Spectroelectrochemistry for determination of the redox potential in heme enzymes: Dye-decolorizing peroxidases.

Dye-decolorizing peroxidases (DyPs) are heme-containing enzymes that are structurally unrelated to other peroxidases. Some DyPs show high potential for applications in biotechnology, which critically depends on the stability and redox potential (E°') of the enzyme. Here we provide a comparative analysis of UV-Vis- and surface-enhanced resonance Raman-based spectroelectrochemical methods for determination of the E°' of DyPs from two different organisms, and their variants generated targeting E°' upshift. We show that substituting the highly conserved Arginine in the distal side of the heme pocket by hydrophobic amino acid residues impacts the heme architecture and redox potential of DyPs from the two organisms in a very distinct manner. We demonstrate the advantages and drawbacks of the used spectroelectrochemical approaches, which is relevant for other heme proteins that contain multiple heme centers or spin populations.

Biochemical, Biophysical, and Structural Analysis of an Unusual DyP from the Extremophile Deinococcus radiodurans.

Dye-decolorizing peroxidases (DyPs) are heme proteins with distinct structural properties and substrate specificities compared to classical peroxidases. Here, we demonstrate that DyP from the extremely radiation-resistant bacterium Deinococcus radiodurans is, like some other homologues, inactive at physiological pH. Resonance Raman (RR) spectroscopy confirms that the heme is in a six-coordinated-low-spin (6cLS) state at pH 7.5 and is thus unable to bind hydrogen peroxide. At pH 4.0, the RR spectra of the enzyme reveal the co-existence of high-spin and low-spin heme states, which corroborates catalytic activity towards H2O2 detected at lower pH. A sequence alignment with other DyPs reveals that DrDyP possesses a Methionine residue in position five in the highly conserved GXXDG motif. To analyze whether the presence of the Methionine is responsible for the lack of activity at high pH, this residue is substituted with a Glycine. UV-vis and RR spectroscopies reveal that the resulting DrDyPM190G is also in a 6cLS spin state at pH 7.5, and thus the Methionine does not affect the activity of the protein. The crystal structures of DrDyP and DrDyPM190G, determined to 2.20 and 1.53 Å resolution, respectively, nevertheless reveal interesting insights. The high-resolution structure of DrDyPM190G, obtained at pH 8.5, shows that one hydroxyl group and one water molecule are within hydrogen bonding distance to the heme and the catalytic Asparagine and Arginine. This strong ligand most likely prevents the binding of the H2O2 substrate, reinforcing questions about physiological substrates of this and other DyPs, and about the possible events that can trigger the removal of the hydroxyl group conferring catalytic activity to DrDyP.

Evaluation of a dye-decolorizing peroxidase from Comamonas serinivorans for lignin valorization potentials.

Although dye-decolourising peroxidases (DyPs) are well-known for lignin degradation, a comprehensive understanding of their mechanism remains unclear. Therefore, studying the mechanism of lignin degradation by DyPs is necessary for industrial applications and enzyme engineering. In this study, a dye-decolourising peroxidase (CsDyP) gene from C. serinivorans was heterologously expressed and studied for its lignin degradation potential. Molecular docking analysis predicted the binding of 2, 2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), veratryl alcohol (VA), 2, 6-dimethylphenol (2, 6- DMP), guaiacol (GUA), and lignin to the substrate-binding pocket of CsDyP. Evaluation of the enzymatic properties showed that CsDyP requires pH 4.0 and 30 °C for optimal activity and has a high affinity for ABTS. In addition, CsDyP is stable over a wide range of temperatures and pH and can tolerate 5.0 mM organic solvents. Low NaCl concentrations promoted CsDyP activity. Further, CsDyP significantly reduced the chemical oxygen demand decolourised alkali lignin (AL) and milled wood lignin (MWL). CsDyP targets the ß-O-4, CO, and CC bonds linking lignin's G, S, and H units to depolymerize and produce aromatic compounds. Overall, this study delivers valuable insights into the lignin degradation mechanism of CsDyP, which can benefit its industrial applications and lignin valorization.

Heterologous expression, purification and structural features of native Dictyostelium discoideum dye-decolorizing peroxidase bound to a natively incorporated heme.

The Dictyostelium discoideum dye-decolorizing peroxidase (DdDyP) is a newly discovered peroxidase, which belongs to a unique class of heme peroxidase family that lacks homology to the known members of plant peroxidase superfamily. DdDyP catalyzes the H2O2-dependent oxidation of a wide-spectrum of substrates ranging from polycyclic dyes to lignin biomass, holding promise for potential industrial and biotechnological applications. To study the molecular mechanism of DdDyP, highly pure and functional protein with a natively incorporated heme is required, however, obtaining a functional DyP-type peroxidase with a natively bound heme is challenging and often requires addition of expensive biosynthesis precursors. Alternatively, a heme in vitro reconstitution approach followed by a chromatographic purification step to remove the excess heme is often used. Here, we show that expressing the DdDyP peroxidase in ×2 YT enriched medium at low temperature (20°C), without adding heme supplement or biosynthetic precursors, allows for a correct native incorporation of heme into the apo-protein, giving rise to a stable protein with a strong Soret peak at 402 nm. Further, we crystallized and determined the native structure of DdDyP at a resolution of 1.95 Å, which verifies the correct heme binding and its geometry. The structural analysis also reveals a binding of two water molecules at the distal site of heme plane bridging the catalytic residues (Arg239 and Asp149) of the GXXDG motif to the heme-Fe(III) via hydrogen bonds. Our results provide new insights into the geometry of native DdDyP active site and its implication on DyP catalysis.

Periplasmic expression of Pseudomonas fluorescens peroxidase Dyp1B and site-directed mutant Dyp1B enzymes enhances polymeric lignin degradation activity in Pseudomonas putida KT2440.

Expression of lignin-oxidising Pseudomonas fluorescens Dyp1B in the periplasm of Pseudomonas putida KT2440, using a tat fusion construct, was found to lead to enhanced whole cell activity for oxidation of DCP and polymeric lignin substrates. Four amino acid residues predicted to lie at the manganese ion binding site of Pseudomonas fluorescens peroxidase Dyp1B were investigated using site-directed mutagenesis. Mutants H127R and S223A showed 2-fold and 4-fold higher kcat for Mn(II) oxidation respectively, and mutant S223A showed 2-fold enhanced production of low molecular weight phenolic products from a polymeric soda lignin. The mutant Pfl Dyp1B genes were expressed as tat fusions to investigate their effect on lignin oxidation by P. putida KT2440.

Comparison of Laccases and Hemeproteins Systems in Bioremediation of Organic Pollutants.

AIM: Laccases and peroxidases have attracted great interest for industrial and environmental applications. These enzymes have a broad substrate range and a robust oxidizing ability. Moreover, using mediators or co-oxidants makes it possible to increase their catalytic activity and extend their substrate scope to more resistant chemical structures. BACKGROUND: Fungal laccases and ligninolytic peroxidases, mainly lignin and manganese peroxidases, are the privileged oxidoreductases for bioremediation processes. Nonetheless, an increasing diversity of laccases and peroxidase-type enzymes has been proposed for environmental technologies. OBJECTIVE: This article aims to provide an overview of these enzymes and compare their applicability in the degradation of organic pollutants. METHODS: Fundamental properties of the proteins are covered and applications towards polycyclic aromatic hydrocarbons (PAHs) and pesticides are specially focused. RESULTS: Laccases are multicopper oxidases initially studied for applications in the pulp and paper industry but able to oxidize a variety of environmentally concerning compounds. Relying on O2, laccases do not require peroxides nor auxiliary agents, like Mn2+, although suitable redox mediators are needed to attack the more recalcitrant pollutants (e.g., PAHs). True and pseudo-peroxidases use a stronger oxidant (H2O2) and the redox chemistry at the heme site generates high potential species that allow the oxidation of dyes and some pesticides. CONCLUSION: Lately, research efforts have been directed to enzyme discovery, testing with micropollutants, and improving biocatalysts' stability by immobilization and protein engineering. Further understanding of the effects of natural media components and solvents on the enzymes might lead to competitive enzymatic treatments of highly toxic media.

Transformation of Reactive Blue 19 by a recombinant peroxidase DyP.

Wastewater containing recalcitrant dyes causes environmental problems. A new superfamily of heme-containing peroxidases, dye-decolorizing peroxidases (DyPs), has been found to decolorize different kinds of dyes, especial anthraquinone dyes efficiently. However, the mechanism of dyes degradation by DyPs has not been fully understood and the toxicity of dye degradation intermediates by DyPs catalysis to microbes is unclear. In this study, a purified recombinant Thermobifida fusca DyP (TfuDyP) in E. coli BL21(DE3) was used to treat Reactive Blue 19 (RB19), an anthraquinone dye. The reaction intermediates analyzed by ultra performance liquid chromatography/mass spectroscopy (UPLC-MS) indicated the initial site of TfuDyP attack on RB19. In addition, it was found that both RB19 and its incomplete degradation products inhibited the growth of Bacillus subtilis. These findings provided a novel understanding of DyPs catalysis to anthraquinone dyes.

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

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.

Loops around the Heme Pocket Have a Critical Role in the Function and Stability of BsDyP from Bacillus subtilis.

Bacillus subtilis BsDyP belongs to class I of the dye-decolorizing peroxidase (DyP) family of enzymes and is an interesting biocatalyst due to its high redox potential, broad substrate spectrum and thermostability. This work reports the optimization of BsDyP using directed evolution for improved oxidation of 2,6-dimethoxyphenol, a model lignin-derived phenolic. After three rounds of evolution, one variant was identified displaying 7-fold higher catalytic rates and higher production yields as compared to the wild-type enzyme. The analysis of X-ray structures of the wild type and the evolved variant showed that the heme pocket is delimited by three long conserved loop regions and a small α helix where, incidentally, the mutations were inserted in the course of evolution. One loop in the proximal side of the heme pocket becomes more flexible in the evolved variant and the size of the active site cavity is increased, as well as the width of its mouth, resulting in an enhanced exposure of the heme to solvent. These conformational changes have a positive functional role in facilitating electron transfer from the substrate to the enzyme. However, they concomitantly resulted in decreasing the enzyme's overall stability by 2 kcal mol-1, indicating a trade-off between functionality and stability. Furthermore, the evolved variant exhibited slightly reduced thermal stability compared to the wild type. The obtained data indicate that understanding the role of loops close to the heme pocket in the catalysis and stability of DyPs is critical for the development of new and more powerful biocatalysts: loops can be modulated for tuning important DyP properties such as activity, specificity and stability.

A New Ciboria sp. for Soil Mycoremediation and the Bacterial Contribution to the Depletion of Total Petroleum Hydrocarbons.

A Ciboria sp. strain (Phylum Ascomycota) was isolated from hydrocarbon-polluted soil of an abandoned oil refinery in Italy. The strain was able to utilize diesel oil as a sole carbon source for growth. Laboratory-scale experiments were designed to evaluate the use of this fungal strain for treatment of the polluted soil. The concentration of total petroleum hydrocarbons (TPH) in the soil was 8,538 mg/kg. Mesocosms containing the contaminated soil were inoculated with the fungal strain at 1 or 7%, on a fresh weight base ratio. After 90 days of incubation, the depletion of TPH contamination was of 78% with the 1% inoculant, and 99% with the 7% inoculant. 16S rDNA and ITS metabarcoding of the bacterial and fungal communities was performed in order to evaluate the potential synergism between fungi and bacteria in the bioremediation process. The functional metagenomic prediction indicated Arthrobacter, Dietzia, Brachybacerium, Brevibacterium, Gordonia, Leucobacter, Lysobacter, and Agrobacterium spp. as generalist saprophytes, essential for the onset of hydrocarbonoclastic specialist bacterial species, identified as Streptomyces, Nocardoides, Pseudonocardia, Solirubrobacter, Parvibaculum, Rhodanobacter, Luteiomonas, Planomicrobium, and Bacillus spp., involved in the TPH depletion. The fungal metabolism accelerated the onset of specialist over generalist bacteria. The capacity of the Ciboria sp. to deplete TPH in the soil in treatment was also ascertained.

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

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

Exploring the Diversity of Fungal DyPs in Mangrove Soils to Produce and Characterize Novel Biocatalysts.

The functional diversity of the New Caledonian mangrove sediments was examined, observing the distribution of fungal dye-decolorizing peroxidases (DyPs), together with the complete biochemical characterization of the main DyP. Using a functional metabarcoding approach, the diversity of expressed genes encoding fungal DyPs was investigated in surface and deeper sediments, collected beneath either Avicennia marina or Rhizophora stylosa trees, during either the wet or the dry seasons. The highest DyP diversity was observed in surface sediments beneath the R. stylosa area during the wet season, and one particular operational functional unit (OFU1) was detected as the most abundant DyP isoform. This OFU was found in all sediment samples, representing 51-100% of the total DyP-encoding sequences in 70% of the samples. The complete cDNA sequence corresponding to this abundant DyP (OFU 1) was retrieved by gene capture, cloned, and heterologously expressed in Pichia pastoris. The recombinant enzyme, called DyP1, was purified and characterized, leading to the description of its physical-chemical properties, its ability to oxidize diverse phenolic substrates, and its potential to decolorize textile dyes; DyP1 was more active at low pH, though moderately stable over a wide pH range. The enzyme was very stable at temperatures up to 50 °C, retaining 60% activity after 180 min incubation. Its ability to decolorize industrial dyes was also tested on Reactive Blue 19, Acid Black, Disperse Blue 79, and Reactive Black 5. The effect of hydrogen peroxide and sea salt on DyP1 activity was studied and compared to what is reported for previously characterized enzymes from terrestrial and marine-derived fungi.

Understanding molecular enzymology of porphyrin-binding α + ß barrel proteins - One fold, multiple functions.

There is a high functional diversity within the structural superfamily of porphyrin-binding dimeric α + ß barrel proteins. In this review we aim to analyze structural constraints of chlorite dismutases, dye-decolorizing peroxidases and coproheme decarboxylases in detail. We identify regions of structural variations within the highly conserved fold, which are most likely crucial for functional specificities. The loop linking the two ferredoxin-like domains within one subunit can be of different sequence lengths and can adopt various structural conformations, consequently defining the shape of the substrate channels and the respective active site architectures. The redox cofactor, heme b or coproheme, is oriented differently in either of the analyzed enzymes. By thoroughly dissecting available structures and discussing all available results in the context of the respective functional mechanisms of each of these redox-active enzymes, we highlight unsolved mechanistic questions in order to spark future research in this field.

Class P dye-decolorizing peroxidase gene: Degenerated primers design and phylogenetic analysis.

Dye-decolorizing peroxidases (DyPs) were classified as a new family of heme peroxidase in 2007. Produced by various bacteria, they are the first bacterial enzymes known able to degrade lignin and dyes, for which their application in waste treatment and pretreatment of lignocellulosic biomass could be envisaged. In this work, a PCR primer pair was created and tested that enabled the detection and quantification of a wide range of bacterial genes of P class DyP in complex matrices. In addition, a phylogenetic tree was built with all available sequences of DyP genes available, offering a first overview of their presence in the bacteria kingdom.

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

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.

Oxidation and nitration of mononitrophenols by a DyP-type peroxidase.

Substantial conversion of nitrophenols, typical high-redox potential phenolic substrates, by heme peroxidases has only been reported for lignin peroxidase (LiP) so far. But also a dye-decolorizing peroxidase of Auricularia auricula-judae (AauDyP) was found to be capable of acting on (i) ortho-nitrophenol (oNP), (ii) meta-nitrophenol (mNP) and (iii) para-nitrophenol (pNP). The pH dependency for pNP oxidation showed an optimum at pH 4.5, which is typical for phenol conversion by DyPs and other heme peroxidases. In the case of oNP and pNP conversion, dinitrophenols (2,4-DNP and 2,6-DNP) were identified as products and for pNP additionally p-benzoquinone. Moreover, indications were found for the formation of random polymerization products originating from initially formed phenoxy radical intermediates. Nitration was examined using (15)N-labeled pNP and Na(14)NO2 as an additional source of nitro-groups. Products were identified by HPLC-MS, and mass-to-charge ratios were evaluated to clarify the origin of nitro-groups. The additional nitrogen in DNPs formed during enzymatic conversion was found to originate both from (15)N-pNP and (14)NO2Na. Based on these results, a hypothetical reaction scheme and a catalytically responsible confine of the enzyme's active site are postulated.

Basidiomycete DyPs: Genomic diversity, structural-functional aspects, reaction mechanism and environmental significance.

The first enzyme with dye-decolorizing peroxidase (DyP) activity was described in 1999 from an arthroconidial culture of the fungus Bjerkandera adusta. However, the first DyP sequence had been deposited three years before, as a peroxidase gene from a culture of an unidentified fungus of the family Polyporaceae (probably Irpex lacteus). Since the first description, fewer than ten basidiomycete DyPs have been purified and characterized, but a large number of sequences are available from genomes. DyPs share a general fold and heme location with chlorite dismutases and other DyP-type related proteins (such as Escherichia coli EfeB), forming the CDE superfamily. Taking into account the lack of an evolutionary relationship with the catalase-peroxidase superfamily, the observed heme pocket similarities must be considered as a convergent type of evolution to provide similar reactivity to the enzyme cofactor. Studies on the Auricularia auricula-judae DyP showed that high-turnover oxidation of anthraquinone type and other DyP substrates occurs via long-range electron transfer from an exposed tryptophan (Trp377, conserved in most basidiomycete DyPs), whose catalytic radical was identified in the H2O2-activated enzyme. The existence of accessory oxidation sites in DyP is suggested by the residual activity observed after site-directed mutagenesis of the above tryptophan. DyP degradation of substituted anthraquinone dyes (such as Reactive Blue 5) most probably proceeds via typical one-electron peroxidase oxidations and product breakdown without a DyP-catalyzed hydrolase reaction. Although various DyPs are able to break down phenolic lignin model dimers, and basidiomycete DyPs also present marginal activity on nonphenolic dimers, a significant contribution to lignin degradation is unlikely because of the low activity on high redox-potential substrates.

The toolbox of Auricularia auricula-judae dye-decolorizing peroxidase - Identification of three new potential substrate-interaction sites.

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.

Radical formation on a conserved tyrosine residue is crucial for DyP activity.

Dye-decolorizing peroxidases (DyPs) are able to cleave bulky anthraquinone dyes. The recently published crystal structure of AauDyPI reveals that a direct oxidation in the distal heme cavity can be excluded for most DyP substrates. It is shown that a surface-exposed tyrosine residue acts as a substrate interaction site for bulky substrates. This amino acid is conserved in eucaryotic DyPs but is missing in the structurally related chlorite dismutases (Clds). Dye-decolorizing peroxidases of procaryotic origin equally possess a conserved tyrosine in the same region of the polypeptide albeit not at the homologous position.