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1.
Proc Natl Acad Sci U S A ; 120(10): e2214076120, 2023 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-36848567

RESUMO

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.


Assuntos
Lentinula , Filogenia , Ásia Oriental , Tailândia
2.
J Fungi (Basel) ; 7(6)2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071235

RESUMO

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 H2O2-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.

3.
J Fungi (Basel) ; 7(5)2021 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-33919051

RESUMO

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.

4.
Int J Mol Sci ; 22(5)2021 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-33807844

RESUMO

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-ß- 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.


Assuntos
Catalase/química , Corantes/química , Proteínas Fúngicas/química , Fungos/enzimologia , Lignina/química , Peroxidase/química
5.
Mol Biol Evol ; 38(4): 1428-1446, 2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33211093

RESUMO

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.


Assuntos
Agaricales/genética , Genoma Fúngico , Lignina/metabolismo , Peroxidases/genética , Filogenia , Agaricales/enzimologia , Ecossistema , Família Multigênica , Peroxidases/metabolismo
6.
DNA Res ; 27(2)2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32531032

RESUMO

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.


Assuntos
Desidrogenases de Carboidrato/genética , Proteínas Fúngicas/genética , Lignina/genética , Pycnoporus/enzimologia , Desidrogenases de Carboidrato/metabolismo , Celulose/metabolismo , Proteínas Fúngicas/metabolismo , Genoma Fúngico , Lignina/metabolismo , Filogenia , Pycnoporus/classificação , Pycnoporus/genética , Madeira/metabolismo , Madeira/microbiologia
7.
Arch Biochem Biophys ; 668: 23-28, 2019 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-31095936

RESUMO

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).


Assuntos
Proteínas Fúngicas/química , Nitrofenóis/química , Peroxidases/química , Triptofano/química , Basidiomycota/enzimologia , Domínio Catalítico , Escherichia coli/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Cinética , Mutagênese Sítio-Dirigida , Nitrofenóis/metabolismo , Oxirredução , Peroxidases/genética , Peroxidases/metabolismo , Pleurotus/enzimologia , Ligação Proteica
8.
Chemistry ; 25(11): 2708-2712, 2019 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-30566756

RESUMO

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 Fe3+ . 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.


Assuntos
Proteínas Fúngicas/química , Lignina/química , Peroxidases , Lignina/metabolismo , Peroxidases/química , Peroxidases/metabolismo
9.
Biotechnol Biofuels ; 11: 201, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30061923

RESUMO

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.

10.
Proc Natl Acad Sci U S A ; 115(25): 6428-6433, 2018 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-29866821

RESUMO

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.


Assuntos
Lignina/metabolismo , Peroxidases/metabolismo , Evolução Biológica , Ciclo do Carbono/fisiologia , Proteínas Fúngicas/metabolismo , Fungos/metabolismo , Oxirredução , Filogenia , Polímeros/metabolismo , Polyporales/metabolismo
11.
Biotechnol Adv ; 35(6): 815-831, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-28624475

RESUMO

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 H2O2 as the source of oxygen and final electron acceptor. Flavo-oxidases are involved in both lignin and cellulose decay generating H2O2 that activates peroxidases and generates hydroxyl radical. The group of copper oxidoreductases also includes other H2O2 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 D3, 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.


Assuntos
Biotransformação , Lacase/química , Oxirredutases/química , Dinitrocresóis/química , Fungos/química , Fungos/enzimologia , Heme/química , Heme/genética , Lacase/genética , Lignina/química , Lignina/genética , Oxirredução , Oxirredutases/classificação , Oxirredutases/genética , Peroxidases/química , Peroxidases/genética
12.
J Phys Chem B ; 121(16): 3946-3954, 2017 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-28375014

RESUMO

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.


Assuntos
Peroxidases/metabolismo , Pleurotus/enzimologia , Sequência de Aminoácidos , Transporte de Elétrons , Lignina/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Oxirredução , Peroxidases/química , Peroxidases/genética , Pleurotus/química , Pleurotus/genética , Especificidade por Substrato
13.
Biotechnol Biofuels ; 10: 67, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28331543

RESUMO

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 (Mn3+ 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 Mn2+-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.

14.
Biochem J ; 473(13): 1917-28, 2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-27118867

RESUMO

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.


Assuntos
Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Peroxidase/química , Peroxidase/metabolismo , Estabilidade Enzimática , Proteínas Fúngicas/genética , Concentração de Íons de Hidrogênio , Peroxidase/genética , Pleurotus/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
15.
Biotechnol Biofuels ; 9: 49, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26933449

RESUMO

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 ß-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.

16.
Biotechnol Biofuels ; 8: 216, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26692083

RESUMO

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.

17.
PLoS One ; 10(10): e0140984, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26496708

RESUMO

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.


Assuntos
Peroxidase/química , Peroxidase/metabolismo , Peroxidases/química , Peroxidases/metabolismo , Cristalografia por Raios X , Estabilidade Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Concentração de Íons de Hidrogênio , Peroxidase/genética , Peroxidases/genética , Pleurotus/química , Pleurotus/genética , Pleurotus/metabolismo
18.
J Phys Chem B ; 119(43): 13583-92, 2015 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-26120933

RESUMO

Peroxide-activated Auricularia auricula-judae dye-decolorizing peroxidase (DyP) forms a mixed Trp377 and Tyr337 radical, the former being responsible for oxidation of the typical DyP substrates (Linde et al. Biochem. J., 2015, 466, 253-262); however, a pure tryptophanyl radical EPR signal is detected at pH 7 (where the enzyme is inactive), in contrast with the mixed signal observed at pH for optimum activity, pH 3. On the contrary, the presence of a second tyrosine radical (at Tyr147) is deduced by a multifrequency EPR study of a variety of simple and double-directed variants (including substitution of the above and other tryptophan and tyrosine residues) at different freezing times after their activation by H2O2 (at pH 3). This points out that subsidiary long-range electron-transfer pathways enter into operation when the main pathway(s) is removed by directed mutagenesis, with catalytic efficiencies progressively decreasing. Finally, self-reduction of the Trp377 neutral radical is observed when reaction time (before freezing) is increased in the absence of reducing substrates (from 10 to 60 s). Interestingly, the tryptophanyl radical is stable in the Y147S/Y337S variant, indicating that these two tyrosine residues are involved in the self-reduction reaction.


Assuntos
Basidiomycota/metabolismo , Corantes/metabolismo , Peroxidases/metabolismo , Basidiomycota/química , Domínio Catalítico , Corantes/química , Espectroscopia de Ressonância de Spin Eletrônica , Transporte de Elétrons , Modelos Moleculares , Oxirredução , Peroxidases/química , Teoria Quântica
19.
Appl Microbiol Biotechnol ; 99(21): 8927-42, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25967658

RESUMO

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.


Assuntos
Corantes/metabolismo , Peroxidases/genética , Peroxidases/metabolismo , Pleurotus/enzimologia , Sequência de Aminoácidos , Clonagem Molecular , DNA Fúngico/química , DNA Fúngico/genética , Estabilidade Enzimática , Escherichia coli/genética , Expressão Gênica , Concentração de Íons de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Oxirredução , Peroxidases/química , Pleurotus/genética , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Temperatura
20.
PLoS One ; 10(4): e0124750, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25923713

RESUMO

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.


Assuntos
Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Fungos/enzimologia , Peroxidases/química , Peroxidases/metabolismo , Biocatálise , Cristalografia por Raios X , Proteínas Fúngicas/genética , Heme/química , Peróxido de Hidrogênio/metabolismo , Cinética , Lignina/metabolismo , Mutagênese Sítio-Dirigida , Oxirredução , Peroxidases/genética , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação
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