peroxidase from tobacco: Molecular analysis and tissue-specific expression.

Plant peroxidases play a major role in lignin formation and wound healing and are believed to be involved in auxin catabolism and defense to pathogen attack. The function of the anionic peroxidase isozymes is best understood in tobacco. These isozymes catalyze the formation of the lignin polymer and form rigid cross-links between lignin, cellulose, and extensin in the secondary plant cell wall. We report the purification of the anionic peroxidase isozymes from tobacco and their partial amino acid sequence. An oligonucleotide probe deduced from the amino acid sequence was used to screen a tobacco leaf cDNA library and a 1200-base-pair cDNA clone was isolated and sequenced in its entirety. The predicted amino acid sequence revealed a 22-amino acid signal peptide and a 302-amino acid mature protein (M(r), 32,311). The amino acid sequence was compared to that of the cationic peroxidases from horseradish and turnip and was found to be 52% and 46% homologous, respectively. By RNA blot analysis, the messenger for the tobacco isozyme was found to be abundant in stem tissue while expressed at very low levels in leaf and root tissue. Four distinguishable copies of the gene were found on genomic DNA blots. The gene copy number may reflect the allotetraploid nature of Nicotiana tabacum.

Journal Article↗
PubMed1991

Proton NMR investigation into the basis for the relatively high redox potential of lignin peroxidase.

Lignin peroxidase shares several structural features with the well-studied horseradish peroxidase and cytochrome c peroxidase but carries a higher redox potential. Here the heme domain of lignin peroxidase and the lignin peroxidase cyanide adduct was examined by 1HNMR spectroscopy, including nuclear Overhauser effect and two-dimensional measurements, and the findings were compared with those for horseradish peroxidase and cytochrome c peroxidase. Structural information was obtained on the orientation of the heme vinyl and propionate groups and the proximal and distal histidines. The shifts of the epsilon1 proton of the proximal histidine were found to be empirically related to the Fe3+/Fe2+ redox potentials.

Journal Article↗
PubMed1995

Oxidation of guaiacol by lignin peroxidase. Role of veratryl alcohol.

We have investigated the lignin peroxidase-catalyzed oxidation of guaiacol and the role of veratryl alcohol in this reaction by steady-state and pre-steady-state methods. Pre-steady-state kinetic analyses demonstrated that guaiacol is a good substrate for both compounds I and II, the two- and one-electron oxidized enzyme intermediates, respectively, of lignin peroxidase. The rate constant for the reaction with compound I is 1.2 x 10(6) M-1s-1. The reaction of guaiacol with compound II exhibits a Kd of 64 microM and a first-order rate constant of 17 s-1. Oxidation of guaiacol leads to tetraguaiacol formation. This reaction exhibits classical Michaelis-Menten kinetics with a Km of 160 microM and a kcat of 7.7 s-1. Veratryl alcohol, a secondary metabolite of ligninolytic fungi, is capable of mediating the oxidation of guaiacol. This was shown by steady-state inhibition studies. Guaiacol completely inhibited the oxidation of veratryl alcohol, whereas veratryl alcohol had no corresponding inhibitory effect on guaiacol oxidation. In fact, at low guaiacol concentrations, veratryl alcohol stimulated the rate of guaiacol oxidation. These results collectively demonstrate that veratryl alcohol can serve as a mediator for phenolic substrates in the lignin peroxidase reaction.

Basidiomycota↗
PubMed1967

Biosynthesis of phenolic compounds in first internodes of sorghum: lignin and related products.

Lignin biosynthesis in excised tissues of Sorghum vulgare variety Wheatland milo incubated in air with and without prior infiltration with H(2)O was presumably limited by H(2)O(2) production and was dependent upon an endogenous substrate, probably starch. In solution culture without shaking, this conversion of endogenous material was partially blocked at some step prior to p-hydroxycinnamic acid. The synthesis was light independent and continued protein synthesis was not required. The accumulation of lignin products was paralleled by an increase in dhurrin, alkaline sensitive esters of p-hydroxycinnamic, ferulic and sinapic acids, and flavin coenzymes, especially flavin-adenine dinucleotide. There was no detectable evidence of competition for substrates with other phenols such as anthocyanins or with the growth of adventitious roots. There was evidence, however, of mechanisms limiting lignification in the first internode in the intact seedling. Comparisons are made with lignin production in comparable tissues of Phleum.

Journal Article↗
PubMed1990

The Role of Auxin and Gibberellin in Controlling Lignin Formation in Primary Phloem Fibers and in Xylem of Coleus blumei Stems.

The hypothesis that auxin (IAA) and gibberellic acid (GA(3)) control the formation of lignin is confirmed for the primary phloem fibers and for the secondary xylem in the stem of Coleus blumel Benth. Indoleacetic acid alone, or a combination of high IAA/low GA(3) (w/w), induced short phloem fibers with thick secondary walls, that contained lignin rich in syringyl units (high ratio of syringyl/guaiacyl). On the other hand, a combination of high GA(3)/low IAA (w/w), which promoted the differentiation of long phloem fibers with thin walls, decreased the relative content of the syringyl units (low syringyl/guaiacyl ratio). In the secondary xylem, these hormonal treatments yielded only slight changes in the noncondensed monomeric guaiacyl units, confirming the relative stability of the guaiacyl lignification pattern in this tissue. In the xylem, indoleacetic acid alone, or a combination of high IAA/low GA(3) induced lignin poor in syringyl units (low syringyl/guaiacyl ratio). A combination of high GA(3)/low IAA promoted a relatively slight increase in syringyl yield, indicating greater responsiveness of the syringyl lignification pattern to growth regulators. The possible functional and technological significance of our results is discussed.

Journal Article↗
PubMed1989

Genetic factors influencing lignin peroxidase activity in Phanerochaete chrysosporium ME446.

Haploid recombinant progeny of Phanerochaete chrysosporium ME446, genome compositions of which had been defined by RFLP-mapping, vary in their idiophasic behaviour. This allowed us to formulate a model of the sequence of idiophasic activities. One component of this variation, the amount of lignin peroxidase activity, is independent of the allele distributions of the lignin peroxidase gene clusters, but correlates with the allele distribution of another locus. This locus appears to control the spread of the lignin peroxidase-active state within the idiophasic mycelial mat and may be the mating-type locus. The successful determination of linkage relied on analysis of chromosome intervals rather than linkage to single markers; this approach should be generally useful for analysing quantitative characters by RFLP mapping.

Alleles↗
PubMed1977

Preparation of specifically labeled C-(lignin)- and C-(cellulose)-lignocelluloses and their decomposition by the microflora of soil.

Microbial decomposition of lignocellulose in soil was studied using radioisotope techniques. Natural lignocelluloses containing C in either their lignin or cellulose (glucan) components were prepared by feeding plants l-[U-C]phenylalanine or d-[U-C]glucose, respectively, through their cut stems. Detailed chemical and chromatographic characterization of labeled lignocelluloses from three hardwood and three softwood species showed that those labeled by the [C]glucose incorporation method contained specifically labeled cellulosic components, whereas those labeled by the [C]phenylalanine incorporation method contained specifically labeled lignin components. Microbial degradation of these differentially labeled lignocelluloses was followed by monitoring CO(2) evolution from selected soil samples incubated with known amounts of radiolabeled lignocelluloses. The lignin components of the six woods were shown to be decomposed in soil 4 to 10 times more slowly than their cellulosic components. These rates of mineralization were comparable to the generalized patterns previously reported in the literature. The present technique, however, was thought to be simpler, more sensitive, and less prone to interference than methods previously available.

Journal Article↗
PubMed1979

Biodegradation of C-labeled model and cornstalk lignins, phenols, model phenolase humic polymers, and fungal melanins as influenced by a readily available carbon source and soil.

After 6 months of incubation in a fertile neutral sandy loam, about 48% of the ring carbons and 2-carbons and 60% of the OCH(3) carbons of specifically labeled coniferyl alcohol had evolved as CO(2). After 1 year, corresponding values were 55 and 65%. When coniferyl alcohol units were linked into model and cornstalk lignins, about 23% of the ring carbons and 2-carbons and 39% of the OCH(3) carbons had evolved as CO(2) after 6 months. After 1 year, corresponding values were about 28 and 46%. The addition of orange leaves (0.5%, wt/wt) after 6 months did not significantly increase the evolution of CO(2). Addition of orange leaves (0.5%, wt/wt) with specifically C-labeled pyrocatechol, coumaryl alcohol, model lignins, humic acid-type phenolic polymers and of uniformly C-labeled fungal melanins did not increase labeled C losses or C losses from the orange leaves. Decomposition of protein and pyrocatechol linked into model humic acid polymers, coniferyl alcohol C in model lignins, and Eurotium echinulatum melanin in six soils varied from 2 to 14%. Significant differences in C losses were related to soils and were not influenced by orange leaf applications.

Journal Article↗
PubMed1981

Polyguaiacol: a useful model polymer for lignin biodegradation research.

A polymer of ring-labeled [C]o-methoxyphenol ([C]guaiacol) was prepared by peroxidase-H(2)O(2)-catalyzed oxidation of the C-labeled monomeric compound. The ring-labeled [C]polyguaiacol contained 67.71% carbon, 5.09% hydrogen, 27.49% oxygen, 25.44% methoxyl, and 8.60% phenolic hydroxyl. The polymer had an average molecular weight of between 5,000 and 15,000, as determined by gel chromatography. A schematic representation of the polymer, similar to previously published structures of polyguaiacols, was devised to meet these and other analytical parameters. The polymer is primarily composed of o-o and p-p-linked guaiacol moieties, with an occasional o-p-biphenyl link and some p-diphenoquinone structures. An approximate molecular formula is [C(49)O(14)H(31)](n), where n =/> 5.8. Its C(6) formula is C(6)H(2.3)O(0.3) (OH)(0.7)(OCH(3))(1.0). Polyguaiacol has many of the characteristics of a synthetic lignin. It is easier and less expensive to prepare than standard synthetic lignins (dehydrogenation polymers of coniferyl alcohol). It is degraded ([C]polyguaiacol --> CO(2)) by the lignolytic system of the white-rot fungus Phanaerochaete chrysosporium. It is suggested that [C]polyguaiacol may be of value as a substrate for lignin biodegradation research.

Journal Article↗
PubMed1981

Nutritional Regulation of Lignin Degradation by Phanerochaete chrysosporium.

Previous studies have shown that a lignin-degrading system appears in cultures of the white rot fungus Phanerochaete chrysosporium in response to nitrogen starvation, apparently as part of secondary metabolism. We examined the influence of limiting carbohydrate, sulfur, or phosphorus and the effect of varying the concentrations of four trace metals, Ca, and Mg. Limitation of carbohydrate or sulfur but not limitation of phosphorus triggered ligninolytic activity. When only carbohydrate was limiting, supplementary carbohydrate caused a transient repression of activity. In carbohydrate-limited cultures, ligninolytic activity appeared when the supplied carbohydrate was depleted, and this activity was associated with a decrease in mycelial dry weight. The amount of lignin degraded depended on the amount of carbohydrate provided, which determined the amount of mycelium produced during primary growth. Carbohydrate-limited cultures synthesized only small amounts of the secondary metabolite veratryl alcohol compared with nitrogen-limited cultures. l-Glutamate sharply repressed ligninolytic activity in carbohydrate-starved cultures, but NH(4) did not. Ligninolytic activity was also triggered in cultures supplied with 37 muM sulfur as the only limiting nutrient. The balance of trace metals, Mg, and Ca was important for lignin degradation.

Journal Article↗