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Deadwood provides habitat for fungi and serves diverse ecological functions in forests. We already have profound knowledge of fungal assembly processes, physiological and enzymatic activities, and resulting physico-chemical changes during deadwood decay. However, in situ detection and identification methods, fungal origins, and a mechanistic understanding of the main lignocellulolytic enzymes are lacking. This study used metaproteomics to detect the main extracellular lignocellulolytic enzymes in 12 tree species in a temperate forest that have decomposed for 8 ½ years. Mainly white-rot (and few brown-rot) Basidiomycota were identified as the main wood decomposers, with Armillaria as the dominant genus; additionally, several soft-rot xylariaceous Ascomycota were identified. The key enzymes involved in lignocellulolysis included manganese peroxidase, peroxide-producing alcohol oxidases, laccase, diverse glycoside hydrolases (cellulase, glucosidase, xylanase), esterases, and lytic polysaccharide monooxygenases. The fungal community and enzyme composition differed among the 12 tree species. Ascomycota species were more prevalent in angiosperm logs than in gymnosperm logs. Regarding lignocellulolysis as a function, the extracellular enzyme toolbox acted simultaneously and was interrelated (e.g. peroxidases and peroxide-producing enzymes were strongly correlated), highly functionally redundant, and present in all logs. In summary, our in situ study provides comprehensive and detailed insight into the enzymatic machinery of wood-inhabiting fungi in temperate tree species. These findings will allow us to relate changes in environmental factors to lignocellulolysis as an ecosystem function in the future., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Fungal dye-decolorizing peroxidases (DyPs) have found applications in the treatment of dye-contaminated industrial wastes or to improve biomass digestibility. Their roles in fungal biology are uncertain, although it has been repeatedly suggested that they could participate in lignin degradation and/or modification. Using a comprehensive set of 162 fully sequenced fungal species, we defined seven distinct fungal DyP clades on basis of a sequence similarity network. Sequences from one of these clades clearly diverged from all others, having on average the lower isoelectric points and hydropathy indices, the highest number of N-glycosylation sites, and N-terminal sequence peptides for secretion. Putative proteins from this clade are absent from brown-rot and ectomycorrhizal species that have lost the capability of degrading lignin enzymatically. They are almost exclusively present in white-rot and other saprotrophic Basidiomycota that digest lignin enzymatically, thus lending support for a specific role of DyPs from this clade in biochemical lignin modification. Additional nearly full-length fungal DyP genes were isolated from the environment by sequence capture by hybridization; they all belonged to the clade of the presumably secreted DyPs and to another related clade. We suggest focusing our attention on the presumably intracellular DyPs from the other clades, which have not been characterized thus far and could represent enzyme proteins with novel catalytic properties. KEY POINTS: • A fungal DyP phylogeny delineates seven main sequence clades. • Putative extracellular DyPs form a single clade of Basidiomycota sequences. • Extracellular DyPs are associated to white-rot fungi., (© 2022. The Author(s).)

Dye-decolorizing peroxidases (DyPs) such as AauDyPI from the fungus Auricularia auricula-judae are able to oxidize substrates of different kinds and sizes. A crystal structure of an AauDyPI-imidazole complex gives insight into the binding patterns of organic molecules within the heme cavity of a DyP. Several small N-containing heterocyclic aromatics are shown to bind in the AauDyPI heme cavity, hinting to susceptibility of DyPs to azole-based inhibitors similar to cytochromes P450. Imidazole is confirmed as a competitive inhibitor with regard to peroxide binding. In contrast, bulky substrates such as anthraquinone dyes are converted at the enzyme surface. In the crystal structure a substrate analog, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), binds to a tyrosine-rich hollow harboring Y25, Y147, and Y337. Spin trapping with a nitric oxide donor uncovers Y229 as an additional tyrosine-based radical center in AauDyPI. Multi-frequency EPR spectroscopy further reveals the presence of at least one intermediate tryptophanyl radical center in activated AauDyPI with W377 as the most likely candidate., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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.)

Fungal secretory peroxidases mediate fundamental ecological functions in the conversion and degradation of plant biomass. Many of these enzymes have strong oxidizing activities towards aromatic compounds and are involved in the degradation of plant cell wall (lignin) and humus. They comprise three major groups: class II peroxidases (including lignin peroxidase, manganese peroxidase, versatile peroxidase and generic peroxidase), dye-decolorizing peroxidases, and heme-thiolate peroxidases (e.g. unspecific/aromatic peroxygenase, chloroperoxidase). Here, we have repeatedly observed a widespread expression of all major peroxidase groups in leaf and needle litter across a range of forest ecosystems (e.g. Fagus, Picea, Acer, Quercus, and Populus spp.), which are widespread in Europe and North America. Manganese peroxidases and unspecific peroxygenases were found expressed in all nine investigated forest sites, and dye-decolorizing peroxidases were observed in five of the nine sites, thereby indicating biological significance of these enzymes for fungal physiology and ecosystem processes. Transcripts of selected secretory peroxidase genes were also analyzed in pure cultures of several litter-decomposing species and other fungi. Using this information, we were able to match, in environmental litter samples, two manganese peroxidase sequences to Mycena galopus and Mycena epipterygia and one unspecific peroxygenase transcript to Mycena galopus, suggesting an important role of this litter- and coarse woody debris-dwelling genus in the disintegration and transformation of litter aromatics and organic matter formation.

Catalytic and physicochemical properties of representative fungal dye-decolorizing peroxidases (DyPs) of wood- (WRF) and litter-decomposing white-rot fungi (LDF) are summarized and compared, including one recombinant Mycetinis scorodonius DyP (rMscDyP; LDF), the wild-type Auricularia auricula-judae DyP (AauDyP; WRF), and two new DyPs secreted by the jelly fungi Exidia glandulosa (EglDyP; WRF) and Mycena epipterygia (MepDyP; LDF). Homogeneous preparations of these DyPs were obtained after different steps of fast protein liquid chromatography, and they increase the total number of characterized fungal DyP proteins to eight. The peptide sequences of AauDyP, MepDyP, and EglDyP showed highest homologies (52-56%) to the DyPs of M. scorodonius. Five out of the eight characterized fungal DyPs were used to evaluate their catalytic properties compared to classic fungal and plant heme peroxidases, namely lignin peroxidase of Phanerochaete chrysosporium (PchLiP; WRF), versatile peroxidase of Bjerkandera adusta (BadVP; WRF), and generic peroxidases of Coprinopsis cinerea (CiP) and Glycine max (soybean peroxidase=SBP). All DyPs tested possess unique properties regarding the stability at low pH values: 50-90% enzymatic activity remained after 4-h exposition at pH 2.5, and the oxidation of nonphenolic aromatic substrates (lignin model compounds) was optimal below pH 3. Furthermore, all DyPs efficiently oxidized recalcitrant dyes (e.g., Azure B) as well as the phenolic substrate 2,6-dimethoxyphenol. Thus, DyPs combine features of different peroxidases on the functional level and may be part of the biocatalytic system secreted by fungi for the oxidation of lignin and/or toxic aromatic compounds.

Dye-decolorizing peroxidases (DyPs) belong to the large group of heme peroxidases. They utilize hydrogen peroxide to catalyze oxidations of various organic compounds. AauDyPI from Auricularia auricula-judae (fungi) was crystallized, and its crystal structure was determined at 2.1 Å resolution. The mostly helical structure also shows a β-sheet motif typical for DyPs and Cld (chlorite dismutase)-related structures and includes the complete polypeptide chain. At the distal side of the heme molecule, a flexible aspartate residue (Asp-168) plays a key role in catalysis. It guides incoming hydrogen peroxide toward the heme iron and mediates proton rearrangement in the process of Compound I formation. Afterward, its side chain changes its conformation, now pointing toward the protein backbone. We propose an extended functionality of Asp-168, which acts like a gatekeeper by altering the width of the heme cavity access channel. Chemical modifications of potentially redox-active amino acids show that a tyrosine is involved in substrate interaction. Using spin-trapping experiments, a transient radical on the surface-exposed Tyr-337 was identified as the oxidation site for bulky substrates. A possible long-range electron transfer pathway from the surface of the enzyme to the redox cofactor (heme) is discussed.

The degradation of lignocellulose and the secretion of extracellular oxidoreductases were investigated in beech-wood (Fagus sylvatica) microcosms using 11 representative fungi of four different ecophysiological and taxonomic groups causing: (1) classic white rot of wood (e.g. Phlebia radiata), (2) 'nonspecific' wood rot (e.g. Agrocybe aegerita), (3) white rot of leaf litter (Stropharia rugosoannulata) or (4) soft rot of wood (e.g. Xylaria polymorpha). All strong white rotters produced manganese-oxidizing peroxidases as the key enzymes of ligninolysis (75-2200 mU g(-1)), whereas lignin peroxidase activity was not detectable in the wood extracts. Interestingly, activities of two recently discovered peroxidases - aromatic peroxygenase and a manganese-independent peroxidase of the DyP-type - were detected in the culture extracts of A. aegerita (up to 125 mU g(-1)) and Auricularia auricula-judae (up to 400 mU g(-1)), respectively. The activity of classic peroxidases correlated to some extent with the removal of wood components (e.g. Klason lignin) and the release of small water-soluble fragments (0.5-1.0 kDa) characterized by aromatic constituents. In contrast, laccase activity correlated with the formation of high-molecular mass fragments (30-200 kDa). The differences observed in the degradation patterns allow to distinguish the rot types caused by basidiomycetes and ascomycetes and may be suitable for following the effects of oxidative key enzymes (ligninolytic peroxidases vs. laccases, role of novel peroxidases) during wood decay., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Reaction of fatty acids, fatty alcohols, alkanes, sterols, sterol esters and triglycerides with the so-called aromatic peroxygenase from Agrocybe aegerita was investigated using GC-MS. Regioselective hydroxylation of C(12)-C(20) saturated/unsaturated fatty acids was observed at the ω-1 and ω-2 positions (except myristoleic acid only forming the ω-2 derivative). Minor hydroxylation at ω and ω-3 to ω-5 positions was also observed. Further oxidized products were detected, including keto, dihydroxylated, keto-hydroxy and dicarboxylic fatty acids. Fatty alcohols also yielded hydroxy or keto derivatives of the corresponding fatty acid. Finally, alkanes gave, in addition to alcohols at positions 2 or 3, dihydroxylated derivatives at both sides of the molecule; and sterols showed side-chain hydroxylation. No derivatives were found for fatty acids esterified with sterols or forming triglycerides, but methyl esters were ω-1 or ω-2 hydroxylated. Reactions using H(2)(18)O(2) established that peroxide is the source of the oxygen introduced in aliphatic hydroxylations. These studies also indicated that oxidation of alcohols to carbonyl and carboxyl groups is produced by successive hydroxylations combined with one dehydration step. We conclude that the A. aegerita peroxygenase not only oxidizes aromatic compounds but also catalyzes the stepwise oxidation of aliphatic compounds by hydrogen peroxide, with different hydroxylated intermediates., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The jelly fungus Auricularia auricula-judae produced an enzyme with manganese-independent peroxidase activity during growth on beech wood (approximately 300 U l(-1)). The same enzymatic activity was detected and produced at larger scale in agitated cultures comprising of liquid, plant-based media (e.g. tomato juice suspensions) at levels up to 8,000 U l(-1). Two pure peroxidase forms (A. auricula-judae peroxidase (AjP I and AjP II) could be obtained from respective culture liquids by three chromatographic steps. Spectroscopic and electrophoretic analyses of the purified proteins revealed their heme and peroxidase nature. The N-terminal amino acid sequence of AjP matched well with sequences of fungal enzymes known as "dye-decolorizing peroxidases". Homology was found to the N-termini of peroxidases from Marasmius scorodonius (up to 86%), Thanatephorus cucumeris (60%), and Termitomyces albuminosus (60%). Both enzyme forms catalyzed not only the conversion of typical peroxidase substrates such as 2,6-dimethoxyphenol and 2,2'-azino-bis(3-ethylthiazoline-6-sulfonate) but also the decolorization of the high-redox potential dyes Reactive Blue 5 and Reactive Black 5, whereas manganese(II) ions (Mn(2+)) were not oxidized. Most remarkable, however, is the finding that both AjPs oxidized nonphenolic lignin model compounds (veratryl alcohol; adlerol, a nonphenolic beta-O-4 lignin model dimer) at low pH (maximum activity at pH 1.4), which indicates a certain ligninolytic activity of dye-decolorizing peroxidases.

Heme-thiolate haloperoxidases are undoubtedly the most versatile biocatalysts of the hemeprotein family and share catalytic properties with at least three further classes of heme-containing oxidoreductases, namely, classic plant and fungal peroxidases, cytochrome P450 monooxygenases, and catalases. For a long time, only one enzyme of this type--the chloroperoxidase (CPO) of the ascomycete Caldariomyces fumago--has been known. The enzyme is commercially available as a fine chemical and catalyzes the unspecific chlorination, bromination, and iodation (but no fluorination) of a variety of electrophilic organic substrates via hypohalous acid as actual halogenating agent. In the absence of halide, CPO resembles cytochrome P450s and epoxidizes and hydroxylates activated substrates such as organic sulfides and olefins; aromatic rings, however, are not susceptible to CPO-catalyzed oxygen-transfer. Recently, a second fungal haloperoxidase of the heme-thiolate type has been discovered in the agaric mushroom Agrocybe aegerita. The UV-Vis adsorption spectrum of the isolated enzyme shows little similarity to that of CPO but is almost identical to a resting-state P450. The Agrocybe aegerita peroxidase (AaP) has strong brominating as well as weak chlorinating and iodating activities, and catalyzes both benzylic and aromatic hydroxylations (e.g., of toluene and naphthalene). AaP and related fungal peroxidases could become promising biocatalysts in biotechnological applications because they seemingly fill the gap between CPO and P450 enzymes and act as "self-sufficient" peroxygenases. From the environmental point of view, the existence of a halogenating mushroom enzyme is interesting because it could be linked to the multitude of halogenated compounds known from these organisms.

Lignin mineralization activity of three basidiomycetous litter-decomposing fungi (LDF) was studied with humus layer samples taken from a boreal forest soil. The total Pb concentration in the samples was 32,000 mg kg(-1) and water soluble Pb 67 mg kg(-1). Synthetic lignin mineralization by Collybia dryophila and Clitocybe (Lepista) nebularis was strongly inhibited, whereas Stropharia coronilla was more tolerant to Pb stress in soil and liquid cultures. Purified laccases maintained their activity and purified MnPs remained partly active up to a concentration of 1450 mg Pb l(-1). High concentrations of Pb inhibited the growth of LDF and affected the activity of ligninolytic enzymes, but the extent of inhibition varied among different LDF species. In consequence, Pb contamination in soil may have a negative impact on recycling of organic carbon.

Culture liquids of the litter-decomposing basidiomycete Stropharia coronilla showed pro-oxidant activity promoting the peroxidation of linoleic acid. This activity depended on the presence of manganese peroxidase (MnP) in the fungal culture. Pro-oxidant activity maxima coincided with maximum MnP activities during the separation of extracellular proteins by anion-exchange chromatography. Purified MnP1 showed substantial pro-oxidant activity in the presence of acetate and Mn2+ ions, even without the addition of hydrogen peroxide. A non-phenolic beta-O-4 lignin model compound [LMC; 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)-1,3-dihydroxypropane] was partially oxidized in an in vitro reaction system developing MnP-dependent lipid peroxidation. The chelating organic acids malonate and tartrate noticeably inhibited both the peroxidation of linoleic acid and the conversion of LMC in the system. The major product of the LMC oxidation was 1-(3,4-dimethoxyphenyl)-1-oxo-2-(2-methoxyphenoxy)-3-hydroxypropane; in addition, small amounts of 3,4-dimethoxybenzaldehyde (veratraldehyde) and 3,4-dimethoxybenzoic (veratric) acid were detected. Thus, MnP-initiated lipid peroxidation may be involved in the degradation of recalcitrant non-phenolic lignin substructures by litter-decomposing fungi similar to MnPs of wood-decaying fungi.

The litter-decomposing basidiomycete Stropharia coronilla, which preferably colonizes grasslands, was found to be capable of metabolizing and mineralizing benzo[a]pyrene (BaP) in liquid culture. Manganese(II) ions (Mn(2+)) supplied at a concentration of 200 micro M stimulated considerably both the conversion and the mineralization of BaP; the fungus metabolized and mineralized about four and twelve times, respectively, more of the BaP in the presence of supplemental Mn(2+) than in the basal medium. This stimulating effect could be attributed to the ligninolytic enzyme manganese peroxidase (MnP), whose activity increased after the addition of Mn(2+). Crude and purified MnP from S. coronilla oxidized BaP efficiently in a cell-free reaction mixture (in vitro), a process which was enhanced by the surfactant Tween 80. Thus, 100 mg of BaP liter(-1) was converted in an in vitro reaction solution containing 1 U of MnP ml(-1) within 24 h. A clear indication was found that BaP-1,6-quinone was formed as a transient metabolite, which disappeared over the further course of the reaction. The treatment of a mixture of 16 different polycyclic aromatic hydrocarbons (PAHs) selected by the U.S. Environmental Protection Agency as model standards for PAH analysis (total concentration, 320 mg liter(-1)) with MnP resulted in concentration decreases of 10 to 100% for the individual compounds, and again the stimulating effect of Tween 80 was observed. Probably due to their lower ionization potentials, poorly bioavailable, high-molecular-mass PAHs such as BaP, benzo(g,h,i)perylene, and indeno(1,2,3-c,d)pyrene were converted to larger extents than low-molecular-mass ones (e.g., phenanthrene and fluoranthene).

The basidiomycete Collybia dryophila K209, which colonizes forest soil, was found to decompose a natural humic acid isolated from pine-forest litter (LHA) and a synthetic (14)C-labeled humic acid ((14)C-HA) prepared from [U-(14)C]catechol in liquid culture. Degradation resulted in the formation of polar, lower-molecular-mass fulvic acid (FA) and carbon dioxide. HA decomposition was considerably enhanced in the presence of Mn(2+) (200 microM), leading to 75% conversion of LHA and 50% mineralization of (14)C-HA (compared to 60% and 20%, respectively, in the absence of Mn(2+)). There was a strong indication that manganese peroxidase (MnP), the production of which was noticeably increased in Mn(2+)-supplemented cultures, was responsible for this effect. The enzyme was produced as a single protein with a pI of 4.7 and a molecular mass of 44 kDa. During solid-state cultivation, C. dryophila released substantial amounts of water-soluble FA (predominantly of 0.9 kDa molecular mass) from insoluble litter material. The results indicate that basidiomycetes such as C. dryophila which colonize forest litter and soil are involved in humus turnover by their recycling of high-molecular-mass humic substances. Extracellular MnP seems to be a key enzyme in the conversion process.

Agrocybe praecox is a litter-decomposing Basidiomycota species of the order Agaricales, and is frequently found in forests and open woodlands. A. praecox grows in leaf-litter and the upper soil and is able to colonize bark mulch and wood chips. It produces extracellular manganese peroxidase (MnP) activities and mineralizes synthetic lignin. In this study, the A. praecox MnP1 isozyme was purified, cloned and enzymatically characterized. The enzyme catalysed the oxidation of Mn 2+ to Mn 3+ , which is the specific reaction for manganese-dependent class II heme-peroxidases, in the presence of malonate as chelator with an activity maximum at pH 4.5; detectable activity was observed even at pH 7.0. The coding sequence of the mnp1 gene demonstrates a short-type of MnP protein with a slightly modified Mn 2+ binding site. Thus, A. praecox MnP1 may represent a novel group of atypical short-MnP enzymes. In lignocellulose-containing cultures composed of cereal bran or forest litter, transcription of mnp1 gene was followed by quantitative real-time RT-PCR. On spruce needle litter, mnp1 expression was more abundant than on leaf litter after three weeks cultivation. However, the expression was constitutive in wheat and rye bran cultures. Our data show that the atypical MnP of A. praecox is able to catalyse Mn 2+ oxidation, which suggests its involvement in lignocellulose decay by this litter-decomposer. [ABSTRACT FROM AUTHOR]