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Angel T Martinez - One of the best experts on this subject based on the ideXlab platform.
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peroxidase evolution in white rot fungi follows wood lignin evolution in plants
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Ivan Ayusofernandez, Jorge Rencoret, Ana Gutierrez, Francisco J Ruizduenas, Angel T MartinezAbstract:A comparison of sequenced Agaricomycotina genomes suggests that efficient degradation of wood lignin was associated with the appearance of secreted Peroxidases with a solvent-exposed catalytic tryptophan. This hypothesis is experimentally demonstrated here by resurrecting ancestral fungal Peroxidases, after sequence reconstruction from genomes of extant white-rot Polyporales, and evaluating their oxidative attack on the lignin polymer by state-of-the-art analytical techniques. Rapid stopped-flow estimation of the transient-state constants for the 2 successive one-electron transfers from lignin to the peroxide-activated enzyme (k2app and k3app) showed a progressive increase during peroxidase evolution (up to 50-fold higher values for the rate-limiting k3app). The above agreed with 2-dimensional NMR analyses during steady-state treatments of hardwood lignin, showing that its degradation (estimated from the normalized aromatic signals of lignin units compared with a control) and syringyl-to-guaiacyl ratio increased with the enzyme evolutionary distance from the first peroxidase ancestor. More interestingly, the stopped-flow estimations of electron transfer rates also showed how the most recent peroxidase ancestors that already incorporated the exposed tryptophan into their molecular structure (as well as the extant lignin peroxidase) were comparatively more efficient at oxidizing hardwood (angiosperm) lignin, while the most ancestral “tryptophanless” enzymes were more efficient at abstracting electrons from softwood (conifer) lignin. A time calibration of the ancestry of Polyporales Peroxidases localized the appearance of the first peroxidase with a solvent-exposed catalytic tryptophan to 194 ± 70 Mya, coincident with the diversification of angiosperm plants characterized by the appearance of dimethoxylated syringyl lignin units.
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lignin degrading Peroxidases from genome of selective ligninolytic fungus ceriporiopsis subvermispora
Journal of Biological Chemistry, 2012Co-Authors: Elena Fernandezfueyo, Francisco J Ruizduenas, Maria Jesus Martinez, Yuta Miki, Kenneth E Hammel, Angel T MartinezAbstract:The white-rot fungus Ceriporiopsis subvermispora delignifies lignocellulose with high selectivity, but until now it has appeared to lack the specialized Peroxidases, termed lignin Peroxidases (LiPs) and versatile Peroxidases (VPs), that are generally thought important for ligninolysis. We screened the recently sequenced C. subvermispora genome for genes that encode Peroxidases with a potential ligninolytic role. A total of 26 peroxidase genes was apparent after a structural-functional classification based on homology modeling and a search for diagnostic catalytic amino acid residues. In addition to revealing the presence of nine heme-thiolate peroxidase superfamily members and the unexpected absence of the dye-decolorizing peroxidase superfamily, the search showed that the C. subvermispora genome encodes 16 class II enzymes in the plant-fungal-bacterial peroxidase superfamily, where LiPs and VPs are classified. The 16 encoded enzymes include 13 putative manganese Peroxidases and one generic peroxidase but most notably two Peroxidases containing the catalytic tryptophan characteristic of LiPs and VPs. We expressed these two enzymes in Escherichia coli and determined their substrate specificities on typical LiP/VP substrates, including nonphenolic lignin model monomers and dimers, as well as synthetic lignin. The results show that the two newly discovered C. subvermispora Peroxidases are functionally competent LiPs and also suggest that they are phylogenetically and catalytically intermediate between classical LiPs and VPs. These results offer new insight into selective lignin degradation by C. subvermispora.
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pleurotus ostreatus heme Peroxidases an in silico analysis from the genome sequence to the enzyme molecular structure
Comptes Rendus Biologies, 2011Co-Authors: Francisco J Ruizduenas, Elena Fernandez, Maria Jesus Martinez, Angel T MartinezAbstract:An exhaustive screening of the Pleurotus ostreatus genome was performed to search for nucleotide sequences of heme Peroxidases in this white-rot fungus, which could be useful for different biotechnological applications. After sequence identification and manual curation of the corresponding genes and cDNAs, the deduced amino acid sequences were converted into structural homology models. A comparative study of these sequences and their structural models with those of known fungal Peroxidases revealed the complete inventory of heme Peroxidases of this fungus. This consists of cytochrome c peroxidase and ligninolytic Peroxidases, including manganese peroxidase and versatile peroxidase but not lignin peroxidase, as representative of the “classical” superfamily of plant, fungal, and bacterial Peroxidases; and members of two relatively “new” peroxidase superfamilies, namely heme-thiolate Peroxidases, here described for the first time in a fungus from the genus Pleurotus, and dye-decolorizing Peroxidases, already known in P. ostreatus but still to be thoroughly explored and characterized.
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molecular biology and structure function of lignin degrading heme Peroxidases
Enzyme and Microbial Technology, 2002Co-Authors: Angel T MartinezAbstract:Abstract Three Peroxidases involved in lignin degradation are produced by white-rot fungi. Lignin peroxidase (LiP) is characterized by oxidation of high redox-potential aromatic compounds (including veratryl alcohol) whereas manganese peroxidase (MnP) requires Mn 2+ to complete the catalytic cycle and forms Mn 3+ chelates acting as diffusing oxidizers. Pleurotus and Bjerkandera versatile peroxidase (VP) is able to oxidize Mn 2+ as well as non-phenolic aromatic compounds, phenols and dyes. Phanerochaete chrysosporium has two gene families including ten LiP-type and three MnP-type genes coding different isoenzymes expressed during secondary metabolism. Two VP genes have been recently cloned from Pleurotus eryngii. Phanerochaete chrysosporium MnP and P. eryngii VP are induced by H 2 O 2 , being Mn 2+ involved in regulation of their transcript levels. At least eighteen more ligninolytic peroxidase genes have been cloned from other white-rot fungi. Protein sequence comparison reveals that typical MnP from P. chrysosporium and two other fungi (showing a longer C-terminal tail) are separated from other ligninolytic Peroxidases, which form two main groups including P. chrysosporium LiP and Pleurotus Peroxidases respectively. LiP and MnP crystal structures and VP theoretical molecular models are available. The high redox potential of ligninolytic Peroxidases seems related to the distance between heme iron and proximal histidine, and the ability of MnP to oxidize Mn 2+ is due to a Mn-binding site formed by three acidic residues near the internal heme propionate. Pleurotus eryngii VP show higher sequence and structural affinities with P. chrysosporium LiP than MnP, but includes a Mn-binding site accounting for its ability to oxidize Mn 2+ . The functionality of this site was demonstrated by site-directed mutagenesis of MnP and VP. All fungal Peroxidases, which exhibit similar topology (11–12 helices) and folding, also include binding sites for two structural Ca 2+ . Veratryl alcohol was first modeled near LiP heme, but evidence for oxidation at the protein surface via a long-range electron transfer pathway has accumulated. Chemical and site-directed mutagenesis modification confirmed that an exposed tryptophan is involved in veratryl alcohol oxidation however, multiple sites could be responsible for oxidation of different aromatic substrates and dyes by these Peroxidases.
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molecular characterization of a novel peroxidase isolated from the ligninolytic fungus pleurotus eryngii
Molecular Microbiology, 1999Co-Authors: Francisco J Ruizduenas, Maria Jesus Martinez, Angel T MartinezAbstract:Summary A haem peroxidase different from other microbial, plant and animal Peroxidases is described. The enzyme is secreted as two isoforms by dikaryotic Pleurotus eryngii in peptone-containing liquid medium. The corresponding gene, which presents 15 introns and encodes a 361-amino-acid protein with a 30-aminoacid signal peptide, was isolated as two alleles corresponding to the two isoforms. The alleles differ in three amino acid residues and in a seven nucleotide deletion affecting a single metal response element in the promoter. When compared with Phanerochaete chrysosporium Peroxidases, the new enzyme appears closer to lignin peroxidase (LiP) than to Mn-dependent peroxidase (MnP) isoenzymes (58‐60% and 55% identity respectively). The molecular model built using crystal structures of three fungal Peroxidases as templates, also showed high structural affinity with LiP (Ca-distance 1.2 A ˚). However, this peroxidase includes aM n 2 ˛ binding site formed by three acidic residues (E36, E40 and D175) near the haem internal propionate, which accounts for the ability to oxidize Mn 2˛ . Its capability to oxidize aromatic substrates could involve interactions with aromatic residues at the edge of the haem channel. Another possibility is long-range electron transfer, e.g. from W164, which occupies the same position of LiP W171 recently reported as involved in the catalytic cycle of LiP.
Karen G. Welinder - One of the best experts on this subject based on the ideXlab platform.
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structural diversity and transcription of class iii Peroxidases from arabidopsis thaliana
FEBS Journal, 2002Co-Authors: Karen G. Welinder, Annemarie F Justesen, Inger V H Kjaersgard, Rikke Beck Jensen, Soren K Rasmussen, Hans M Jespersen, Laurent DurouxAbstract:Understanding peroxidase function in plants is complicated by the lack of substrate specificity, the high number of genes, their diversity in structure and our limited knowledge of peroxidase gene transcription and translation. In the present study we sequenced expressed sequence tags (ESTs) encoding novel heme-containing class III Peroxidases from Arabidopsis thaliana and annotated 73 full-length genes identified in the genome. In total, transcripts of 58 of these genes have now been observed. The expression of individual peroxidase genes was assessed in organ-specific EST libraries and compared to the expression of 33 peroxidase genes which we analyzed in whole plants 3, 6, 15, 35 and 59 days after sowing. Expression was assessed in root, rosette leaf, stem, cauline leaf, flower bud and cell culture tissues using the gene-specific and highly sensitive reverse transcriptase-polymerase chain reaction (RT-PCR). We predicted that 71 genes could yield stable proteins folded similarly to horseradish peroxidase (HRP). The putative mature Peroxidases derived from these genes showed 28-94% amino acid sequence identity and were all targeted to the endoplasmic reticulum by N-terminal signal peptides. In 20 Peroxidases these signal peptides were followed by various N-terminal extensions of unknown function which are not present in HRP. Ten Peroxidases showed a C-terminal extension indicating vacuolar targeting. We found that the majority of peroxidase genes were expressed in root. In total, class III Peroxidases accounted for an impressive 2.2% of root ESTs. Rather few Peroxidases showed organ specificity. Most importantly, genes expressed constitutively in all organs and genes with a preference for root represented structurally diverse Peroxidases (< 70% sequence identity). Furthermore, genes appearing in tandem showed distinct expression profiles. The alignment of 73 Arabidopsis peroxidase sequences provides an easy access to the identification of orthologous Peroxidases in other plant species and will provide a common platform for combining knowledge of peroxidase structure and function relationships obtained in various species.
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from sequence analysis of three novel ascorbate Peroxidases from arabidopsis thaliana to structure function and evolution of seven types of ascorbate peroxidase
Biochemical Journal, 1997Co-Authors: Hans M Jespersen, Inger V H Kjaersgard, Lars Ostergaard, Karen G. WelinderAbstract:Ascorbate Peroxidases are haem proteins that efficiently scavenge H2O2 in the cytosol and chloroplasts of plants. Database analyses retrieved 52 expressed sequence tags coding for Arabidopsis thaliana ascorbate Peroxidases. Complete sequencing of non-redundant clones revealed three novel types in addition to the two cytosol types described previously in Arabidopsis. Analysis of sequence data available for all plant ascorbate Peroxidases resulted in the following classification: two types of cytosol soluble ascorbate peroxidase designated cs1 and cs2; three types of cytosol membrane-bound ascorbate peroxidase, namely cm1, bound to microbodies via a C-terminal membrane-spanning segment, and cm2 and cm3, both of unknown location; two types of chloroplast ascorbate peroxidase with N-terminal transit sequences, the stromal ascorbate peroxidase (chs), and the thylakoid-bound ascorbate peroxidase showing a C-terminal transmembrane segment and designated cht. Further comparison of the patterns of conserved residues and the crystal structure of pea ascorbate peroxidase showed that active site residues are conserved, and three peptide segments implicated in interaction with reducing substrate are similar, excepting cm2 and cm3 types. A change of Phe-175 in cytosol types to Trp-175 in chloroplast types might explain the greater ascorbate specificity of chloroplast compared with cytosol ascorbate Peroxidases. Residues involved in homodimeric subunit interaction are conserved only in cs1, cs2 and cm1 types. The proximal cation (K+)-binding site observed in pea ascorbate peroxidase seems to be conserved. In addition, cm1, cm2, cm3, chs and cht ascorbate Peroxidases contain Asp-43, Asn-57 and Ser-59, indicative of a distal monovalent cation site. The data support the hypothesis that present-day Peroxidases evolved by an early gene duplication event.
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Superfamily of plant, fungal and bacterial Peroxidases
Current Opinion in Structural Biology, 1992Co-Authors: Karen G. WelinderAbstract:Abstract New peroxidase structures have significantly increased our understanding of the evolutionary and functional relationships within the plant peroxidase superfamily. Three distantly related structural classes have emerged: mitochondrial yeast cytochrome c peroxidase, chloroplast and cytosol ascorbate Peroxidases, and gene duplicated bacterial peroxidase (class I); secretory fungal Peroxidases (class II); and, classical, secretory plant Peroxidases (class III).
Christophe Dunand - One of the best experts on this subject based on the ideXlab platform.
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ascorbate peroxidase related apx r is a new heme containing protein functionally associated with ascorbate peroxidase but evolutionarily divergent
New Phytologist, 2011Co-Authors: Fernanda Lazzarotto, Christophe Dunand, Felipe Karam Teixeira, Silvia Barcellos Rosa, Claudia Lemelle Fernandes, Adilton V Fontenele, Joaquim Albenisio Gomes Silveira, Hugo Verli, Rogerio MargisAbstract:• Peroxidases are involved in several important processes, such as development and responses to environmental cues. In higher plants, most Peroxidases are encoded by large, multigenic families that mainly originated from gene and chromosomal duplications. • Using phylogenetic, genomic and functional analyses, we have identified and characterized a new class of putative heme Peroxidases, called ascorbate peroxidase-related (APx-R), which arose specifically in the lineage of plants. • The APx-R protein is structurally related to the ascorbate Peroxidases, although the active site contains many conserved substitutions. Unlike all other plant Peroxidases, which are encoded by gene families, APx-R is encoded by a single-copy gene in virtually all the species analyzed. APx-R proteins are targeted to the chloroplast and can physically interact with chloroplastic APx proteins. APx-R-knockdown rice (Oryza sativa) plants presented delayed development and a disturbed steady state of the antioxidant system compared with wild type. Moreover, the accumulation of APx-R transcripts was modulated by drought, UV irradiation, cold, and aluminum exposure in rice, suggesting the involvement of APx-R in the environmental stress response. • Our results reveal the existence of a new class of heme peroxidase which seems to play a role in the antioxidant system in plants, probably by modulating the activity of chloroplastic APx proteins.
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specific functions of individual class iii peroxidase genes
Journal of Experimental Botany, 2009Co-Authors: Claudia Cosio, Christophe DunandAbstract:In higher plants, class III Peroxidases exist as large multigene families (e.g. 73 genes in Arabidopsis thaliana). The diversity of processes catalysed by Peroxidases as well as the large number of their genes suggests the possibility of a functional specialization of each isoform. In addition, the fact that peroxidase promoter sequences are very divergent and that protein sequences contain both highly conserved domains and variable regions supports this hypothesis. However, two difficulties are associated with the study of the function of specific peroxidase genes: (i) the modification of the expression of a single peroxidase gene often results in no visible mutant phenotype, because it is compensated by redundant genes; and (ii) Peroxidases show low substrate specificity in vitro resulting in an unreliable indication of peroxidase specific activity unless complementary data are available. The generalization of molecular biology approaches such as whole transcriptome analysis and recombinant DNA combined with biochemical approaches provide unprecedented tools for overcoming these difficulties. This review highlights progress made with these new techniques for identifying the specific function of individual class III peroxidase genes taking as an example the model plant A. thaliana, as well as discussing some other plants.
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the class iii peroxidase multigenic family in rice and its evolution in land plants
Phytochemistry, 2004Co-Authors: Filippo Passardi, Claude Penel, David Longet, Christophe DunandAbstract:Plant Peroxidases (class III Peroxidases, E.C. 1.11.1.7) are secreted glycoproteins known to be involved in the mechanism of cell elongation, in cell wall construction and differentiation, and in the defense against pathogens. They usually form large multigenic families in angiosperms. The recent completion of rice (Oryza sativa japonica c.v. Nipponbare) genome sequencing allowed drawing up the full inventory of the genes encoding class III Peroxidases in this plant. We found 138 peroxidase genes distributed among the 12 rice chromosomes. In contrast to several other gene families studied so far, peroxidase genes are twice as numerous in rice as in Arabidopsis. This large number of genes results from various duplication events that were tentatively traced back using a phylogenetic tree based on the alignment of conserved amino acid sequences. We also searched for peroxidase encoding genes in the major phyla of plant kingdom. In addition to gymnosperms and angiosperms, sequences were found in liverworts, mosses and ferns, but not in unicellular green algae. Two rice and one Arabidopsis peroxidase genes appeared to be rather close to the only known sequence from the liverwort Marchantia polymorpha. The possible relationship of these Peroxidases with the putative ancestor of peroxidase genes is discussed, as well as the connection between the development of the class III peroxidase multigenic family and the emergence of the first land plants.
Martin Hofrichter - One of the best experts on this subject based on the ideXlab platform.
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Widespread Occurrence of Expressed Fungal Secretory Peroxidases in Forest Soils
2016Co-Authors: Harald Kellner, Donald R. Zak, Patricia Luis, Marek J. Pecyna, Florian Barbi, Danuta Kapturska, Martin HofrichterAbstract:Fungal secretory Peroxidases mediate fundamental ecological functions in the conversion and degradation of plant biomass. Many of these enzymes have strong oxidizing activities towards aromatic compounds and are involved in the degradation of plant cell wall (lignin) and humus. They comprise three major groups: class II Peroxidases (including lignin peroxidase, manganese peroxidase, versatile peroxidase and generic peroxidase), dye-decolorizing Peroxidases, and heme-thiolate Peroxidases (e.g. unspecific/aromatic peroxygenase, chloroperoxidase). Here, we have repeatedly observed a widespread expression of all major peroxidase groups in leaf and needle litter across a range of forest ecosystems (e.g. Fagus, Picea, Acer, Quercus, and Populus spp.), which are widespread in Europe and North America. Manganese Peroxidases and unspecific peroxygenases were found expressed in all nine investigated forest sites, and dye-decolorizing Peroxidases were observed in five of the nine sites, thereby indicating biological significance of these enzymes for fungal physiology and ecosystem processes. Transcripts of selected secretory peroxidase genes were also analyzed in pure cultures of several litter-decomposing species and other fungi. Using this information, we were able to match, in environmental litter samples, two manganese peroxidase sequences to Mycena galopus and Mycena epipterygia and one unspecific peroxygenase transcript to Mycena galopus, suggesting an important role of this litter- and coarse woody debris-dwelling genus in the disintegratio
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Widespread Occurrence of Expressed Fungal Secretory Peroxidases in Forest Soils
PLoS ONE, 2014Co-Authors: Harald Kellner Mail, Patricia Luis, Marek J. Pecyna, Florian Barbi, Danuta Kapturska, Dirk Krüger, Donald R. Zak, Roland Marmeisse, Micheline Vandenbol, Martin HofrichterAbstract:Fungal secretory Peroxidases mediate fundamental ecological functions in the conversion and degradation of plant biomass. Many of these enzymes have strong oxidizing activities towards aromatic compounds and are involved in the degradation of plant cell wall (lignin) and humus. They comprise three major groups: class II Peroxidases (including lignin peroxidase, manganese peroxidase, versatile peroxidase and generic peroxidase), dye-decolorizing Peroxidases, and heme-thiolate Peroxidases (e.g. unspecific/aromatic peroxygenase, chloroperoxidase). Here, we have repeatedly observed a widespread expression of all major peroxidase groups in leaf and needle litter across a range of forest ecosystems (e.g. Fagus, Picea, Acer, Quercus, and Populus spp.), which are widespread in Europe and North America. Manganese Peroxidases and unspecific peroxygenases were found expressed in all nine investigated forest sites, and dye-decolorizing Peroxidases were observed in five of the nine sites, thereby indicating biological significance of these enzymes for fungal physiology and ecosystem processes. Transcripts of selected secretory peroxidase genes were also analyzed in pure cultures of several litter-decomposing species and other fungi. Using this information, we were able to match, in environmental litter samples, two manganese peroxidase sequences to Mycena galopus and Mycena epipterygia and one unspecific peroxygenase transcript to Mycena galopus, suggesting an important role of this litter- and coarse woody debris-dwelling genus in the disintegration and transformation of litter aromatics and organic matter formation.
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dyp like Peroxidases of the jelly fungus auricularia auricula judae oxidize nonphenolic lignin model compounds and high redox potential dyes
Applied Microbiology and Biotechnology, 2010Co-Authors: Christiane Liers, Marek J. Pecyna, René Ullrich, Caroline Bobeth, Martin HofrichterAbstract:The jelly fungus Auricularia auricula-judae pro- duced an enzyme with manganese-independent peroxidase activity during growth on beech wood (∼300 U l −1 ). The same enzymatic activity was detected and produced at larger scale in agitated cultures comprising of liquid, plant-based media (e.g. tomato juice suspensions) at levels up to 8,000 U l −1 . Two pure peroxidase forms (A. auricula-judae peroxidase (AjP I and AjP II) could be obtained from respective culture liquids by three chromatographic steps. Spectroscopic and electrophoretic analyses of the purified proteins revealed their heme and peroxidase nature. The N-terminal amino acid sequence of AjP matched well with sequences of fungal enzymes known as "dye-decolorizing Peroxidases". Homology was found to the N-termini of Peroxidases from Marasmius scorodonius (up to 86%), Thanatephorus cucumeris (60%), and Termitomyces albuminosus (60%). Both enzyme forms catalyzed not only the conversion of typical peroxidase substrates such as 2,6-dimethoxyphenol and 2,2'-azino-bis(3-ethylthiazoline-6- sulfonate) but also the decolorization of the high-redox potential dyes Reactive Blue 5 and Reactive Black 5, whereas manganese(II) ions (Mn 2+ ) were not oxidized. Most remarkable, however, is the finding that both AjPs oxidized nonphenolic lignin model compounds (veratryl alcohol; adlerol, a nonphenolic β-O-4 lignin model dimer) at low pH (maximum activity at pH 1.4), which indicates a certain ligninolytic activity of dye-decolorizing Peroxidases.
Francisco J Ruizduenas - One of the best experts on this subject based on the ideXlab platform.
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peroxidase evolution in white rot fungi follows wood lignin evolution in plants
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Ivan Ayusofernandez, Jorge Rencoret, Ana Gutierrez, Francisco J Ruizduenas, Angel T MartinezAbstract:A comparison of sequenced Agaricomycotina genomes suggests that efficient degradation of wood lignin was associated with the appearance of secreted Peroxidases with a solvent-exposed catalytic tryptophan. This hypothesis is experimentally demonstrated here by resurrecting ancestral fungal Peroxidases, after sequence reconstruction from genomes of extant white-rot Polyporales, and evaluating their oxidative attack on the lignin polymer by state-of-the-art analytical techniques. Rapid stopped-flow estimation of the transient-state constants for the 2 successive one-electron transfers from lignin to the peroxide-activated enzyme (k2app and k3app) showed a progressive increase during peroxidase evolution (up to 50-fold higher values for the rate-limiting k3app). The above agreed with 2-dimensional NMR analyses during steady-state treatments of hardwood lignin, showing that its degradation (estimated from the normalized aromatic signals of lignin units compared with a control) and syringyl-to-guaiacyl ratio increased with the enzyme evolutionary distance from the first peroxidase ancestor. More interestingly, the stopped-flow estimations of electron transfer rates also showed how the most recent peroxidase ancestors that already incorporated the exposed tryptophan into their molecular structure (as well as the extant lignin peroxidase) were comparatively more efficient at oxidizing hardwood (angiosperm) lignin, while the most ancestral “tryptophanless” enzymes were more efficient at abstracting electrons from softwood (conifer) lignin. A time calibration of the ancestry of Polyporales Peroxidases localized the appearance of the first peroxidase with a solvent-exposed catalytic tryptophan to 194 ± 70 Mya, coincident with the diversification of angiosperm plants characterized by the appearance of dimethoxylated syringyl lignin units.
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lignin degrading Peroxidases from genome of selective ligninolytic fungus ceriporiopsis subvermispora
Journal of Biological Chemistry, 2012Co-Authors: Elena Fernandezfueyo, Francisco J Ruizduenas, Maria Jesus Martinez, Yuta Miki, Kenneth E Hammel, Angel T MartinezAbstract:The white-rot fungus Ceriporiopsis subvermispora delignifies lignocellulose with high selectivity, but until now it has appeared to lack the specialized Peroxidases, termed lignin Peroxidases (LiPs) and versatile Peroxidases (VPs), that are generally thought important for ligninolysis. We screened the recently sequenced C. subvermispora genome for genes that encode Peroxidases with a potential ligninolytic role. A total of 26 peroxidase genes was apparent after a structural-functional classification based on homology modeling and a search for diagnostic catalytic amino acid residues. In addition to revealing the presence of nine heme-thiolate peroxidase superfamily members and the unexpected absence of the dye-decolorizing peroxidase superfamily, the search showed that the C. subvermispora genome encodes 16 class II enzymes in the plant-fungal-bacterial peroxidase superfamily, where LiPs and VPs are classified. The 16 encoded enzymes include 13 putative manganese Peroxidases and one generic peroxidase but most notably two Peroxidases containing the catalytic tryptophan characteristic of LiPs and VPs. We expressed these two enzymes in Escherichia coli and determined their substrate specificities on typical LiP/VP substrates, including nonphenolic lignin model monomers and dimers, as well as synthetic lignin. The results show that the two newly discovered C. subvermispora Peroxidases are functionally competent LiPs and also suggest that they are phylogenetically and catalytically intermediate between classical LiPs and VPs. These results offer new insight into selective lignin degradation by C. subvermispora.
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pleurotus ostreatus heme Peroxidases an in silico analysis from the genome sequence to the enzyme molecular structure
Comptes Rendus Biologies, 2011Co-Authors: Francisco J Ruizduenas, Elena Fernandez, Maria Jesus Martinez, Angel T MartinezAbstract:An exhaustive screening of the Pleurotus ostreatus genome was performed to search for nucleotide sequences of heme Peroxidases in this white-rot fungus, which could be useful for different biotechnological applications. After sequence identification and manual curation of the corresponding genes and cDNAs, the deduced amino acid sequences were converted into structural homology models. A comparative study of these sequences and their structural models with those of known fungal Peroxidases revealed the complete inventory of heme Peroxidases of this fungus. This consists of cytochrome c peroxidase and ligninolytic Peroxidases, including manganese peroxidase and versatile peroxidase but not lignin peroxidase, as representative of the “classical” superfamily of plant, fungal, and bacterial Peroxidases; and members of two relatively “new” peroxidase superfamilies, namely heme-thiolate Peroxidases, here described for the first time in a fungus from the genus Pleurotus, and dye-decolorizing Peroxidases, already known in P. ostreatus but still to be thoroughly explored and characterized.
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molecular characterization of a novel peroxidase isolated from the ligninolytic fungus pleurotus eryngii
Molecular Microbiology, 1999Co-Authors: Francisco J Ruizduenas, Maria Jesus Martinez, Angel T MartinezAbstract:Summary A haem peroxidase different from other microbial, plant and animal Peroxidases is described. The enzyme is secreted as two isoforms by dikaryotic Pleurotus eryngii in peptone-containing liquid medium. The corresponding gene, which presents 15 introns and encodes a 361-amino-acid protein with a 30-aminoacid signal peptide, was isolated as two alleles corresponding to the two isoforms. The alleles differ in three amino acid residues and in a seven nucleotide deletion affecting a single metal response element in the promoter. When compared with Phanerochaete chrysosporium Peroxidases, the new enzyme appears closer to lignin peroxidase (LiP) than to Mn-dependent peroxidase (MnP) isoenzymes (58‐60% and 55% identity respectively). The molecular model built using crystal structures of three fungal Peroxidases as templates, also showed high structural affinity with LiP (Ca-distance 1.2 A ˚). However, this peroxidase includes aM n 2 ˛ binding site formed by three acidic residues (E36, E40 and D175) near the haem internal propionate, which accounts for the ability to oxidize Mn 2˛ . Its capability to oxidize aromatic substrates could involve interactions with aromatic residues at the edge of the haem channel. Another possibility is long-range electron transfer, e.g. from W164, which occupies the same position of LiP W171 recently reported as involved in the catalytic cycle of LiP.