Search

Multicopper oxidases (MCOs) comprise different types of enzymes widely distributed in nature with quite diverse functions. Laccases are the most interesting MCOs from a biotechnological point of view, particularly those secreted by ligninolytic Basidiomycota fungi due to their versatility to oxidize lignin and a variety of aromatic substrates. The term "laccase" has been broadly (but sometimes erroneously) applied due to their low sequence homology and some overlapping activities with other MCO groups. We examined the distribution and phylogenetic relationships of MCOs in Basidiomycota fungi aiming to provide a complete and precise picture of the different MCO types across the division, including fungal orders phylogenetically distant from those typically studied. The phylogenetic tree revealed eight clusters of MCOs, each sharing common sequence/structural features. With this information we classified the MCOs in eight groups and described their distinctive amino acid residues. These eight MCO types are: laccases (LAC), ferroxidases (FOX), laccase-ferroxidases (LAC-FOX), ascorbate oxidases (AO), fungal pigment MCOs, and three new groups of laccase-like enzymes or "atypical laccases" related to but different from laccases sensu stricto, namely novel laccases (NLAC), new MCO (NMCO) and new laccases with potential ferroxidase activity (NLF). Accordingly, MCOs already described in the literature were reclassified into the updated groups., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 The Author(s). Published by Elsevier Inc. All rights reserved.)

Fungal enzyme systems for the degradation of plant cell wall lignin, consisting of, among others, laccases and lignin-active peroxidases, are well characterized. Additionally, fungi and bacteria contain dye-decolorizing peroxidases (DyP), which are also capable of oxidizing and modifying lignin constituents. Studying DyP activity on lignocellulose poses challenges due to the heterogeneity of the substrate and the lack of continuous kinetic methods. In this study, we report the kinetic parameters of bacterial DyP from Amycolatopsis 75iv2 and fungal DyP from Auricularia auricula-judae on insoluble plant materials and kraft lignin by monitoring the depletion of the cosubstrate of the peroxidases with a H 2 O 2 sensor. In the reactions with spruce, both enzymes showed similar kinetics. On kraft lignin, the catalytic rate of bacterial DyP reached 30 ± 2 s -1 , whereas fungal DyP was nearly 3 times more active (81 ± 7 s -1 ). Importantly, the real-time measurement of H 2 O 2 allowed the assessment of continuous activity for both enzymes, revealing a previously unreported exceptionally high stability under turnover conditions. Bacterial DyP performed 24,000 turnovers of H 2 O 2 , whereas the fungal DyP achieved 94,000 H 2 O 2 turnovers in 1 h with a remaining activity of 40 and 80%, respectively. Using mass spectrometry, the depletion of the cosubstrate H 2 O 2 was shown to correlate with product formation, validating the amperometric method., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Background: Manganese peroxidases (MnPs) are, together with lignin peroxidases and versatile peroxidases, key elements of the enzymatic machineries secreted by white-rot fungi to degrade lignin, thus providing access to cellulose and hemicellulose in plant cell walls. A recent genomic analysis of 52 Agaricomycetes species revealed the existence of novel MnP subfamilies differing in the amino-acid residues that constitute the manganese oxidation site. Following this in silico analysis, a comprehensive structure-function study is needed to understand how these enzymes work and contribute to transform the lignin macromolecule., Results: Two MnPs belonging to the subfamilies recently classified as MnP-DGD and MnP-ESD-referred to as Ape-MnP1 and Cst-MnP1, respectively-were identified as the primary peroxidases secreted by the Agaricales species Agrocybe pediades and Cyathus striatus when growing on lignocellulosic substrates. Following heterologous expression and in vitro activation, their biochemical characterization confirmed that these enzymes are active MnPs. However, crystal structure and mutagenesis studies revealed manganese coordination spheres different from those expected after their initial classification. Specifically, a glutamine residue (Gln333) in the C-terminal tail of Ape-MnP1 was found to be involved in manganese binding, along with Asp35 and Asp177, while Cst-MnP1 counts only two amino acids (Glu36 and Asp176), instead of three, to function as a MnP. These findings led to the renaming of these subfamilies as MnP-DDQ and MnP-ED and to re-evaluate their evolutionary origin. Both enzymes were also able to directly oxidize lignin-derived phenolic compounds, as seen for other short MnPs. Importantly, size-exclusion chromatography analyses showed that both enzymes cause changes in polymeric lignin in the presence of manganese, suggesting their relevance in lignocellulose transformation., Conclusions: Understanding the mechanisms used by basidiomycetes to degrade lignin is of particular relevance to comprehend carbon cycle in nature and to design biotechnological tools for the industrial use of plant biomass. Here, we provide the first structure-function characterization of two novel MnP subfamilies present in Agaricales mushrooms, elucidating the main residues involved in catalysis and demonstrating their ability to modify the lignin macromolecule., (© 2024. The Author(s).)

Lentinula is a broadly distributed group of fungi that contains the cultivated shiitake mushroom, L. edodes . We sequenced 24 genomes representing eight described species and several unnamed lineages of Lentinula from 15 countries on four continents. Lentinula comprises four major clades that arose in the Oligocene, three in the Americas and one in Asia-Australasia. To expand sampling of shiitake mushrooms, we assembled 60 genomes of L. edodes from China that were previously published as raw Illumina reads and added them to our dataset. Lentinula edodes sensu lato (s. lat.) contains three lineages that may warrant recognition as species, one including a single isolate from Nepal that is the sister group to the rest of L. edodes s. lat., a second with 20 cultivars and 12 wild isolates from China, Japan, Korea, and the Russian Far East, and a third with 28 wild isolates from China, Thailand, and Vietnam. Two additional lineages in China have arisen by hybridization among the second and third groups. Genes encoding cysteine sulfoxide lyase ( lecsl ) and γ-glutamyl transpeptidase ( leggt ), which are implicated in biosynthesis of the organosulfur flavor compound lenthionine, have diversified in Lentinula . Paralogs of both genes that are unique to Lentinula ( lecsl 3 and leggt 5b) are coordinately up-regulated in fruiting bodies of L. edodes . The pangenome of L. edodes s. lat. contains 20,308 groups of orthologous genes, but only 6,438 orthogroups (32%) are shared among all strains, whereas 3,444 orthogroups (17%) are found only in wild populations, which should be targeted for conservation.

The study of evolution is limited by the techniques available to do so. Aside from the use of the fossil record, molecular phylogenetics can provide a detailed characterization of evolutionary histories using genes, genomes and proteins. However, these tools provide scarce biochemical information of the organisms and systems of interest and are therefore very limited when they come to explain protein evolution. In the past decade, this limitation has been overcome by the development of ancestral sequence reconstruction (ASR) methods. ASR allows the subsequent resurrection in the laboratory of inferred proteins from now extinct organisms, becoming an outstanding tool to study enzyme evolution. Here we review the recent advances in ASR methods and their application to study fungal evolution, with special focus on wood-decay fungi as essential organisms in the global carbon cycling., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ayuso-Fernández, Molpeceres, Camarero, Ruiz-Dueñas and Martínez.)

In the present work, lignin-like fractions were isolated from several ancestral plants -including moss ( Hypnum cupressiforme and Polytrichum commune ), lycophyte ( Selaginella kraussiana ), horsetail ( Equisetum palustre ), fern ( Nephrolepis cordifolia and Pteridium aquilinum ), cycad ( Cycas revoluta ), and gnetophyte ( Ephedra fragilis ) species- and structurally characterized by pyrolysis-gas chromatography-mass spectrometry (Py-GC/MS) and two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy. Py-GC/MS yielded marker compounds characteristic of lignin units, except in the H. cupressiforme , P. commune and E. palustre "lignins," where they were practically absent. Additional structural information on the other five samples was obtained from 2D-NMR experiments displaying intense correlations signals of guaiacyl (G) units in the fern and cycad lignins, along with smaller amounts of p -hydroxyphenyl (H) units. Interestingly, the lignins from the lycophyte S. kraussiana and the gnetophyte E. fragilis were not only composed of G- and H-lignin units but they also incorporated significant amounts of the syringyl (S) units characteristic of angiosperms, which appeared much later in plant evolution, most probably due to convergent evolution. The latter finding is also supported by the abundance of syringol derivatives after the Py-GC/MS analyses of these two samples. Regarding lignin structure, β- O -4' alkyl-aryl ethers were the most abundant substructures, followed by condensed β-5' phenylcoumarans and β-β' resinols (and dibenzodioxocins in the fern and cycad lignins). The highest percentages of alkyl-aryl ether structures correlated with the higher S/G ratio in the S. Kraussiana and E. fragilis lignin-like fractions. More interestingly, apart from the typical monolignol-derived lignin units (H, G and S), other structures, assigned to flavonoid compounds never reported before in natural lignins (such as amentoflavone, apigenin, hypnogenol B, kaempferol, and naringenin), could also be identified in the HSQC spectra of all the lignin-like fractions analyzed. With this purpose, in vitro synthesized coniferyl-naringenin and coniferyl-apigenin dehydrogenation polymers were used as standards. These flavonoids were abundant in H. cupressiforme appearing as the only constituents of the moss lignin-like fraction (including 84% of dimeric hypnogenol B) and their abundance decreased in those of S. Kraussiana (with amentoflavone and naringenin representing 14% of the total aromatic units), and the two ancient gymnosperms (0.4-1.2%) and ferns (0-0.7%)., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rencoret, Gutiérrez, Marques, del Río, Tobimatsu, Lam, Pérez-Boada, Ruiz-Dueñas, Barrasa and Martínez.)

Lignin biodegradation has been extensively studied in white-rot fungi, which largely belong to order Polyporales. Among the enzymes that wood-rotting polypores secrete, lignin peroxidases (LiPs) have been labeled as the most efficient. Here, we characterize a similar enzyme (ApeLiP) from a fungus of the order Agaricales (with ~13,000 described species), the soil-inhabiting mushroom Agrocybe pediades . X-ray crystallography revealed that ApeLiP is structurally related to Polyporales LiPs, with a conserved heme-pocket and a solvent-exposed tryptophan. Its biochemical characterization shows that ApeLiP can oxidize both phenolic and non-phenolic lignin model-compounds, as well as different dyes. Moreover, using stopped-flow rapid spectrophotometry and 2D-NMR, we demonstrate that ApeLiP can also act on real lignin. Characterization of a variant lacking the above tryptophan residue shows that this is the oxidation site for lignin and other high redox-potential substrates, and also plays a role in phenolic substrate oxidation. The reduction potentials of the catalytic-cycle intermediates were estimated by stopped-flow in equilibrium reactions, showing similar activation by H 2 O 2 , but a lower potential for the rate-limiting step (compound-II reduction) compared to other LiPs. Unexpectedly, ApeLiP was stable from acidic to basic pH, a relevant feature for application considering its different optima for oxidation of phenolic and nonphenolic compounds.

Pleurotus eryngii is a grassland-inhabiting fungus of biotechnological interest due to its ability to colonize non-woody lignocellulosic material. Genomic, transcriptomic, exoproteomic, and metabolomic analyses were combined to explain the enzymatic aspects underlaying wheat-straw transformation. Up-regulated and constitutive glycoside-hydrolases, polysaccharide-lyases, and carbohydrate-esterases active on polysaccharides, laccases active on lignin, and a surprisingly high amount of constitutive/inducible aryl-alcohol oxidases (AAOs) constituted the suite of extracellular enzymes at early fungal growth. Higher enzyme diversity and abundance characterized the longer-term growth, with an array of oxidoreductases involved in depolymerization of both cellulose and lignin, which were often up-regulated since initial growth. These oxidative enzymes included lytic polysaccharide monooxygenases (LPMOs) acting on crystalline polysaccharides, cellobiose dehydrogenase involved in LPMO activation, and ligninolytic peroxidases (mainly manganese-oxidizing peroxidases), together with highly abundant H 2 O 2 -producing AAOs. Interestingly, some of the most relevant enzymes acting on polysaccharides were appended to a cellulose-binding module. This is potentially related to the non-woody habitat of P. eryngii (in contrast to the wood habitat of many basidiomycetes). Additionally, insights into the intracellular catabolism of aromatic compounds, which is a neglected area of study in lignin degradation by basidiomycetes, were also provided. The multiomic approach reveals that although non-woody decay does not result in dramatic modifications, as revealed by detailed 2D-NMR and other analyses, it implies activation of the complete set of hydrolytic and oxidative enzymes characterizing lignocellulose-decaying basidiomycetes.

Agaricomycetes fungi responsible for decay of wood and other lignocellulosic substrates constitute a valuable source of lignin-degrading enzymes. Among these enzymes, laccases (multi-copper oxidases) present remarkable biotechnological potential as environmentally friendly biocatalysts able to oxidize a wide range of aromatic compounds using oxygen as the only requirement. Laccases from saprotrophic Agaricales species have been much less studied than laccases from Polyporales, despite the fact that the former fungi are excellent sources of laccases. Here, the gene of a novel laccase of Agrocybe pediades , that is secreted by the fungus during lignocellulose degradation, was synthesised de novo and expressed in Saccharomyces cerevisiae using an improved signal peptide previously obtained and enzyme directed evolution. The characterization of the new laccase variants provided new insights on the contribution of different amino acid residues to modulate laccase production, catalytic activity or optimal pH. The selected double-mutated variant also showed interesting properties as a biocatalyst, such as the ability to oxidise a wide range of substrates, including high-redox potential mediators and recalcitrant organic dyes, improved activity at neutral pH and high tolerance to inhibitors. Finally, we demonstrate the existence of three N -glycosylation sites in the laccase and their distinct effect on the secretion or catalytic activity of the enzyme.

A dye-decolorizing peroxidase (DyP) from Irpex lacteus was cloned and heterologously expressed as inclusion bodies in Escherichia coli . The protein was purified in one chromatographic step after its in vitro activation. It was active on ABTS, 2,6-dimethoxyphenol (DMP), and anthraquinoid and azo dyes as reported for other fungal DyPs, but it was also able to oxidize Mn 2+ (as manganese peroxidases and versatile peroxidases) and veratryl alcohol (VA) (as lignin peroxidases and versatile peroxidases). This corroborated that I. lacteus DyPs are the only enzymes able to oxidize high redox potential dyes, VA and Mn +2 . Phylogenetic analysis grouped this enzyme with other type D-DyPs from basidiomycetes. In addition to its interest for dye decolorization, the results of the transformation of softwood and hardwood lignosulfonates suggest a putative biological role of this enzyme in the degradation of phenolic lignin.

The functional diversity of the New Caledonian mangrove sediments was examined, observing the distribution of fungal dye-decolorizing peroxidases (DyPs), together with the complete biochemical characterization of the main DyP. Using a functional metabarcoding approach, the diversity of expressed genes encoding fungal DyPs was investigated in surface and deeper sediments, collected beneath either Avicennia marina or Rhizophora stylosa trees, during either the wet or the dry seasons. The highest DyP diversity was observed in surface sediments beneath the R. stylosa area during the wet season, and one particular operational functional unit (OFU1) was detected as the most abundant DyP isoform. This OFU was found in all sediment samples, representing 51-100% of the total DyP-encoding sequences in 70% of the samples. The complete cDNA sequence corresponding to this abundant DyP (OFU 1) was retrieved by gene capture, cloned, and heterologously expressed in Pichia pastoris . The recombinant enzyme, called DyP1, was purified and characterized, leading to the description of its physical-chemical properties, its ability to oxidize diverse phenolic substrates, and its potential to decolorize textile dyes; DyP1 was more active at low pH, though moderately stable over a wide pH range. The enzyme was very stable at temperatures up to 50 °C, retaining 60% activity after 180 min incubation. Its ability to decolorize industrial dyes was also tested on Reactive Blue 19, Acid Black, Disperse Blue 79, and Reactive Black 5. The effect of hydrogen peroxide and sea salt on DyP1 activity was studied and compared to what is reported for previously characterized enzymes from terrestrial and marine-derived fungi.

As actors of global carbon cycle, Agaricomycetes (Basidiomycota) have developed complex enzymatic machineries that allow them to decompose all plant polymers, including lignin. Among them, saprotrophic Agaricales are characterized by an unparalleled diversity of habitats and lifestyles. Comparative analysis of 52 Agaricomycetes genomes (14 of them sequenced de novo) reveals that Agaricales possess a large diversity of hydrolytic and oxidative enzymes for lignocellulose decay. Based on the gene families with the predicted highest evolutionary rates-namely cellulose-binding CBM1, glycoside hydrolase GH43, lytic polysaccharide monooxygenase AA9, class-II peroxidases, glucose-methanol-choline oxidase/dehydrogenases, laccases, and unspecific peroxygenases-we reconstructed the lifestyles of the ancestors that led to the extant lignocellulose-decomposing Agaricomycetes. The changes in the enzymatic toolkit of ancestral Agaricales are correlated with the evolution of their ability to grow not only on wood but also on leaf litter and decayed wood, with grass-litter decomposers as the most recent eco-physiological group. In this context, the above families were analyzed in detail in connection with lifestyle diversity. Peroxidases appear as a central component of the enzymatic toolkit of saprotrophic Agaricomycetes, consistent with their essential role in lignin degradation and high evolutionary rates. This includes not only expansions/losses in peroxidase genes common to other basidiomycetes but also the widespread presence in Agaricales (and Russulales) of new peroxidases types not found in wood-rotting Polyporales, and other Agaricomycetes orders. Therefore, we analyzed the peroxidase evolution in Agaricomycetes by ancestral-sequence reconstruction revealing several major evolutionary pathways and mapped the appearance of the different enzyme types in a time-calibrated species tree., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

We aim to clarify the ligninolytic capabilities of dye-decolorizing peroxidases (DyPs) from bacteria and fungi, compared to fungal lignin peroxidase (LiP) and versatile peroxidase (VP). With this purpose, DyPs from Amycolatopsis sp., Thermomonospora curvata , and Auricularia auricula-judae , VP from Pleurotus eryngii , and LiP from Phanerochaete chrysosporium were produced, and their kinetic constants and reduction potentials determined. Sharp differences were found in the oxidation of nonphenolic simple (veratryl alcohol, VA) and dimeric (veratrylglycerol-β- guaiacyl ether, VGE) lignin model compounds, with LiP showing the highest catalytic efficiencies (around 15 and 200 s -1 ·mM -1 for VGE and VA, respectively), while the efficiency of the A. auricula-judae DyP was 1-3 orders of magnitude lower, and no activity was detected with the bacterial DyPs. VP and LiP also showed the highest reduction potential (1.28-1.33 V) in the rate-limiting step of the catalytic cycle (i.e., compound-II reduction to resting enzyme), estimated by stopped-flow measurements at the equilibrium, while the T. curvata DyP showed the lowest value (1.23 V). We conclude that, when using realistic enzyme doses, only fungal LiP and VP, and in much lower extent fungal DyP, oxidize nonphenolic aromatics and, therefore, have the capability to act on the main moiety of the native lignin macromolecule.

White-rot (WR) fungi are pivotal decomposers of dead organic matter in forest ecosystems and typically use a large array of hydrolytic and oxidative enzymes to deconstruct lignocellulose. However, the extent of lignin and cellulose degradation may vary between species and wood type. Here, we combined comparative genomics, transcriptomics and secretome proteomics to identify conserved enzymatic signatures at the onset of wood-decaying activity within the Basidiomycota genus Pycnoporus. We observed a strong conservation in the genome structures and the repertoires of protein-coding genes across the four Pycnoporus species described to date, despite the species having distinct geographic distributions. We further analysed the early response of P. cinnabarinus, P. coccineus and P. sanguineus to diverse (ligno)-cellulosic substrates. We identified a conserved set of enzymes mobilized by the three species for breaking down cellulose, hemicellulose and pectin. The co-occurrence in the exo-proteomes of H2O2-producing enzymes with H2O2-consuming enzymes was a common feature of the three species, although each enzymatic partner displayed independent transcriptional regulation. Finally, cellobiose dehydrogenase-coding genes were systematically co-regulated with at least one AA9 lytic polysaccharide monooxygenase gene, indicative of enzymatic synergy in vivo. This study highlights a conserved core white-rot fungal enzymatic mechanism behind the wood-decaying process., (© The Author(s) 2020. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.)

Fungal plant pathogens remain a serious threat to the sustainable agriculture and forestry, despite the extensive efforts undertaken to control their spread. White root rot disease is threatening rubber tree (Hevea brasiliensis) plantations throughout South and Southeast Asia and Western Africa, causing tree mortality and severe yield losses. Here, we report the complete genome sequence of the basidiomycete fungus Rigidoporus microporus, a causative agent of the disease. Our phylogenetic analysis confirmed the position of R. microporus among the members of Hymenochaetales, an understudied group of basidiomycetes. Our analysis further identified pathogen's genes with a predicted role in the decay of plant cell wall polymers, in the utilization of latex components and in interspecific interactions between the pathogen and other fungi. We also detected putative horizontal gene transfer events in the genome of R. microporus. The reported first genome sequence of a tropical rubber tree pathogen R. microporus should contribute to the better understanding of how the fungus is able to facilitate wood decay and nutrient cycling as well as tolerate latex and utilize resinous extractives.

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 ( k 2app and k 3app ) showed a progressive increase during peroxidase evolution (up to 50-fold higher values for the rate-limiting k 3app ). 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., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Dye-decolorizing peroxidase (DyP) from Auricularia auricula-judae and versatile peroxidase (VP) from Pleurotus eryngii oxidize the three mononitrophenol isomers. Both enzymes have been overexpressed in Escherichia coli and in vitro activated. Despite their very different three-dimensional structures, the nitrophenol oxidation site is located at a solvent-exposed aromatic residue in both DyP (Trp377) and VP (Trp164), as revealed by liquid chromatography coupled to mass spectrometry and kinetic analyses of nitrophenol oxidation by the native enzymes and their tryptophan-less variants (the latter showing 10-60 fold lower catalytic efficiencies)., (Copyright © 2019. Published by Elsevier Inc.)

To investigate how ligninolytic peroxidases acquired the uniquely high redox potential they show today, their ancestors were resurrected and characterized. Unfortunately, the transient Compounds I (CI) and II (CII) from peroxide activation of the enzyme resting state (RS) are unstable. Therefore, the reduction potentials (E°') of the three redox couples (CI/RS, CI/CII and CII/RS) were estimated (for the first time in a ligninolytic peroxidase) from equilibrium concentrations analyzed by stopped-flow UV/Vis spectroscopy. Interestingly, the E°' of rate-limiting CII reduction to RS increased 70 mV from the common peroxidase ancestor to extant lignin peroxidase (LiP), and the same boost was observed for CI/RS and CI/CII, albeit with higher E°' values. A straightforward correlation was found between the E°' value and the progressive displacement of the proximal histidine Hϵ1 chemical shift in the NMR spectra, due to the higher paramagnetic effect of the heme Fe 3+ . More interestingly, the E°' and NMR data also correlated with the evolutionary time, revealing that ancestral peroxidases increased their reduction potential in the evolution to LiP thanks to molecular rearrangements in their heme pocket during the last 400 million years., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Plant biomass conversion for green chemistry and bio-energy is a current challenge for a modern sustainable bioeconomy. The complex polyaromatic lignin polymers in raw biomass feedstocks (i.e., agriculture and forestry by-products) are major obstacles for biomass conversions. White-rot fungi are wood decayers able to degrade all polymers from lignocellulosic biomass including cellulose, hemicelluloses, and lignin. The white-rot fungus Polyporus brumalis efficiently breaks down lignin and is regarded as having a high potential for the initial treatment of plant biomass in its conversion to bio-energy. Here, we describe the extraordinary ability of P. brumalis for lignin degradation using its enzymatic arsenal to break down wheat straw, a lignocellulosic substrate that is considered as a biomass feedstock worldwide., Results: We performed integrative multi-omics analyses by combining data from the fungal genome, transcriptomes, and secretomes. We found that the fungus possessed an unexpectedly large set of genes coding for Class II peroxidases involved in lignin degradation (19 genes) and GMC oxidoreductases/dehydrogenases involved in generating the hydrogen peroxide required for lignin peroxidase activity and promoting redox cycling of the fungal enzymes involved in oxidative cleavage of lignocellulose polymers (36 genes). The examination of interrelated multi-omics patterns revealed that eleven Class II Peroxidases were secreted by the fungus during fermentation and eight of them where tightly co-regulated with redox cycling enzymatic partners., Conclusion: As a peculiar feature of P. brumalis , we observed gene family extension, up-regulation and secretion of an abundant set of versatile peroxidases and manganese peroxidases, compared with other Polyporales species. The orchestrated secretion of an abundant set of these delignifying enzymes and redox cycling enzymatic partners could contribute to the delignification capabilities of the fungus. Our findings highlight the diversity of wood decay mechanisms present in Polyporales and the potentiality of further exploring this taxonomic order for enzymatic functions of biotechnological interest.

The resurrection of ancestral enzymes of now-extinct organisms (paleogenetics) is a developing field that allows the study of evolutionary hypotheses otherwise impossible to be tested. In the present study, we target fungal peroxidases that play a key role in lignin degradation, an essential process in the carbon cycle and often a limiting step in biobased industries. Ligninolytic peroxidases are secreted by wood-rotting fungi, the origin of which was recently established in the Carboniferous period associated with the appearance of these enzymes. These first peroxidases were not able to degrade lignin directly and used diffusible metal cations to attack its phenolic moiety. The phylogenetic analysis of the peroxidases of Polyporales, the order in which most extant wood-rotting fungi are included, suggests that later in evolution these enzymes would have acquired the ability to degrade nonphenolic lignin using a tryptophanyl radical interacting with the bulky polymer at the surface of the enzyme. Here, we track this powerful strategy for lignin degradation as a phenotypic trait in fungi and show that it is not an isolated event in the evolution of Polyporales. Using ancestral enzyme resurrection, we study the molecular changes that led to the appearance of the same surface oxidation site in two distant peroxidase lineages. By characterization of the resurrected enzymes, we demonstrate convergent evolution at the amino acid level during the evolution of these fungi and track the different changes leading to phylogenetically distant ligninolytic peroxidases from ancestors lacking the ability to degrade nonphenolic lignin., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Fungi produce heme-containing peroxidases and peroxygenases, flavin-containing oxidases and dehydrogenases, and different copper-containing oxidoreductases involved in the biodegradation of lignin and other recalcitrant compounds. Heme peroxidases comprise the classical ligninolytic peroxidases and the new dye-decolorizing peroxidases, while heme peroxygenases belong to a still largely unexplored superfamily of heme-thiolate proteins. Nevertheless, basidiomycete unspecific peroxygenases have the highest biotechnological interest due to their ability to catalyze a variety of regio- and stereo-selective monooxygenation reactions with H 2 O 2 as the source of oxygen and final electron acceptor. Flavo-oxidases are involved in both lignin and cellulose decay generating H 2 O 2 that activates peroxidases and generates hydroxyl radical. The group of copper oxidoreductases also includes other H 2 O 2 generating enzymes - copper-radical oxidases - together with classical laccases that are the oxidoreductases with the largest number of reported applications to date. However, the recently described lytic polysaccharide monooxygenases have attracted the highest attention among copper oxidoreductases, since they are capable of oxidatively breaking down crystalline cellulose, the disintegration of which is still a major bottleneck in lignocellulose biorefineries, along with lignin degradation. Interestingly, some flavin-containing dehydrogenases also play a key role in cellulose breakdown by directly/indirectly "fueling" electrons for polysaccharide monooxygenase activation. Many of the above oxidoreductases have been engineered, combining rational and computational design with directed evolution, to attain the selectivity, catalytic efficiency and stability properties required for their industrial utilization. Indeed, using ad hoc software and current computational capabilities, it is now possible to predict substrate access to the active site in biophysical simulations, and electron transfer efficiency in biochemical simulations, reducing in orders of magnitude the time of experimental work in oxidoreductase screening and engineering. What has been set out above is illustrated by a series of remarkable oxyfunctionalization and oxidation reactions developed in the frame of an intersectorial and multidisciplinary European RTD project. The optimized reactions include enzymatic synthesis of 1-naphthol, 25-hydroxyvitamin D 3 , drug metabolites, furandicarboxylic acid, indigo and other dyes, and conductive polyaniline, terminal oxygenation of alkanes, biomass delignification and lignin oxidation, among others. These successful case stories demonstrate the unexploited potential of oxidoreductases in medium and large-scale biotransformations., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Combining a computational analysis with site-directed mutagenesis, we have studied the long-range electron transfer pathway in versatile and lignin peroxidases, two enzymes of biotechnological interest that play a key role for fungal degradation of the bulky lignin molecule in plant biomass. The in silico study established two possible electron transfer routes starting at the surface tryptophan residue previously identified as responsible for oxidation of the bulky lignin polymer. Moreover, in both enzymes, a second buried tryptophan residue appears as a top electron transfer carrier, indicating the prevalence of one pathway. Site-directed mutagenesis of versatile peroxidase (from Pleurotus eryngii) allowed us to corroborate the computational analysis and the role played by the buried tryptophan (Trp244) and a neighbor phenylalanine residue (Phe198), together with the surface tryptophan, in the electron transfer. These three aromatic residues are highly conserved in all the sequences analyzed (up to a total of 169). The importance of the surface (Trp171) and buried (Trp251) tryptophan residues in lignin peroxidase has been also confirmed by directed mutagenesis of the Phanerochaete chrysosporium enzyme. Overall, the combined procedure identifies analogous electron transfer pathways in the long-range oxidation mechanism for both ligninolytic peroxidases, constituting a good example of how computational analysis avoids making extensive trial-error mutagenic experiments.

Background: Floudas et al. ( Science 336: 1715) established that lignin-degrading fungi appeared at the end of Carboniferous period associated with the production of the first ligninolytic peroxidases. Here, the subsequent evolution of these enzymes in Polyporales, where most wood-rotting fungi are included, is experimentally recreated using genomic information., Results: With this purpose, we analyzed the evolutionary pathway leading to the most efficient lignin-degrading peroxidases characterizing Polyporales species. After sequence reconstruction from 113 genes of ten sequenced genomes, the main enzyme intermediates were resurrected and characterized. Biochemical changes were analyzed together with predicted sequences and structures, to understand how these enzymes acquired the ability to degrade lignin and how this ability changed with time. The most probable first peroxidase in Polyporales would be a manganese peroxidase (Mn 3+ oxidizing phenolic lignin) that did not change substantially until the appearance of an exposed tryptophan (oxidizing nonphenolic lignin) originating an ancestral versatile peroxidase. Later, a quick evolution, with loss of the Mn 2+ -binding site, generated the first lignin peroxidase that evolved to the extant form by improving the catalytic efficiency. Increased stability at acidic pH, which strongly increases the oxidizing power of these enzymes, was observed paralleling the appearance of the exposed catalytic tryptophan., Conclusions: We show how the change in peroxidase catalytic activities meant an evolutionary exploration for more efficient ways of lignin degradation by fungi, a key step for carbon recycling in land ecosystems. The study provides ancestral enzymes with a potential biotechnological interest for the sustainable production of fuels and chemicals in a biomass-based economy.

Background: Despite claims as key enzymes in enzymatic delignification, very scarce information on the reaction rates between the ligninolytic versatile peroxidase (VP) and lignin peroxidase (LiP) and the lignin polymer is available, due to methodological difficulties related to lignin heterogeneity and low solubility., Results: Two water-soluble sulfonated lignins (from Picea abies and Eucalyptus grandis ) were chemically characterized and used to estimate single electron-transfer rates to the H 2 O 2 -activated Pleurotus eryngii VP (native enzyme and mutated variant) transient states (compounds I and II bearing two- and one-electron deficiencies, respectively). When the rate-limiting reduction of compound II was quantified by stopped-flow rapid spectrophotometry, from fourfold (softwood lignin) to over 100-fold (hardwood lignin) lower electron-transfer efficiencies ( k 3app values) were observed for the W164S variant at surface Trp164, compared with the native VP. These lignosulfonates have ~20-30 % phenolic units, which could be responsible for the observed residual activity. Therefore, methylated (and acetylated) samples were used in new stopped-flow experiments, where negligible electron transfer to the W164S compound II was found. This revealed that the residual reduction of W164S compound II by native lignin was due to its phenolic moiety. Since both native lignins have a relatively similar phenolic moiety, the higher W164S activity on the softwood lignin could be due to easier access of its mono-methoxylated units for direct oxidation at the heme channel in the absence of the catalytic tryptophan. Moreover, the lower electron transfer rates from the derivatized lignosulfonates to native VP suggest that peroxidase attack starts at the phenolic lignin moiety. In agreement with the transient-state kinetic data, very low structural modification of lignin, as revealed by size-exclusion chromatography and two-dimensional nuclear magnetic resonance, was obtained during steady-state treatment (up to 24 h) of native lignosulfonates with the W164S variant compared with native VP and, more importantly, this activity disappeared when nonphenolic lignosulfonates were used., Conclusions: We demonstrate for the first time that the surface tryptophan conserved in most LiPs and VPs (Trp164 of P. eryngii VPL) is strictly required for oxidation of the nonphenolic moiety, which represents the major and more recalcitrant part of the lignin polymer.

A variant of high biotechnological interest (called 2-1B) was obtained by directed evolution of the Pleurotus eryngii VP (versatile peroxidase) expressed in Saccharomyces cerevisiae [García-Ruiz, González-Pérez, Ruiz-Dueñas, Martínez and Alcalde (2012) Biochem. J. 441: , 487-498]. 2-1B shows seven mutations in the mature protein that resulted in improved functional expression, activity and thermostability, along with a remarkable stronger alkaline stability (it retains 60% of the initial activity after 120 h of incubation at pH 9 compared with complete inactivation of the native enzyme after only 1 h). The latter is highly demanded for biorefinery applications. In the present study we investigate the structural basis behind the enhanced alkaline stabilization of this evolved enzyme. In order to do this, several VP variants containing one or several of the mutations present in 2-1B were expressed in Escherichia coli, and their alkaline stability and biochemical properties were determined. In addition, the crystal structures of 2-1B and one of the intermediate variants were solved and carefully analysed, and molecular dynamics simulations were carried out. We concluded that the introduction of three basic residues in VP (Lys-37, Arg-39 and Arg-330) led to new connections between haem and helix B (where the distal histidine residue is located), and formation of new electrostatic interactions, that avoided the hexa-co-ordination of the haem iron. These new structural determinants stabilized the haem and its environment, helping to maintain the structural enzyme integrity (with penta-co-ordinated haem iron) under alkaline conditions. Moreover, the reinforcement of the solvent-exposed area around Gln-305 in the proximal side, prompted by the Q202L mutation, further enhanced the stability., (© 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.)

Background: Pleurotus ostreatus is the second edible mushroom worldwide, and a model fungus for delignification applications, with the advantage of growing on woody and nonwoody feedstocks. Its sequenced genome is available, and this gave us the opportunity to perform proteomic studies to identify the enzymes overproduced in lignocellulose cultures., Results: Monokaryotic P. ostreatus (PC9) was grown with poplar wood or wheat straw as the sole C/N source and the extracellular proteins were analyzed, together with those from glucose medium. Using nano-liquid chromatography coupled to tandem mass spectrometry of whole-protein hydrolyzate, over five-hundred proteins were identified. Thirty-four percent were unique of the straw cultures, while only 15 and 6 % were unique of the glucose and poplar cultures, respectively (20 % were produced under the three conditions, and additional 19 % were shared by the two lignocellulose cultures). Semi-quantitative analysis showed oxidoreductases as the main protein type both in the poplar (39 % total abundance) and straw (31 %) secretomes, while carbohydrate-active enzymes (CAZys) were only slightly overproduced (14-16 %). Laccase 10 (LACC10) was the main protein in the two lignocellulose secretomes (10-14 %) and, together with LACC2, LACC9, LACC6, versatile peroxidase 1 (VP1), and manganese peroxidase 3 (MnP3), were strongly overproduced in the lignocellulose cultures. Seven CAZys were also among the top-50 proteins, but only CE16 acetylesterase was overproduced on lignocellulose. When the woody and nonwoody secretomes were compared, GH1 and GH3 β-glycosidases were more abundant on poplar and straw, respectively and, among less abundant proteins, VP2 was overproduced on straw, while VP3 was only found on poplar. The treated lignocellulosic substrates were analyzed by two-dimensional nuclear magnetic resonance (2D NMR), and a decrease of lignin relative to carbohydrate signals was observed, together with the disappearance of some minor lignin substructures, and an increase of sugar reducing ends., Conclusions: Oxidoreductases are strongly induced when P. ostreatus grows on woody and nonwoody lignocellulosic substrates. One laccase occupied the first position in both secretomes, and three more were overproduced together with one VP and one MnP, suggesting an important role in lignocellulose degradation. Preferential removal of lignin vs carbohydrates was shown by 2D NMR, in agreement with the above secretomic results.

Background: White-rot basidiomycete fungi are potent degraders of plant biomass, with the ability to mineralize all lignocellulose components. Recent comparative genomics studies showed that these fungi use a wide diversity of enzymes for wood degradation. Deeper functional analyses are however necessary to understand the enzymatic mechanisms leading to lignocellulose breakdown. The Polyporale fungus Pycnoporus coccineus BRFM310 grows well on both coniferous and deciduous wood. In the present study, we analyzed the early response of the fungus to softwood (pine) and hardwood (aspen) feedstocks and tested the effect of the secreted enzymes on lignocellulose deconstruction., Results: Transcriptomic and proteomic analyses revealed that P. coccineus grown separately on pine and aspen displayed similar sets of transcripts and enzymes implicated in lignin and polysaccharide degradation. In particular, the expression of lignin-targeting oxidoreductases, such as manganese peroxidases, increased upon cultivation on both woods. The sets of enzymes secreted during growth on both pine and aspen were more efficient in saccharide release from pine than from aspen, and characterization of the residual solids revealed polysaccharide conversion on both pine and aspen fiber surfaces., Conclusion: The combined analysis of soluble sugars and solid residues showed the suitability of P. coccineus secreted enzymes for softwood degradation. Analyses of solubilized products and residual surface chemistries of enzyme-treated wood samples pointed to differences in fiber penetration by different P. coccineus secretomes. Accordingly, beyond the variety of CAZymes identified in P. coccineus genome, transcriptome and secretome, we discuss several parameters such as the abundance of manganese peroxidases and the potential role of cytochrome P450s and pectin degradation on the efficacy of fungi for softwood conversion.

Two phylogenetically divergent genes of the new family of dye-decolorizing peroxidases (DyPs) were found during comparison of the four DyP genes identified in the Pleurotus ostreatus genome with over 200 DyP genes from other basidiomycete genomes. The heterologously expressed enzymes (Pleos-DyP1 and Pleos-DyP4, following the genome nomenclature) efficiently oxidize anthraquinoid dyes (such as Reactive Blue 19), which are characteristic DyP substrates, as well as low redox-potential dyes (such as 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) and substituted phenols. However, only Pleos-DyP4 oxidizes the high redox-potential dye Reactive Black 5, at the same time that it displays high thermal and pH stability. Unexpectedly, both enzymes also oxidize Mn(2+) to Mn(3+), albeit with very different catalytic efficiencies. Pleos-DyP4 presents a Mn(2+) turnover (56 s(-1)) nearly in the same order of the two other Mn(2+)-oxidizing peroxidase families identified in the P. ostreatus genome: manganese peroxidases (100 s(-1) average turnover) and versatile peroxidases (145 s(-1) average turnover), whose genes were also heterologously expressed. Oxidation of Mn(2+) has been reported for an Amycolatopsis DyP (24 s(-1)) and claimed for other bacterial DyPs, albeit with lower activities, but this is the first time that Mn(2+) oxidation is reported for a fungal DyP. Interestingly, Pleos-DyP4 (together with ligninolytic peroxidases) is detected in the secretome of P. ostreatus grown on different lignocellulosic substrates. It is suggested that generation of Mn(3+) oxidizers plays a role in the P. ostreatus white-rot lifestyle since three different families of Mn(2+)-oxidizing peroxidase genes are present in its genome being expressed during lignocellulose degradation.

Versatile peroxidase (VP) from the white-rot fungus Pleurotus eryngii is a high redox potential peroxidase of biotechnological interest able to oxidize a wide range of recalcitrant substrates including lignin, phenolic and non-phenolic aromatic compounds and dyes. However, the relatively low stability towards pH of this and other fungal peroxidases is a drawback for their industrial application. A strategy based on the comparative analysis of the crystal structures of VP and the highly pH-stable manganese peroxidase (MnP4) from Pleurotus ostreatus was followed to improve the VP pH stability. Several interactions, including hydrogen bonds and salt bridges, and charged residues exposed to the solvent were identified as putatively contributing to the pH stability of MnP4. The eight amino acid residues responsible for these interactions and seven surface basic residues were introduced into VP by directed mutagenesis. Furthermore, two cysteines were also included to explore the effect of an extra disulfide bond stabilizing the distal Ca2+ region. Three of the four designed variants were crystallized and new interactions were confirmed, being correlated with the observed improvement in pH stability. The extra hydrogen bonds and salt bridges stabilized the heme pocket at acidic and neutral pH as revealed by UV-visible spectroscopy. They led to a VP variant that retained a significant percentage of the initial activity at both pH 3.5 (61% after 24 h) and pH 7 (55% after 120 h) compared with the native enzyme, which was almost completely inactivated. The introduction of extra solvent-exposed basic residues and an additional disulfide bond into the above variant further improved the stability at acidic pH (85% residual activity at pH 3.5 after 24 h when introduced separately, and 64% at pH 3 when introduced together). The analysis of the results provides a rational explanation to the pH stability improvement achieved.

Versatile peroxidase (VP) is a high redox-potential peroxidase of biotechnological interest that is able to oxidize phenolic and non-phenolic aromatics, Mn(2+), and different dyes. The ability of VP from Pleurotus eryngii to oxidize water-soluble lignins (softwood and hardwood lignosulfonates) is demonstrated here by a combination of directed mutagenesis and spectroscopic techniques, among others. In addition, direct electron transfer between the peroxidase and the lignin macromolecule was kinetically characterized using stopped-flow spectrophotometry. VP variants were used to show that this reaction strongly depends on the presence of a solvent-exposed tryptophan residue (Trp-164). Moreover, the tryptophanyl radical detected by EPR spectroscopy of H2O2-activated VP (being absent from the W164S variant) was identified as catalytically active because it was reduced during lignosulfonate oxidation, resulting in the appearance of a lignin radical. The decrease of lignin fluorescence (excitation at 355 nm/emission at 400 nm) during VP treatment under steady-state conditions was accompanied by a decrease of the lignin (aromatic nuclei and side chains) signals in one-dimensional and two-dimensional NMR spectra, confirming the ligninolytic capabilities of the enzyme. Simultaneously, size-exclusion chromatography showed an increase of the molecular mass of the modified residual lignin, especially for the (low molecular mass) hardwood lignosulfonate, revealing that the oxidation products tend to recondense during the VP treatment. Finally, mutagenesis of selected residues neighboring Trp-164 resulted in improved apparent second-order rate constants for lignosulfonate reactions, revealing that changes in its protein environment (modifying the net negative charge and/or substrate accessibility/binding) can modulate the reactivity of the catalytic tryptophan., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The first enzyme with dye-decolorizing peroxidase (DyP) activity was described in 1999 from an arthroconidial culture of the fungus Bjerkandera adusta. However, the first DyP sequence had been deposited three years before, as a peroxidase gene from a culture of an unidentified fungus of the family Polyporaceae (probably Irpex lacteus). Since the first description, fewer than ten basidiomycete DyPs have been purified and characterized, but a large number of sequences are available from genomes. DyPs share a general fold and heme location with chlorite dismutases and other DyP-type related proteins (such as Escherichia coli EfeB), forming the CDE superfamily. Taking into account the lack of an evolutionary relationship with the catalase-peroxidase superfamily, the observed heme pocket similarities must be considered as a convergent type of evolution to provide similar reactivity to the enzyme cofactor. Studies on the Auricularia auricula-judae DyP showed that high-turnover oxidation of anthraquinone type and other DyP substrates occurs via long-range electron transfer from an exposed tryptophan (Trp377, conserved in most basidiomycete DyPs), whose catalytic radical was identified in the H2O2-activated enzyme. The existence of accessory oxidation sites in DyP is suggested by the residual activity observed after site-directed mutagenesis of the above tryptophan. DyP degradation of substituted anthraquinone dyes (such as Reactive Blue 5) most probably proceeds via typical one-electron peroxidase oxidations and product breakdown without a DyP-catalyzed hydrolase reaction. Although various DyPs are able to break down phenolic lignin model dimers, and basidiomycete DyPs also present marginal activity on nonphenolic dimers, a significant contribution to lignin degradation is unlikely because of the low activity on high redox-potential substrates., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Ligninolytic peroxidases are enzymes of biotechnological interest due to their ability to oxidize high redox potential aromatic compounds, including the recalcitrant lignin polymer. However, different obstacles prevent their use in industrial and environmental applications, including low stability towards their natural oxidizing-substrate H2O2. In this work, versatile peroxidase was taken as a model ligninolytic peroxidase, its oxidative inactivation by H2O2 was studied and different strategies were evaluated with the aim of improving H2O2 stability. Oxidation of the methionine residues was produced during enzyme inactivation by H2O2 excess. Substitution of these residues, located near the heme cofactor and the catalytic tryptophan, rendered a variant with a 7.8-fold decreased oxidative inactivation rate. A second strategy consisted in mutating two residues (Thr45 and Ile103) near the catalytic distal histidine with the aim of modifying the reactivity of the enzyme with H2O2. The T45A/I103T variant showed a 2.9-fold slower reaction rate with H2O2 and 2.8-fold enhanced oxidative stability. Finally, both strategies were combined in the T45A/I103T/M152F/M262F/M265L variant, whose stability in the presence of H2O2 was improved 11.7-fold. This variant showed an increased half-life, over 30 min compared with 3.4 min of the native enzyme, under an excess of 2000 equivalents of H2O2. Interestingly, the stability improvement achieved was related with slower formation, subsequent stabilization and slower bleaching of the enzyme Compound III, a peroxidase intermediate that is not part of the catalytic cycle and leads to the inactivation of the enzyme.

Dye-decolorizing peroxidase (DyP) of Auricularia auricula-judae has been expressed in Escherichia coli as a representative of a new DyP family, and subjected to mutagenic, spectroscopic, crystallographic and computational studies. The crystal structure of DyP shows a buried haem cofactor, and surface tryptophan and tyrosine residues potentially involved in long-range electron transfer from bulky dyes. Simulations using PELE (Protein Energy Landscape Exploration) software provided several binding-energy optima for the anthraquinone-type RB19 (Reactive Blue 19) near the above aromatic residues and the haem access-channel. Subsequent QM/MM (quantum mechanics/molecular mechanics) calculations showed a higher tendency of Trp-377 than other exposed haem-neighbouring residues to harbour a catalytic protein radical, and identified the electron-transfer pathway. The existence of such a radical in H₂O₂-activated DyP was shown by low-temperature EPR, being identified as a mixed tryptophanyl/tyrosyl radical in multifrequency experiments. The signal was dominated by the Trp-377 neutral radical contribution, which disappeared in the W377S variant, and included a tyrosyl contribution assigned to Tyr-337 after analysing the W377S spectra. Kinetics of substrate oxidation by DyP suggests the existence of high- and low-turnover sites. The high-turnover site for oxidation of RB19 (k(cat) > 200 s⁻¹) and other DyP substrates was assigned to Trp-377 since it was absent from the W377S variant. The low-turnover site/s (RB19 k(cat) ~20 s⁻¹) could correspond to the haem access-channel, since activity was decreased when the haem channel was occluded by the G169L mutation. If a tyrosine residue is also involved, it will be different from Tyr-337 since all activities are largely unaffected in the Y337S variant.

Collectively classified as white-rot fungi, certain basidiomycetes efficiently degrade the major structural polymers of wood cell walls. A small subset of these Agaricomycetes, exemplified by Phlebiopsis gigantea, is capable of colonizing freshly exposed conifer sapwood despite its high content of extractives, which retards the establishment of other fungal species. The mechanism(s) by which P. gigantea tolerates and metabolizes resinous compounds have not been explored. Here, we report the annotated P. gigantea genome and compare profiles of its transcriptome and secretome when cultured on fresh-cut versus solvent-extracted loblolly pine wood. The P. gigantea genome contains a conventional repertoire of hydrolase genes involved in cellulose/hemicellulose degradation, whose patterns of expression were relatively unperturbed by the absence of extractives. The expression of genes typically ascribed to lignin degradation was also largely unaffected. In contrast, genes likely involved in the transformation and detoxification of wood extractives were highly induced in its presence. Their products included an ABC transporter, lipases, cytochrome P450s, glutathione S-transferase and aldehyde dehydrogenase. Other regulated genes of unknown function and several constitutively expressed genes are also likely involved in P. gigantea's extractives metabolism. These results contribute to our fundamental understanding of pioneer colonization of conifer wood and provide insight into the diverse chemistries employed by fungi in carbon cycling processes.

The genome of Ceriporiopsis subvermispora includes 13 manganese peroxidase (MnP) genes representative of the three subfamilies described in ligninolytic fungi, which share an Mn(2+)-oxidation site and have varying lengths of the C-terminal tail. Short, long and extralong MnPs were heterologously expressed and biochemically characterized, and the first structure of an extralong MnP was solved. Its C-terminal tail surrounds the haem-propionate access channel, contributing to Mn(2+) oxidation by the internal propionate, but prevents the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), which is only oxidized by short MnPs and by shortened-tail variants from site-directed mutagenesis. The tail, which is anchored by numerous contacts, not only affects the catalytic properties of long/extralong MnPs but is also associated with their high acidic stability. Cd(2+) binds at the Mn(2+)-oxidation site and competitively inhibits oxidation of both Mn(2+) and ABTS. Moreover, mutations blocking the haem-propionate channel prevent substrate oxidation. This agrees with molecular simulations that position ABTS at an electron-transfer distance from the haem propionates of an in silico shortened-tail form, while it cannot reach this position in the extralong MnP crystal structure. Only small differences exist between the long and the extralong MnPs, which do not justify their classification as two different subfamilies, but they significantly differ from the short MnPs, with the presence/absence of the C-terminal tail extension being implicated in these differences.

An efficient heterologous expression system for Auricularia auricula-judae dye-decolorizing peroxidase (DyP) has been constructed. DNA coding for the mature protein sequence was cloned into the pET23a vector and expressed in Escherichia coli BL21(DE3)pLysS. Recombinant DyP was obtained in high yield as inclusion bodies, and different parameters for its in vitro activation were optimized with a refolding yield of ∼8.5% of the E. coli-expressed DyP. Then, a single chromatographic step allowed the recovery of 17% of the refolded DyP as pure enzyme (1.5mg per liter of culture). The thermal stabilities of wild DyP from A. auricula-judae and recombinant DyP from E. coli expression were similar up to 60°C, but the former was more stable in the 62-70°C range. Stabilities against pH and H2O2 were also measured, and a remarkably high stability at extreme pH values (from pH 2 to 12) was observed. The kinetic constants of recombinant DyP for the oxidation of different substrates were determined and, when compared with those of wild DyP, no important differences were ascertained. Both enzymes showed high affinity for Reactive Blue 19 (anthraquinone dye), Reactive Black 5 (azo dye), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and 2,6-dimethoxyphenol, with similar acidic pH optima and oxidative stabilities. Oxidation of veratryl alcohol and a nonphenolic lignin model dimer were confirmed, although as minor enzymatic activities. Interestingly, two sets of kinetic constants could be obtained for the oxidation of Reactive Blue 19 and other substrates, suggesting the existence of more than one oxidation site in this new peroxidase family., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Pleurotus ostreatus is an important edible mushroom and a model lignin degrading organism, whose genome contains nine genes of ligninolytic peroxidases, characteristic of white-rot fungi. These genes encode six manganese peroxidase (MnP) and three versatile peroxidase (VP) isoenzymes. Using liquid chromatography coupled to tandem mass spectrometry, secretion of four of these peroxidase isoenzymes (VP1, VP2, MnP2 and MnP6) was confirmed when P. ostreatus grows in a lignocellulose medium at 25°C (three more isoenzymes were identified by only one unique peptide). Then, the effect of environmental parameters on the expression of the above nine genes was studied by reverse transcription-quantitative PCR by changing the incubation temperature and medium pH of P. ostreatus cultures pre-grown under the above conditions (using specific primers and two reference genes for result normalization). The cultures maintained at 25°C (without pH adjustment) provided the highest levels of peroxidase transcripts and the highest total activity on Mn(2+) (a substrate of both MnP and VP) and Reactive Black 5 (a VP specific substrate). The global analysis of the expression patterns divides peroxidase genes into three main groups according to the level of expression at optimal conditions (vp1/mnp3>vp2/vp3/mnp1/mnp2/mnp6>mnp4/mnp5). Decreasing or increasing the incubation temperature (to 10°C or 37°C) and adjusting the culture pH to acidic or alkaline conditions (pH 3 and 8) generally led to downregulation of most of the peroxidase genes (and decrease of the enzymatic activity), as shown when the transcription levels were referred to those found in the cultures maintained at the initial conditions. Temperature modification produced less dramatic effects than pH modification, with most genes being downregulated during the whole 10°C treatment, while many of them were alternatively upregulated (often 6h after the thermal shock) and downregulated (12h) at 37°C. Interestingly, mnp4 and mnp5 were the only peroxidase genes upregulated under alkaline pH conditions. The differences in the transcription levels of the peroxidase genes when the culture temperature and pH parameters were changed suggest an adaptive expression according to environmental conditions. Finally, the intracellular proteome was analyzed, under the same conditions used in the secretomic analysis, and the protein product of the highly-transcribed gene mnp3 was detected. Therefore, it was concluded that the absence of MnP3 from the secretome of the P. ostreatus lignocellulose cultures was related to impaired secretion., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

During several forays for ligninolytic fungi in different Spanish native forests, 35 white-rot basidiomycetes growing on dead wood (16 species from 12 genera) and leaf litter (19 species from 10 genera) were selected for their ability to decolorize two recalcitrant aromatic dyes (Reactive Blue 38 and Reactive Black 5) added to malt extract agar medium. In this study, two dye decolorization patterns were observed and correlated with two ecophysiological groups (wood and humus white-rot basidiomycetes) and three taxonomical groups (orders Polyporales, Hymenochaetales and Agaricales). Depending on the above groups, different decolorization zones were observed on the dye-containing plates, being restricted to the colony area or extending to the surrounding medium, which suggested two different decay strategies. These two strategies were related to the ability to secrete peroxidases and laccases inside (white-rot wood Polyporales, Hymenochaetales and Agaricales) and outside (white-rot humus Agaricales) of the fungal colony, as revealed by enzymatic tests performed directly on the agar plates. Similar oxidoreductases production patterns were observed when fungi were grown in the absence of dyes, although the set of enzyme released was different. All these results suggest that the decolorization patterns observed could be related with the existence of two decay strategies developed by white-rot basidiomycetes adapted to wood and leaf litter decay in the field., (Published by Elsevier Inc.)

Background: Ligninolytic peroxidases are divided into three families: manganese peroxidases (MnPs), lignin peroxidases (LiPs), and versatile peroxidases (VPs). The latter two are able to degrade intact lignins, as shown using nonphenolic lignin model compounds, with VP oxidizing the widest range of recalcitrant substrates. One of the main limiting issues for the use of these two enzymes in lignocellulose biorefineries (for delignification and production of cellulose-based products or modification of industrial lignins to added-value products) is their progressive inactivation under acidic pH conditions, where they exhibit the highest oxidative activities., Results: In the screening of peroxidases from basidiomycete genomes, one MnP from Ceriporiopsis subvermispora was found to have a remarkable acidic stability. The crystal structure of this enzyme recently became available and, after comparison with Pleurotus ostreatus VP and Phanerochaete chrysosporium LiP structures, it was used as a robust scaffold to engineer a stable VP by introducing an exposed catalytic tryptophan, with different protein environments. The variants obtained largely maintain the acidic stability and strong Mn(2+)-oxidizing activity of the parent enzyme, and the ability to oxidize veratryl alcohol and Reactive Black 5 (two simple VP substrates) was introduced. The engineered peroxidases present more acidic optimal pH than the best VP from P. ostreatus, enabling higher catalytic efficiency oxidizing lignins, by lowering the reaction pH, as shown using a nonphenolic model dimer., Conclusions: A peroxidase that degrades lignin at very acidic pH could be obtained by engineering an exposed catalytic site, able to oxidize the bulky and recalcitrant lignin polymers, in a different peroxidase type selected because of its high stability at acidic pH. The potential of this type of engineered peroxidases as industrial biocatalysts in lignocellulose biorefineries is strongly enhanced by the possibility to perform the delignification (or lignin modification) reactions under extremely acidic pH conditions (below pH 2), resulting in enhanced oxidative power of the enzymes.

Background: Saprophytic filamentous fungi are ubiquitous micro-organisms that play an essential role in photosynthetic carbon recycling. The wood-decayer Pycnoporus cinnabarinus is a model fungus for the study of plant cell wall decomposition and is used for a number of applications in green and white biotechnology., Results: The 33.6 megabase genome of P. cinnabarinus was sequenced and assembled, and the 10,442 predicted genes were functionally annotated using a phylogenomic procedure. In-depth analyses were carried out for the numerous enzyme families involved in lignocellulosic biomass breakdown, for protein secretion and glycosylation pathways, and for mating type. The P. cinnabarinus genome sequence revealed a consistent repertoire of genes shared with wood-decaying basidiomycetes. P. cinnabarinus is thus fully equipped with the classical families involved in cellulose and hemicellulose degradation, whereas its pectinolytic repertoire appears relatively limited. In addition, P. cinnabarinus possesses a complete versatile enzymatic arsenal for lignin breakdown. We identified several genes encoding members of the three ligninolytic peroxidase types, namely lignin peroxidase, manganese peroxidase and versatile peroxidase. Comparative genome analyses were performed in fungi displaying different nutritional strategies (white-rot and brown-rot modes of decay). P. cinnabarinus presents a typical distribution of all the specific families found in the white-rot life style. Growth profiling of P. cinnabarinus was performed on 35 carbon sources including simple and complex substrates to study substrate utilization and preferences. P. cinnabarinus grew faster on crude plant substrates than on pure, mono- or polysaccharide substrates. Finally, proteomic analyses were conducted from liquid and solid-state fermentation to analyze the composition of the secretomes corresponding to growth on different substrates. The distribution of lignocellulolytic enzymes in the secretomes was strongly dependent on growth conditions, especially for lytic polysaccharide mono-oxygenases., Conclusions: With its available genome sequence, P. cinnabarinus is now an outstanding model system for the study of the enzyme machinery involved in the degradation or transformation of lignocellulosic biomass.

Background: The genome of Pleurotus ostreatus, an important edible mushroom and a model ligninolytic organism of interest in lignocellulose biorefineries due to its ability to delignify agricultural wastes, was sequenced with the purpose of identifying and characterizing the enzymes responsible for lignin degradation., Results: Heterologous expression of the class II peroxidase genes, followed by kinetic studies, enabled their functional classification. The resulting inventory revealed the absence of lignin peroxidases (LiPs) and the presence of three versatile peroxidases (VPs) and six manganese peroxidases (MnPs), the crystal structures of two of them (VP1 and MnP4) were solved at 1.0 to 1.1 Å showing significant structural differences. Gene expansion supports the importance of both peroxidase types in the white-rot lifestyle of this fungus. Using a lignin model dimer and synthetic lignin, we showed that VP is able to degrade lignin. Moreover, the dual Mn-mediated and Mn-independent activity of P. ostreatus MnPs justifies their inclusion in a new peroxidase subfamily. The availability of the whole POD repertoire enabled investigation, at a biochemical level, of the existence of duplicated genes. Differences between isoenzymes are not limited to their kinetic constants. Surprising differences in their activity T50 and residual activity at both acidic and alkaline pH were observed. Directed mutagenesis and spectroscopic/structural information were combined to explain the catalytic and stability properties of the most interesting isoenzymes, and their evolutionary history was analyzed in the context of over 200 basidiomycete peroxidase sequences., Conclusions: The analysis of the P. ostreatus genome shows a lignin-degrading system where the role generally played by LiP has been assumed by VP. Moreover, it enabled the first characterization of the complete set of peroxidase isoenzymes in a basidiomycete, revealing strong differences in stability properties and providing enzymes of biotechnological interest.

The genomes of three representative Polyporales (Bjerkandera adusta, Phlebia brevispora and a member of the Ganoderma lucidum complex) were sequenced to expand our knowledge on the diversity of ligninolytic and related peroxidase genes in this Basidiomycota order that includes most wood-rotting fungi. The survey was completed by analyzing the heme-peroxidase genes in the already available genomes of seven more Polyporales species representing the antrodia, gelatoporia, core polyporoid and phlebioid clades. The study confirms the absence of ligninolytic peroxidase genes from the manganese peroxidase (MnP), lignin peroxidase (LiP) and versatile peroxidase (VP) families, in the brown-rot fungal genomes (all of them from the antrodia clade), which include only a limited number of predicted low redox-potential generic peroxidase (GP) genes. When members of the heme-thiolate peroxidase (HTP) and dye-decolorizing peroxidase (DyP) superfamilies (up to a total of 64 genes) also are considered, the newly sequenced B. adusta appears as the Polyporales species with the highest number of peroxidase genes due to the high expansion of both the ligninolytic peroxidase and DyP (super)families. The evolutionary relationships of the 111 genes for class-II peroxidases (from the GP, MnP, VP, LiP families) in the 10 Polyporales genomes is discussed including the existence of different MnP subfamilies and of a large and homogeneous LiP cluster, while different VPs mainly cluster with short MnPs. Finally, ancestral state reconstructions showed that a putative MnP gene, derived from a primitive GP that incorporated the Mn(II)-oxidation site, is the precursor of all the class-II ligninolytic peroxidases. Incorporation of an exposed tryptophan residue involved in oxidative degradation of lignin in a short MnP apparently resulted in evolution of the first VP. One of these ancient VPs might have lost the Mn(II)-oxidation site being at the origin of all the LiP enzymes, which are found only in species of the order Polyporales.

LiP (lignin peroxidase) from Trametopsis cervina has an exposed catalytic tyrosine residue (Tyr181) instead of the tryptophan conserved in other lignin-degrading peroxidases. Pristine LiP showed a lag period in VA (veratryl alcohol) oxidation. However, VA-LiP (LiP after treatment with H2O2 and VA) lacked this lag, and H2O2-LiP (H2O2-treated LiP) was inactive. MS analyses revealed that VA-LiP includes one VA molecule covalently bound to the side chain of Tyr181, whereas H2O2-LiP contains a hydroxylated Tyr181. No adduct is formed in the Y171N variant. Molecular docking showed that VA binding is favoured by sandwich π stacking with Tyr181 and Phe89. EPR spectroscopy after peroxide activation of the pre-treated LiPs showed protein radicals other than the tyrosine radical found in pristine LiP, which were assigned to a tyrosine-VA adduct radical in VA-LiP and a dihydroxyphenyalanine radical in H2O2-LiP. Both radicals are able to oxidize large low-redox-potential substrates, but H2O2-LiP is unable to oxidize high-redox-potential substrates. Transient-state kinetics showed that the tyrosine-VA adduct strongly promotes (>100-fold) substrate oxidation by compound II, the rate-limiting step in catalysis. The novel activation mechanism is involved in ligninolysis, as demonstrated using lignin model substrates. The present paper is the first report on autocatalytic modification, resulting in functional alteration, among class II peroxidases.

Versatile peroxidase shares with manganese peroxidase and lignin peroxidase the ability to oxidize Mn(2+) and high redox potential aromatic compounds, respectively. Moreover, it is also able to oxidize phenols (and low redox potential dyes) at two catalytic sites, as shown by biphasic kinetics. A high efficiency site (with 2,6-dimethoxyphenol and p-hydroquinone catalytic efficiencies of ∼70 and ∼700 s(-1) mM(-1), respectively) was localized at the same exposed Trp-164 responsible for high redox potential substrate oxidation (as shown by activity loss in the W164S variant). The second site, characterized by low catalytic efficiency (∼3 and ∼50 s(-1) mM(-1) for 2,6-dimethoxyphenol and p-hydroquinone, respectively) was localized at the main heme access channel. Steady-state and transient-state kinetics for oxidation of phenols and dyes at the latter site were improved when side chains of residues forming the heme channel edge were removed in single and multiple variants. Among them, the E140G/K176G, E140G/P141G/K176G, and E140G/W164S/K176G variants attained catalytic efficiencies for oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) at the heme channel similar to those of the exposed tryptophan site. The heme channel enlargement shown by x-ray diffraction of the E140G, P141G, K176G, and E140G/K176G variants would allow a better substrate accommodation near the heme, as revealed by the up to 26-fold lower K(m) values (compared with native VP). The resulting interactions were shown by the x-ray structure of the E140G-guaiacol complex, which includes two H-bonds of the substrate with Arg-43 and Pro-139 in the distal heme pocket (at the end of the heme channel) and several hydrophobic interactions with other residues and the heme cofactor.

Wood is a major pool of organic carbon that is highly resistant to decay, owing largely to the presence of lignin. The only organisms capable of substantial lignin decay are white rot fungi in the Agaricomycetes, which also contains non-lignin-degrading brown rot and ectomycorrhizal species. Comparative analyses of 31 fungal genomes (12 generated for this study) suggest that lignin-degrading peroxidases expanded in the lineage leading to the ancestor of the Agaricomycetes, which is reconstructed as a white rot species, and then contracted in parallel lineages leading to brown rot and mycorrhizal species. Molecular clock analyses suggest that the origin of lignin degradation might have coincided with the sharp decrease in the rate of organic carbon burial around the end of the Carboniferous period.

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.

Efficient lignin depolymerization is unique to the wood decay basidiomycetes, collectively referred to as white rot fungi. Phanerochaete chrysosporium simultaneously degrades lignin and cellulose, whereas the closely related species, Ceriporiopsis subvermispora, also depolymerizes lignin but may do so with relatively little cellulose degradation. To investigate the basis for selective ligninolysis, we conducted comparative genome analysis of C. subvermispora and P. chrysosporium. Genes encoding manganese peroxidase numbered 13 and five in C. subvermispora and P. chrysosporium, respectively. In addition, the C. subvermispora genome contains at least seven genes predicted to encode laccases, whereas the P. chrysosporium genome contains none. We also observed expansion of the number of C. subvermispora desaturase-encoding genes putatively involved in lipid metabolism. Microarray-based transcriptome analysis showed substantial up-regulation of several desaturase and MnP genes in wood-containing medium. MS identified MnP proteins in C. subvermispora culture filtrates, but none in P. chrysosporium cultures. These results support the importance of MnP and a lignin degradation mechanism whereby cleavage of the dominant nonphenolic structures is mediated by lipid peroxidation products. Two C. subvermispora genes were predicted to encode peroxidases structurally similar to P. chrysosporium lignin peroxidase and, following heterologous expression in Escherichia coli, the enzymes were shown to oxidize high redox potential substrates, but not Mn(2+). Apart from oxidative lignin degradation, we also examined cellulolytic and hemicellulolytic systems in both fungi. In summary, the C. subvermispora genetic inventory and expression patterns exhibit increased oxidoreductase potential and diminished cellulolytic capability relative to P. chrysosporium.

The VPs (versatile peroxidases) secreted by white-rot fungi are involved in the natural decay of lignin. In the present study, a fusion gene containing the VP from Pleurotus eryngii was subjected to six rounds of directed evolution, achieving a level of secretion in Saccharomyces cerevisiae (21 mg/l) as yet unseen for any ligninolytic peroxidase. The evolved variant for expression harboured four mutations and increased its total VP activity 129-fold. The signal leader processing by the STE13 protease at the Golgi compartment changed as a consequence of overexpression, retaining the additional N-terminal sequence Glu-Ala-Glu-Ala that enhanced secretion. The engineered N-terminally truncated variant displayed similar biochemical properties to those of the non-truncated counterpart in terms of kinetics, stability and spectroscopic features. Additional cycles of evolution raised the T50 8°C and significantly increased the enzyme's stability at alkaline pHs. In addition, the Km for H2O2 was enhanced up to 15-fold while the catalytic efficiency was maintained, and there was an improvement in peroxide stability (with half-lives for H2O2 of 43 min at a H2O2/enzyme molar ratio of 4000:1). Overall, the directed evolution approach described provides a set of strategies for selecting VPs with improvements in secretion, activity and stability.