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On the Track of Long-Range Electron Transfer in B-Type Dye-Decolorizing Peroxidases: Identification of a Tyrosyl Radical by Computational Prediction and Electron Paramagnetic Resonance Spectroscopy.

ABSTRACT: The catalytic activity of dye-decolorizing peroxidases (DyPs) toward bulky substrates, including anthraquinone dyes, phenolic lignin model compounds, or 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), is in strong contrast to their sterically restrictive active site. In two of the three known subfamilies (A- and C/D-type DyPs), catalytic protein radicals at surface-exposed sites, which are connected to the heme cofactor by electron transfer path(s), have been identified. So far in B-type DyPs, there has been no evidence for protein radical formation after activation by hydrogen peroxide. Interestingly, B-type Klebsiella pneumoniae dye-decolorizing peroxidase (KpDyP) displays a persistent organic radical in the resting state composed of two species that can be distinguished by W-band electron spin echo electron paramagnetic resonance (EPR) spectroscopy. Here, on the basis of a comprehensive mutational and EPR study of computationally predicted tyrosine and tryptophan variants of KpDyP, we demonstrate the formation of tyrosyl radicals (Y247 and Y92) and a radical-stabilizing Y-W dyad between Y247 and W18 in KpDyP, which are unique to enterobacterial B-type DyPs. Y247 is connected to Y92 by a hydrogen bonding network, is solvent accessible in simulations, and is involved in ABTS oxidation. This suggests the existence of long-range electron path(s) in B-type DyPs. The mechanistic and physiological relevance of the reaction mechanism of B-type DyPs is discussed.

SUBMITTER: Nys K

PROVIDER: S-EPMC8154254 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Unexpected diversity of dye-decolorizing peroxidases

Project description:Dye-decolorizing peroxidase (DyP)-type peroxidases are a family of heme-containing peroxidases. Because DyP-type peroxidases can degrade recalcitrant anthraquinone dyes and lignin, their potential applications in the treatment of wastewater containing dyes and lignin degradation are expected. Although many DyP-type peroxidases have been characterized experimentally, most of the reported DyP-type peroxidases are from basidiomycetous fungi and bacteria. Therefore, the taxonomic distribution of the DyP-type peroxidases remains unclear. In this study, we analyzed the phylogenetic tree using all DyP-type peroxidase sequences available in the InterPro database. The findings mainly divided this family into three classes. Metazoa and Archaea also have the genes coding for DyP-type peroxidases, and the sequences belonging to two subclasses have the pyruvate formate lyase or cytochrome P450 domain in addition to the DyP domain. This study reveals differences in the conservation of important residues among classes. The findings will accelerate research on the DyP-type peroxidase family. Highlights • DyP-type peroxidase family is mainly divided into three classes P, I, and V.• Obvious difference of three classes is the amino acid sequence lengths.• Metazoa and Archaea also have the genes coding for DyP-type peroxidases.• Subclasses with domains other than DyP also exist.

| S-EPMC9719856 | 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

Efficient Degradation of 2-Mercaptobenzothiazole and Other Emerging Pollutants by Recombinant Bacterial Dye-Decolorizing Peroxidases.

Project description:In recent years, concerns are being raised about the potential harmful effects of emerging pollutants (EPs) on human and aquatic lives. Extensive research is being conducted on developing efficient remediation strategies to target this new class of toxic pollutants. Studies focused on biological (enzyme-based) methods have shown potential as greener and possibly more economical alternatives to other treatment approaches, such as chemical methods. The current study focused on the use of recombinantly produced novel bacterial peroxidases, namely dye-decolorizing peroxidases (DyPs), to study their effectiveness in degrading a number of diverse EPs. In this context, a sensitive bioanalytical Liquid chromatography-tandem mass spectrometry (LCMSMS)-based method was developed to simultaneously detect a mixture of 31 EPs and to examine their degradability by a panel of seven different recombinant bacterial DyPs (rDyPs). We show that up to 9 of the 31 tested EPs could be degraded by at least one of the DyPs tested. The results also indicated that not all rDyPs behaved similarly in their abilities to degrade EPs, as some rDyPs (such as SviDyP and CboDyP) showed a promising potential to degrade EPs while others (such as ScDyP) were almost ineffective. Additionally, the role of redox mediators for effective emerging pollutant degradation by rDyPs was also examined, which showed dramatic improvement in the DyP-mediated degradation of five different EPs. Detailed analysis of 2-mercaptobenzothiazole degradation by SviDyP showed that six distinct breakdown products were generated. The present study showed for the first time that recombinant bacterial DyPs can be used for wastewater remediation by degrading a range of different EPs.

| S-EPMC8146892 | biostudies-literature

Direct Electrochemical Generation of Catalytically Competent Oxyferryl Species of Classes I and P Dye Decolorizing Peroxidases.

Project description:This work introduces a novel way to obtain catalytically competent oxyferryl species for two different dye-decolorizing peroxidases (DyPs) in the absence of H2O2 or any other peroxide by simply applying a reductive electrochemical potential under aerobic conditions. UV-vis and resonance Raman spectroscopies show that this method yields long-lived compounds II and I for the DyPs from Bacillus subtilis (BsDyP; Class I) and Pseudomonas putida (PpDyP; Class P), respectively. Both electrochemically generated high valent intermediates are able to oxidize ABTS at both acidic and alkaline pH. Interestingly, the electrocatalytic efficiencies obtained at pH 7.6 are very similar to the values recorded for regular catalytic ABTS/H2O2 assays at the optimal pH of the enzymes, ca. 3.7. These findings pave the way for the design of DyP-based electrocatalytic reactors operable in an extended pH range without the need of harmful reagents such as H2O2.

| S-EPMC8653965 | biostudies-literature

Dye-decolorizing peroxidases in <i>Irpex lacteus</i> combining the catalytic properties of heme peroxidases and laccase play important roles in ligninolytic system.

Project description:BackgroundThe white rot fungus Irpex lacteus exhibits a great potential in biopretreatment of lignocellulose as well as in biodegradation of xenobiotic compounds by extracellular ligninolytic enzymes. Among these enzymes, the possible involvement of dye-decolorizing peroxidase (DyP) in lignin degradation is not clear yet.ResultsBased on the extracellular enzyme activities and secretome analysis, I. lacteus CD2 produced DyPs as the main ligninolytic enzymes when grown in Kirk's medium supplemented with lignin. Further transcriptome analysis revealed that induced transcription of genes encoding DyPs was accompanied by the increased expression of transcripts for H2O2-generating enzymes such as alcohol oxidase, pyranose 2-oxidase, and glyoxal oxidases. Meanwhile, accumulation of transcripts for glycoside hydrolase and protease was observed, in agreement with abundant proteins. Moreover, the biochemical analysis of IlDyP2 and IlDyP1 confirmed that DyPs were able to catalyze the oxidation of typical peroxidases substrates ABTS, phenolic lignin compounds DMP, and guaiacol as well as non-phenolic lignin compound, veratryl alcohol. More importantly, IlDyP1 enhanced catalytic activity for veratryl alcohol oxidation in the presence of mediator 1-hydroxybenzotriazole, which was similar to the laccase/1-hydroxybenzotriazole system.ConclusionsThe results proved for the first time that DyPs depolymerized lignin individually, combining catalytic features of different peroxidases on the functional level. Therefore, DyPs may be considered an important part of ligninolytic system in wood-decaying fungi.

| S-EPMC6223037 | 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

Radical sites in Mycobacterium tuberculosis KatG identified using electron paramagnetic resonance spectroscopy, the three-dimensional crystal structure, and electron transfer couplings.

Project description:Catalase-peroxidase (KatG) from Mycobacterium tuberculosis, a Class I peroxidase, exhibits high catalase activity and peroxidase activity with various substrates and is responsible for activation of the commonly used antitubercular drug, isoniazid (INH). KatG readily forms amino acid-based radicals during turnover with alkyl peroxides, and this work focuses on extending the identification and characterization of radicals forming on the millisecond to second time scale. Rapid freeze-quench electron paramagnetic resonance spectroscopy (RFQ-EPR) reveals a change in the structure of the initially formed radical in the presence of INH. Heme pocket binding of the drug and knowledge that KatG[Y229F] lacks this signal provides evidence for radical formation on residue Tyr(229). High field RFQ-EPR spectroscopy confirmed a tryptophanyl radical signal, and new analyses of X-band RFQ-EPR spectra also established its presence. High field EPR spectroscopy also confirmed that the majority radical species is a tyrosyl radical. Site-directed mutagenesis, along with simulations of EPR spectra based on x-ray structural data for particular tyrosine and tryptophan residues, enabled assignments based on predicted hyperfine coupling parameters. KatG mutants W107F, Y229F, and the double mutant W107F/Y229F showed alteration in type and yield of radical species. Results are consistent with formation of a tyrosyl radical reasonably assigned to residue Tyr(229) within the first few milliseconds of turnover. This is followed by a mixture of tyrosyl and tryptophanyl radical species and finally to only a tyrosyl radical on residue Tyr(353), which lies more distant from the heme. The radical processing of enzyme lacking the Trp(107)-Tyr(229)-Met(255) adduct (found as a unique structural feature of catalase-peroxidases) is suggested to be a reasonable assignment of the phenomena.

| S-EPMC1885898 | biostudies-literature

Electron paramagnetic resonance spectroscopy of nitroxide-labeled calmodulin.

Project description:Calmodulin (CaM) is a highly conserved calcium-binding protein consisting of two homologous domains, each of which contains two EF-hands, that is known to bind well over 300 proteins and peptides. In most cases the (Ca(2+))(4-)form of CaM leads to the activation of a key regulatory enzyme or protein in a myriad of biological processes. Using the nitroxide spin-labeling reagent, 3-(2-iodoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyl oxyl, bovine brain CaM was modified at 2-3 methionines with retention of activity as judged by the activation of cyclic nucleotide phosphodiesterase. X-band electron paramagnetic resonance (EPR) spectroscopy was used to measure the spectral changes upon addition of Ca(2+) to the apo-form of spin-labeled protein. A significant loss of spectral intensity, arising primarily from reductions in the heights of the low, intermediate, and high field peaks, accompanied Ca(2+) binding. The midpoint of the Ca(2+)-mediated transition determined by EPR occurred at a higher Ca(2+) concentration than that measured with circular dichroic spectroscopy and enzyme activation. Recent data have indicated that the transition from the apo-state of CaM to the fully saturated form, [(Ca(2+))(4-)CaM], contains a compact intermediate corresponding to [(Ca(2+))(2-)CaM], and the present results suggest that the spin probes are reporting on Ca(2+) binding to the last two sites in the N-terminal domain, i.e. for the [(Ca(2+))(2)-CaM] ? [(Ca(2+))(4-)CaM] transition in which the compact structure becomes more extended. EPR of CaM, spin-labeled at methionines, offers a different approach for studying Ca(2+)-mediated conformational changes and may emerge as a useful technique for monitoring interactions with target proteins.

| S-EPMC4096354 | biostudies-literature

Catalytic surface radical in dye-decolorizing peroxidase: a computational, spectroscopic and site-directed mutagenesis study.

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

| S-EPMC4357238 | biostudies-literature

Very Low-Frequency Broadband Electron Paramagnetic Resonance Spectroscopy of Metalloproteins.

Project description:A previously developed spectrometer for broadband electron paramagnetic resonance (EPR) spectroscopy of dilute randomly oriented systems has been considerably modified to extend the frequency reach down to the hundred MHz range and to boost concentration sensitivity by 1 to 2 orders of magnitude. The instrument is now suitable for the study of biological systems in particular metalloproteins. As a proof of concept, examples from the class of low-spin ferric hemoproteins are studied in terms of frequency-dependent changes in their EPR spectra. Mono-heme cytochrome c EPR is determined by g-strain over a wide frequency range, whereas a combination of unresolved ligand hyperfine interaction and concentration-dependent intermolecular dipolar interaction becomes dominant at very low frequencies. In the four heme containing cytochrome c3, g-strain combines with intramolecular dipolar interaction over the full-studied frequency range of 0.23-12.0 GHz. It is concluded that the point-dipole approach is inappropriate to describe magnetic interactions between low-spin ferric heme systems and that a body of literature on redox interactions in multi-heme proteins will be affected by this conclusion.

| S-EPMC8154605 | biostudies-literature

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