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Mutational and structural analysis of an ancestral fungal dye-decolorizing peroxidase.

ABSTRACT: Dye-decolorizing peroxidases (DyPs) constitute a superfamily of heme-containing peroxidases that are related neither to animal nor to plant peroxidase families. These are divided into four classes (types A, B, C, and D) based on sequence features. The active site of DyPs contains two highly conserved distal ligands, an aspartate and an arginine, the roles of which are still controversial. These ligands have mainly been studied in class A-C bacterial DyPs, largely because no effective recombinant expression systems have been developed for the fungal (D-type) DyPs. In this work, we employ ancestral sequence reconstruction (ASR) to resurrect a D-type DyP ancestor, AncDyPD-b1. Expression of AncDyPD-b1 in Escherichia coli results in large amounts of a heme-containing soluble protein and allows for the first mutagenesis study on the two distal ligands of a fungal DyP. UV-Vis and resonance Raman (RR) spectroscopic analyses, in combination with steady-state kinetics and the crystal structure, reveal fine pH-dependent details about the heme active site structure and show that both the aspartate (D222) and the arginine (R390) are crucial for hydrogen peroxide reduction. Moreover, the data indicate that these two residues play important but mechanistically different roles on the intraprotein long-range electron transfer process. DATABASE: Structural data are available in the PDB database under the accession number 7ANV.

SUBMITTER: Zitare UA

PROVIDER: S-EPMC8248431 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Description of the first fungal dye-decolorizing peroxidase oxidizing manganese(II).

Project description: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.

| S-EPMC4619462 | biostudies-literature

Fungal dye-decolorizing peroxidase diversity: roles in either intra- or extracellular processes.

Project description: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.

| S-EPMC9064869 | biostudies-literature

Comparing Ligninolytic Capabilities of Bacterial and Fungal Dye-Decolorizing Peroxidases and Class-II Peroxidase-Catalases.

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

| S-EPMC7961821 | biostudies-literature

Structural and Biochemical Characterization of a Dye-Decolorizing Peroxidase from <i>Dictyostelium discoideum</i>.

Project description:A novel cytoplasmic dye-decolorizing peroxidase from Dictyostelium discoideum was investigated that oxidizes anthraquinone dyes, lignin model compounds, and general peroxidase substrates such as ABTS efficiently. Unlike related enzymes, an aspartate residue replaces the first glycine of the conserved GXXDG motif in Dictyostelium DyPA. In solution, Dictyostelium DyPA exists as a stable dimer with the side chain of Asp146 contributing to the stabilization of the dimer interface by extending the hydrogen bond network connecting two monomers. To gain mechanistic insights, we solved the Dictyostelium DyPA structures in the absence of substrate as well as in the presence of potassium cyanide and veratryl alcohol to 1.7, 1.85, and 1.6 Å resolution, respectively. The active site of Dictyostelium DyPA has a hexa-coordinated heme iron with a histidine residue at the proximal axial position and either an activated oxygen or CN- molecule at the distal axial position. Asp149 is in an optimal conformation to accept a proton from H2O2 during the formation of compound I. Two potential distal solvent channels and a conserved shallow pocket leading to the heme molecule were found in Dictyostelium DyPA. Further, we identified two substrate-binding pockets per monomer in Dictyostelium DyPA at the dimer interface. Long-range electron transfer pathways associated with a hydrogen-bonding network that connects the substrate-binding sites with the heme moiety are described.

| S-EPMC8230527 | biostudies-literature

First crystal structure of a fungal high-redox potential dye-decolorizing peroxidase: substrate interaction sites and long-range electron transfer.

Project description: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.

| S-EPMC3568178 | biostudies-literature

Mechanistic Insights into Dye-Decolorizing Peroxidase Revealed by Solvent Isotope and Viscosity Effects.

Project description:Dye-decolorizing peroxidases (DyPs) are a family of H2O2-dependent heme peroxidases, which have shown potential applications in lignin degradation and valorization. However, the DyP kinetic mechanism remains underexplored. Using structural biology and solvent isotope (sKIE) and viscosity effects, many mechanistic characteristics have been uncovered for the B-class ElDyP from Enterobacter lignolyticus. Its structure revealed that a water molecule acts as the sixth axial ligand with two channels at diameters of ~3.0 and 8.0 Å leading to the heme center. A conformational change of ERS* to ERS, which have identical spectral characteristics, was proposed as the final step in DyPs' bisubstrate Ping-Pong mechanism. This step is also the rate-determining step in ABTS oxidation. The normal KIE of wild-type ElDyP with D2O2 at pH 3.5 suggested that cmpd 0 deprotonation by the distal aspartate is rate-limiting in the formation of cmpd I, which is more reactive under acidic pH than under neutral or alkaline pH. The viscosity effects and other biochemical methods implied that the reducing substrate binds with cmpd I instead of the free enzyme. The significant inverse sKIEs of kcat/KM and kERS* suggested that the aquo release in DyPs is mechanistically important and may explain the enzyme's adoption of two-electron reduction for cmpd I. The distal aspartate is catalytically more important than the distal arginine and plays key roles in determining DyPs' acidic pH optimum. The kinetic mechanism of D143H-ElDyP was also briefly studied. The results obtained will pave the way for future protein engineering to improve DyPs' lignolytic activity.

| S-EPMC5751952 | biostudies-literature

Identification, heterologous expression and characterization of a dye-decolorizing peroxidase of Pleurotus sapidus.

Project description:The coding sequence of a peroxidase from the secretome of Pleurotus sapidus was cloned from a cDNA library. Bioinformatic analyses revealed an open reading frame of 1551 bp corresponding to a primary translation product of 516 amino acids. The DyP-type peroxidase was heterologously produced in Trichoderma reesei with an activity of 55,000 U L-1. The enzyme was purified from the culture supernatant, biochemically characterized and the kinetic parameters were determined. The enzyme has an N-terminal signal peptide composed of 62 amino acids. Analysis by Blue Native PAGE and activity staining with ABTS, as well as gel filtration chromatography showed the native dimeric state of the enzyme (115 kDa). Analysis of the substrate range revealed that the recombinant enzyme catalyzes, in addition to the conversion of some classic peroxidase substrates such as 2,2'-azino-bis(3-ethylthiazoline-6-sulfonate) and substituted phenols like 2,6-dimethoxyphenol, also the decolorization of the anthraquinonic dye Reactive Blue 5. The enzyme also catalyzes bleaching of natural colorants such as β-carotene and annatto. Surprisingly, β-carotene was transformed in the presence and absence of H2O2 by rPsaDyP, however enzyme activity was increased by the addition of H2O2. This indicates that the rPsaDyP has an oxidase function in addition to a peroxidase activity. As a consequence of the high affinity to the characteristic substrate Reactive Blue 5 the rPsaDyP belongs functionally to the dyp-type peroxidase family.

| S-EPMC5567573 | biostudies-literature

First Dye-Decolorizing Peroxidase from an Ascomycetous Fungus Secreted by <i>Xylaria grammica</i>.

Project description:BackgroundFungal DyP-type peroxidases have so far been described exclusively for basidiomycetes. Moreover, peroxidases from ascomycetes that oxidize Mn2+ ions are yet not known.MethodsWe describe here the physicochemical, biocatalytic, and molecular characterization of a DyP-type peroxidase (DyP, EC 1.11.1.19) from an ascomycetous fungus.ResultsThe enzyme oxidizes classic peroxidase substrates such as 2,6-DMP but also veratryl alcohol and notably Mn2+ to Mn3+ ions, suggesting a physiological function of this DyP in lignin modification. The KM value (49 µM) indicates that Mn2+ ions bind with high affinity to the XgrDyP protein but their subsequent oxidation into reactive Mn3+ proceeds with moderate efficiency compared to MnPs and VPs. Mn2+ oxidation was most effective at an acidic pH (between 4.0 and 5.0) and a hypothetical surface exposed an Mn2+ binding site comprising three acidic amino acids (two aspartates and one glutamate) could be localized within the hypothetical XgrDyP structure. The oxidation of Mn2+ ions is seemingly supported by four aromatic amino acids that mediate an electron transfer from the surface to the heme center.ConclusionsOur findings shed new light on the possible involvement of DyP-type peroxidases in lignocellulose degradation, especially by fungi that lack prototypical ligninolytic class II peroxidases.

| S-EPMC8469222 | biostudies-literature

Comprehensive investigation of a dye-decolorizing peroxidase and a manganese peroxidase from Irpex lacteus F17, a lignin-degrading basidiomycete.

Project description:Irpex lacteus F17 is well-known for its ability to degrade recalcitrant aromatic pollutants, which mainly results from the action of the manganese peroxidase (MnP) that it is able to produce. Recently, the genome sequencing and annotation of this strain provided comprehensive picture of the ligninolytic peroxidase gene family. In addition to revealing the presence of 13 MnPs, genes for five dye-decolorizing peroxidases (DyPs) were also discovered in the I. lacteus F17 genome, which are unrelated to the fungal class II peroxidases. In the present study, amino acid sequences of five DyPs and 13 MnPs, representing two different families of heme peroxidases, were analyzed. Of these, two enzymes, a DyP (Il-DyP4) and a MnP (Il-MnP6) were expressed respectively in Escherichia coli, and were characterized by comparing their molecular models, substrate specificities, and catalytic features. The results showed that Il-DyP4 possessed a higher catalytic efficiency for some representative substrates, and a stronger decolorizing ability to a wide range of synthetic dyes in acidic conditions. Based on electrochemical measurements, Il-DyP4 was found to have a high redox potential of 27 mV at pH 3.5, which was superior to that of Il-MnP6 (- 75 mV), thereby contributing to its ability to oxidize high redox potential substrates, such as veratryl alcohol and polymeric dye Poly R-478. The results highlighted the potential of Il-DyP4 for use in industrial and environmental applications.

| S-EPMC6049852 | biostudies-literature

Efficient Degradation of Zearalenone by Dye-Decolorizing Peroxidase from Streptomyces thermocarboxydus Combining Catalytic Properties of Manganese Peroxidase and Laccase.

Project description:Ligninolytic enzymes, including laccase, manganese peroxidase, and dye-decolorizing peroxidase (DyP), have attracted much attention in the degradation of mycotoxins. Among these enzymes, the possible degradation pathway of mycotoxins catalyzed by DyP is not yet clear. Herein, a DyP-encoding gene, StDyP, from Streptomyces thermocarboxydus 41291 was identified, cloned, and expressed in Escherichia coli BL21/pG-Tf2. The recombinant StDyP was capable of catalyzing the oxidation of the peroxidase substrate 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), phenolic lignin compounds 2,6-dimethylphenol, and guaiacol, non-phenolic lignin compound veratryl alcohol, Mn2+, as well as anthraquinone dye reactive blue 19. Moreover, StDyP was able to slightly degrade zearalenone (ZEN). Most importantly, we found that StDyP combined the catalytic properties of manganese peroxidase and laccase, and could significantly accelerate the enzymatic degradation of ZEN in the presence of their corresponding substrates Mn2+ and 1-hydroxybenzotriazole. Furthermore, the biological toxicities of the main degradation products 15-OH-ZEN and 13-OH-ZEN-quinone might be remarkably removed. These findings suggested that DyP might be a promising candidate for the efficient degradation of mycotoxins in food and feed.

| S-EPMC8473283 | biostudies-literature

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