Heme-thiolate Peroxidases Research Articles

In silico analysis of a heme-thiolate peroxidase gene discovered in an ectomycorrhizal fungus of Carpathian primeval forest: implications for biotechnological applications

In this work, we focus on the identification of novel fungal peroxygenase gene belonging to the peroxidase-peroxygenase superfamily. We applied a metagenomic approach on soil samples from primeval forest and appropriate bioinformatics tools for analysis of obtained genomic DNA sequence. Peroxidases are ubiquitous metalloenzymes that are able to reduce reactive peroxides, mainly hydrogen peroxide, into water, whereas several substrates can be concomitantly oxidized during their catalytic reaction. Our purpose was to collect unique peroxygenase sequence data originating from a preserved biotope for a robust phylogenetic reconstruction of a particular gene family coding for highly versatile heme-thiolate peroxidases that has peculiar yet undiscovered representatives among ectomycorrhizal fungi. We identified unique DNA sequence, 812 bp long, from ectomycorrhizal Suillus species coding for a heme-thiolate peroxidase with 1 typical intron that appears distinctive for Carpathian forests. After translation in corresponding protein sequence 251 amino acids long we could identify typical signatures of this peroxygenase. On the proximal side of heme we found the conserved P-C-P triad responsible for efficient ligation of heme iron thus influencing the reactivity of this peroxidase. On the distal side we recognized the E-H-D-X-S-L motif for interaction with a stabilizing magnesium ion. Maximum likelihood reconstruction of protein phylogeny revealed with a high bootstrap support the presence of a monophyletic HTP4 clade originating in numerous Suillus representatives. Together with sister clades of edible Boletus and poisonous Paxillus containing diverse peroxygenases these newly discovered heme catalyst can be considered for application of oxyfunctionalization of organic molecules.

Unlocking the distinctive enzymatic functions of the early plant biomass deconstructive genes in a brown rot fungus by cell-free protein expression.

Saprotrophic fungi that cause brown rot of woody biomass evolved a distinctive mechanism that relies on reactive oxygen species (ROS) to kick-start lignocellulosic polymers' deconstruction. These ROS agents are generated at incipient decay stages through a series of redox relays that shuttle electrons from fungus's central metabolism to extracellular Fenton chemistry. A list of genes has been suggested encoding the enzyme catalysts of the redox processes involved in ROS's function. However, navigating the functions of the encoded enzymes has been challenging due to the lack of a rapid method for protein synthesis. Here, we employed cell-free expression system to synthesize four redox or degradative enzymes, which were identified, by transcriptomic data, as conserved players of the ROS oxidation phase across brown rot fungal species. All four enzymes were successfully expressed and showed activities that enable confident assignment of function, namely, benzoquinone reductase (BQR), ferric reductase, α-L-arabinofuranosidase (ABF), and heme-thiolate peroxidase (HTP). Detailed analysis of their catalytic features within the context of brown rot environments allowed us to interpret their roles during ROS-driven wood decomposition. Specifically, we validated the functions of BQR as the driver redox enzyme of Fenton cycles and reconstructed its interactions with the co-occurring HTP or laccase and ABF. Taken together, this research demonstrated that the cell-free expression platform is adequate for synthesizing functional fungal enzymes and provided an alternative route for the rapid characterization of fungal proteins, escalating our understanding of the distinctive biocatalyst system for plant biomass conversion.IMPORTANCEBrown rot fungi are efficient wood decomposers in nature, and their unique degradative systems harbor untapped catalysts pursued by the biorefinery and bioremediation industries. While the use of "omics" platforms has recently uncovered the key "oxidative-hydrolytic" mechanisms that allow these fungi to attack lignocellulose, individual protein characterization is lagging behind due to the lack of a robust method for rapid synthesis of crucial fungal enzymes. This work delves into the studies of biochemical functions of brown rot enzymes using a rapid, cell-free expression platform, which allowed the successful depictions of enzymes' catalytic features, their interactions with Fenton chemistry, and their roles played during the incipient stage of brown rot when fungus sets off the reactive oxygen species for oxidative degradation. We expect this research could illuminate cell-free protein expression system's use to fulfill the increasing need for functional studies of fungal enzymes, advancing the discoveries of novel biomass-converting catalysts.

Transcriptomic Analysis of Salt Stress Response in Pleurotus ostreatus

This study was conducted to confirm the expression patterns of genes involved in stress resistance by comparing the expression patterns of genes expressed after sodium chloride (NaCl) treatment in Pleurotus ostreatus (PO) cultivation. To confirm this, as a result of checking different gene expressions for the untreated group and the NaCl 1% and 2% treated group, a total of 12,460 gene expression differences were confirmed. There were 275 and 397 genes with increased expression in the 2.0% and 1.0% NaCl treated group and 400 and 247 genes with reduced expression in the 1.0% and 2.0% NaCl treated group, respectively. Among the genes whose expression was confirmed in DEG, qRT-PCR was performed on six genes to confirm the expression pattern of the genes affecting the mycelium structure. The DEG results showed that a putative aldo-keto reductase of akor3, Alpha-1,4 glucan phosphorylase of PLEOSDRAFT_1058949, and heme-thiolate peroxidase of HTP1 were up-regulated and that glycoside hydrolase family 92 protein of PLEOSDRAFT_1063499 were down-regulated, and the qRT-PCR showed the same results. However, hydrophobin of Hydph16 and pleurotolysin B of plyB were up-regulated in the qRT-PCR results while down-regulated in the DEG results. From the above results, it is judged that NaCl ultimately inhibits growth by inhibiting the formation of the skeleton constituting the mycelium and the physiological activity within the cell.

New insights on unspecific peroxygenases: superfamily reclassification and evolution

BackgroundUnspecific peroxygenases (UPO) (EC 1.11.2.1) represent an intriguing oxidoreductase sub-subclass of heme proteins with peroxygenase and peroxidase activity. With over 300 identified substrates, UPOs catalyze numerous oxidations including 1- or 2- electron oxygenation, selective oxyfunctionalizations, which make them most significant in organic syntheses and potentially attractive as industrial biocatalysts. There are very few UPOs available with distinct properties, notably, MroUPO which shows behavior ranging between UPO and another heme-thiolate peroxidase, called Chloroperoxidase (CPO). It prompted us to search for more UPOs in fungal kingdom which led us to studying their relationship with CPO.ResultsIn this study, we searched for novel UPOs in more than 800 fungal genomes and found 113 putative UPO-encoding sequences distributed in 35 different fungal species (or strains), amongst which single sequence per species were subjected to phylogeny study along with CPOs. Our phylogenetic study show that the UPOs are distributed in Basidiomycota and Ascomycota phyla of fungi. The sequence analysis helped to classify the UPOs into five distinct subfamilies: classic AaeUPO and four new subfamilies with potential new traits. We have also shown that each of these five subfamilies (supported by) have their own signature motifs. Surprisingly, some of the CPOs appeared to be a type of UPOs indicating that they were previously identified incorrectly. Selection pressure was observed on important motifs in UPOs which could have driven their functional divergence. Furthermore, the sites having different evolutionary rates caused by the functional divergence were also identified on some motifs along with the other relevant amino acid residues. Finally, we predicted critical amino acids responsible for the functional divergence in the UPOs and identified some sequence differences among UPOs, CPOs, and MroUPO to predict it’s ranging behavior.ConclusionThis study discovers new UPOs, provides a glimpse of their evolution from CPOs, and presents new insight on their functional divergence. We present a new classification of UPOs and shed new light on its phylogenetics. These different UPOs may exhibit a wide range of characteristics and specificities which may help in various fields of synthetic chemistry and industrial biocatalysts, and may as well lead to an advancement towards the understanding of physiological role of UPOs in fungi.

Fungal Unspecific Peroxygenases Oxidize the Majority of Organic EPA Priority Pollutants.

Unspecific peroxygenases (UPOs) are secreted fungal enzymes with promiscuity for oxygen transfer and oxidation reactions. Functionally, they represent hybrids of P450 monooxygenases and heme peroxidases; phylogenetically they belong to the family of heme-thiolate peroxidases. Two UPOs from the basidiomycetous fungi Agrocybe aegerita (AaeUPO) and Marasmius rotula (MroUPO) converted 35 out of 40 compounds listed as EPA priority pollutants, including chlorinated benzenes and their derivatives, halogenated biphenyl ethers, nitroaromatic compounds, polycyclic aromatic hydrocarbons (PAHs) and phthalic acid derivatives. These oxygenations and oxidations resulted in diverse products and—if at all—were limited for three reasons: (i) steric hindrance caused by multiple substitutions or bulkiness of the compound as such (e.g., hexachlorobenzene or large PAHs), (ii) strong inactivation of aromatic rings (e.g., nitrobenzene), and (iii) low water solubility (e.g., complex arenes). The general outcome of our study is that UPOs can be considered as extracellular counterparts of intracellular monooxygenases, both with respect to catalyzed reactions and catalytic versatility. Therefore, they should be taken into consideration as a relevant biocatalytic detoxification and biodegradation tool used by fungi when confronted with toxins, xenobiotics and pollutants in their natural environments.

Modification of the peroxygenative:peroxidative activity ratio in the unspecific peroxygenase from Agrocybe aegerita by structure-guided evolution.

Unspecific peroxygenase (UPO) is a heme-thiolate peroxidase capable of performing with high-selectivity C-H oxyfunctionalizations of great interest in organic synthesis through its peroxygenative activity. However, the convergence of such activity with an unwanted peroxidative activity encumbers practical applications. In this study, we have modified the peroxygenative:peroxidative activity ratio (P:p ratio) of UPO from Agrocybe aegerita by structure-guided evolution. Several flexible loops (Glu1-Pro35, Gly103-Asp131, Ser226-Gly243, Gln254-Thr276 and Ty293-Arg327) were selected on the basis on their B-factors and ΔΔG values. The full ensemble of segments (43% of UPO sequence) was subjected to focused evolution by the Mutagenic Organized Recombination Process by Homologous IN vivo Grouping (MORPHING) method in Saccharomyces cerevisiae. Five independent mutant libraries were screened in terms of P:p ratio and thermostability. We identified several variants that harbored substitutions at positions 120 and 320 with a strong enhancement in the P:p ratio albeit at the cost of stability. The most thermostable mutant of this process (S226G with an increased T50 of 2°C) was subjected to further combinatorial saturation mutagenesis on Thr120 and Thr320 yielding a collection of variants with modified P:p ratio and recovered stability. Our results seem to indicate the coexistence of several oxidation sites for peroxidative and peroxygenative activities in UPO.

From Alkanes to Carboxylic Acids: Terminal Oxygenation by a Fungal Peroxygenase.

A new heme-thiolate peroxidase catalyzes the hydroxylation of n-alkanes at the terminal position-a challenging reaction in organic chemistry-with H2 O2 as the only cosubstrate. Besides the primary product, 1-dodecanol, the conversion of dodecane yielded dodecanoic, 12-hydroxydodecanoic, and 1,12-dodecanedioic acids, as identified by GC-MS. Dodecanal could be detected only in trace amounts, and 1,12-dodecanediol was not observed, thus suggesting that dodecanoic acid is the branch point between mono- and diterminal hydroxylation. Simultaneously, oxygenation was observed at other hydrocarbon chain positions (preferentially C2 and C11). Similar results were observed in reactions of tetradecane. The pattern of products formed, together with data on the incorporation of (18) O from the cosubstrate H2 (18) O2 , demonstrate that the enzyme acts as a peroxygenase that is able to catalyze a cascade of mono- and diterminal oxidation reactions of long-chain n-alkanes to give carboxylic acids.

Draft Genome Sequence of the Chloroperoxidase-Producing Fungus Caldariomyces fumago Woronichin DSM1256.

We report here the draft genome sequence of the chloroperoxidase (EC 1.11.1.10)-producing ascomycete Caldariomyces fumago. Its genome was assembled into 511 contigs with a total size of 25 Mb. The G+C content is 51.4%, and 9,806 putative protein-coding genes were predicted. Eight heme-thiolate peroxidase genes, including two chloroperoxidase genes, were found.

Fungal unspecific peroxygenases: heme-thiolate proteins that combine peroxidase and cytochrome p450 properties.

Eleven years ago, a secreted heme-thiolate peroxidase with promiscuity for oxygen transfer reactions was discovered in the basidiomycetous fungus, Agrocybe aegerita. The enzyme turned out to be a functional mono-peroxygenase that transferred an oxygen atom from hydrogen peroxide to diverse organic substrates (aromatics, heterocycles, linear and cyclic alkanes/alkenes, fatty acids, etc.). Later similar enzymes were found in other mushroom genera such as Coprinellus and Marasmius. Approximately one thousand putative peroxygenase sequences that form two large clusters can be found in genetic databases and fungal genomes, indicating the widespread occurrence of such enzymes in the whole fungal kingdom including all phyla of true fungi (Eumycota) and certain fungus-like heterokonts (Oomycota). This new enzyme type was classified as unspecific peroxygenase (UPO, EC 1.11.2.1) and placed in a separate peroxidase subclass. Furthermore, UPOs and related heme-thiolate peroxidases such as well-studied chloroperoxidase (CPO) represent a separate superfamily of heme proteins on the phylogenetic level. The reactions catalyzed by UPOs include hydroxylation, epoxidation, O- and N-dealkylation, aromatization, sulfoxidation, N-oxygenation, dechlorination and halide oxidation. In many cases, the product patterns of UPOs resemble those of human cytochrome P450 (P450) monooxygenases and, in fact, combine the catalytic cycle of heme peroxidases with the "peroxide shunt" of P450s. Here, an overview on UPOs is provided with focus on their molecular and catalytic properties.

The ligninolytic peroxidases in the genus Pleurotus: divergence in activities, expression, and potential applications.

Mushrooms of the genus Pleurotus are comprised of cultivated edible ligninolytic fungi with medicinal properties and a wide array of biotechnological and environmental applications. Like other white-rot fungi (WRF), they are able to grow on a variety of lignocellulosic biomass substrates and degrade both natural and anthropogenic aromatic compounds. This is due to the presence of the non-specific oxidative enzymatic systems, which are mainly consisted of lacasses, versatile peroxidases (VPs), and short manganese peroxidases (short-MnPs). Additional, less studied, peroxidase are dye-decolorizing peroxidases (DyPs) and heme-thiolate peroxidases (HTPs). During the past two decades, substantial information has accumulated concerning the biochemistry, structure and function of the Pleurotus ligninolytic peroxidases, which are considered to play a key role in many biodegradation processes. The production of these enzymes is dependent on growth media composition, pH, and temperature as well as the growth phase of the fungus. Mn(2+) concentration differentially affects the expression of the different genes. It also severs as a preferred substrate for these preoxidases. Recently, sequencing of the Pleurotus ostreatus genome was completed, and a comprehensive picture of the ligninolytic peroxidase gene family, consisting of three VPs and six short-MnPs, has been established. Similar enzymes were also discovered and studied in other Pleurotus species. In addition, progress has been made in the development of molecular tools for targeted gene replacement, RNAi-based gene silencing and overexpression of genes of interest. These advances increase the fundamental understanding of the ligninolytic system and provide the opportunity for harnessing the unique attributes of these WRF for applied purposes.

Widespread occurrence of expressed fungal secretory peroxidases in forest soils.

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.

Lignin-degrading peroxidases in Polyporales: an evolutionary survey based on 10 sequenced genomes

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.

A comparative genomic analysis of the oxidative enzymes potentially involved in lignin degradation by Agaricus bisporus

The oxidative enzymatic machinery for degradation of organic substrates in Agaricus bisporus (Ab) is at the core of the carbon recycling mechanisms in this fungus. To date, 156 genes have been tentatively identified as part of this oxidative enzymatic machinery, which includes 26 peroxidase encoding genes, nine copper radical oxidase [including three putative glyoxal oxidase-encoding genes (GLXs)], 12 laccases sensu stricto and 109 cytochrome P450 monooxygenases. Comparative analyses of these enzymes in Ab with those of the white-rot fungus, Phanerochaete chrysosporium, the brown-rot fungus, Postia placenta, the coprophilic litter fungus, Coprinopsis cinerea and the ectomychorizal fungus, Laccaria bicolor, revealed enzyme diversity consistent with adaptation to substrates rich in humic substances and partially degraded plant material. For instance, relative to wood decay fungi, Ab cytochrome P450 genes were less numerous (109 gene models), distributed among distinctive families, and lacked extensive duplication and clustering. Viewed together with P450 transcript accumulation patterns in three tested growth conditions, these observations were consistent with the unique Ab lifestyle. Based on tandem gene arrangements, a certain degree of gene duplication seems to have occurred in this fungus in the copper radical oxidase (CRO) and the laccase gene families. In Ab, high transcript levels and regulation of the heme-thiolate peroxidases, two manganese peroxidases and the three GLX-like genes are likely in response to complex natural substrates, including lignocellulose and its derivatives, thereby suggesting an important role in lignin degradation. On the other hand, the expression patterns of the related CROs suggest a developmental role in this fungus. Based on these observations, a brief comparative genomic overview of the Ab oxidative enzyme machinery is presented.

Agaricus bisporus genome sequence: A commentary

The genomes of two isolates of Agaricus bisporus have been sequenced recently. This soil-inhabiting fungus has a wide geographical distribution in nature and it is also cultivated in an industrialized indoor process ($4.7bn annual worldwide value) to produce edible mushrooms. Previously this lignocellulosic fungus has resisted precise econutritional classification, i.e. into white- or brown-rot decomposers. The generation of the genome sequence and transcriptomic analyses has revealed a new classification, ‘humicolous’, for species adapted to grow in humic-rich, partially decomposed leaf material. The Agaricus biporus genomes contain a collection of polysaccharide and lignin-degrading genes and more interestingly an expanded number of genes (relative to other lignocellulosic fungi) that enhance degradation of lignin derivatives, i.e. heme-thiolate peroxidases and β-etherases. A motif that is hypothesized to be a promoter element in the humicolous adaptation suite is present in a large number of genes specifically up-regulated when the mycelium is grown on humic-rich substrate. The genome sequence of A. bisporus offers a platform to explore fungal biology in carbon-rich soil environments and terrestrial cycling of carbon, nitrogen, phosphorus and potassium.

Genome sequence of the button mushroomAgaricus bisporusreveals mechanisms governing adaptation to a humic-rich ecological niche

Agaricus bisporus is the model fungus for the adaptation, persistence, and growth in the humic-rich leaf-litter environment. Aside from its ecological role, A. bisporus has been an important component of the human diet for over 200 y and worldwide cultivation of the "button mushroom" forms a multibillion dollar industry. We present two A. bisporus genomes, their gene repertoires and transcript profiles on compost and during mushroom formation. The genomes encode a full repertoire of polysaccharide-degrading enzymes similar to that of wood-decayers. Comparative transcriptomics of mycelium grown on defined medium, casing-soil, and compost revealed genes encoding enzymes involved in xylan, cellulose, pectin, and protein degradation are more highly expressed in compost. The striking expansion of heme-thiolate peroxidases and β-etherases is distinctive from Agaricomycotina wood-decayers and suggests a broad attack on decaying lignin and related metabolites found in humic acid-rich environment. Similarly, up-regulation of these genes together with a lignolytic manganese peroxidase, multiple copper radical oxidases, and cytochrome P450s is consistent with challenges posed by complex humic-rich substrates. The gene repertoire and expression of hydrolytic enzymes in A. bisporus is substantially different from the taxonomically related ectomycorrhizal symbiont Laccaria bicolor. A common promoter motif was also identified in genes very highly expressed in humic-rich substrates. These observations reveal genetic and enzymatic mechanisms governing adaptation to the humic-rich ecological niche formed during plant degradation, further defining the critical role such fungi contribute to soil structure and carbon sequestration in terrestrial ecosystems. Genome sequence will expedite mushroom breeding for improved agronomic characteristics.

Lignin-degrading Peroxidases from Genome of Selective Ligninolytic Fungus Ceriporiopsis subvermispora

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.

Pleurotus ostreatus heme peroxidases: An in silico analysis from the genome sequence to the enzyme molecular structure

An exhaustive screening of the Pleurotus ostreatus genome was performed to search for nucleotide sequences of heme peroxidases in this white-rot fungus, which could be useful for different biotechnological applications. After sequence identification and manual curation of the corresponding genes and cDNAs, the deduced amino acid sequences were converted into structural homology models. A comparative study of these sequences and their structural models with those of known fungal peroxidases revealed the complete inventory of heme peroxidases of this fungus. This consists of cytochrome c peroxidase and ligninolytic peroxidases, including manganese peroxidase and versatile peroxidase but not lignin peroxidase, as representative of the “classical” superfamily of plant, fungal, and bacterial peroxidases; and members of two relatively “new” peroxidase superfamilies, namely heme-thiolate peroxidases, here described for the first time in a fungus from the genus Pleurotus, and dye-decolorizing peroxidases, already known in P. ostreatus but still to be thoroughly explored and characterized.

Regioselective oxygenation of fatty acids, fatty alcohols and other aliphatic compounds by a basidiomycete heme-thiolate peroxidase

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 18O 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.