Dye-decolorizing Peroxidase Research Articles

Structural Characterization of Mycobacterium tuberculosis Encapsulin in Complex with Dye-Decolorizing Peroxide

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, the world’s deadliest infectious disease. Mtb uses a variety of mechanisms to evade the human host’s defenses and survive intracellularly. Mtb’s oxidative stress response enables Mtb to survive within activated macrophages, an environment with reactive oxygen species and low pH. Dye-decolorizing peroxidase (DyP), an enzyme involved in Mtb’s oxidative stress response, is encapsulated in a nanocompartment, encapsulin (Enc), and is important for Mtb’s survival in macrophages. Encs are homologs of viral capsids and encapsulate cargo proteins of diverse function, including those involved in iron storage and stress responses. DyP contains a targeting peptide (TP) at its C-terminus that recognizes and binds to the interior of the Enc nanocompartment. Here, we present the crystal structure of the Mtb-Enc•DyP complex and compare it to cryogenic-electron microscopy (cryo-EM) Mtb-Enc structures. Investigation into the canonical pores formed at symmetrical interfaces reveals that the five-fold pore for the Mtb-Enc crystal structure is strikingly different from that observed in cryo-EM structures. We also observe DyP-TP electron density within the Mtb-Enc shell. Finally, investigation into crystallographic small-molecule binding sites gives insight into potential novel avenues by which substrates could enter Mtb-Enc to react with Mtb-DyP.

Dye-Decolorizing Peroxidases Maintain High Stability and Turnover on Kraft Lignin and Lignocellulose Substrates.

Fungal enzyme systems for the degradation of plant cell wall lignin, consisting of, among others, laccases and lignin-active peroxidases, are well characterized. Additionally, fungi and bacteria contain dye-decolorizing peroxidases (DyP), which are also capable of oxidizing and modifying lignin constituents. Studying DyP activity on lignocellulose poses challenges due to the heterogeneity of the substrate and the lack of continuous kinetic methods. In this study, we report the kinetic parameters of bacterial DyP from Amycolatopsis 75iv2 and fungal DyP from Auricularia auricula-judae on insoluble plant materials and kraft lignin by monitoring the depletion of the cosubstrate of the peroxidases with a H2O2 sensor. In the reactions with spruce, both enzymes showed similar kinetics. On kraft lignin, the catalytic rate of bacterial DyP reached 30 ± 2 s-1, whereas fungal DyP was nearly 3 times more active (81 ± 7 s-1). Importantly, the real-time measurement of H2O2 allowed the assessment of continuous activity for both enzymes, revealing a previously unreported exceptionally high stability under turnover conditions. Bacterial DyP performed 24,000 turnovers of H2O2, whereas the fungal DyP achieved 94,000 H2O2 turnovers in 1 h with a remaining activity of 40 and 80%, respectively. Using mass spectrometry, the depletion of the cosubstrate H2O2 was shown to correlate with product formation, validating the amperometric method.

Characterization of the Coriolopsis gallica DyP for Its Potential to Biotransform Various Fluoroquinolones.

Coriolopsis gallica (Cga) is a white-rot fungus renowned for its ability to secrete ligninolytic enzymes that are capable of oxidizing phenolic compounds. This study aimed to investigate the biochemical characteristics of a dye-decolorizing peroxidase named CgaDyP1 and test its ability to biotransform antibiotics. CgaDyP1 was cloned and heterologously expressed in Escherichia coli. We fully characterized the biochemical properties of CgaDyP1 and evaluated its dye-decolorizing potential to confirm that it belongs to the DyP class of enzymes. We also tested its fluoroquinolone antibiotic biotransformation potential for possible biotechnological applications. Alignment of the primary amino acid sequence with DyP homolog sequences showed that CgaDyP1 has high similarity with other fungal DyPs. The recombinant CgaDyP1 exhibited activity on substrates such as ABTS and 2,6-dimethoxyphenol (DMP) with optimal performance at a pH of 3, although activity at pH 2.5, pH 4, and pH5 diminished over time. Thermostability tests indicated that the enzyme remains stable at temperatures between 30 °C and 50 °C and retains 70% of its initial activity after 180 min at 50 °C. Tests on the effect of hydrogen peroxide on CgaDyP1 activity found peak activity at 0.25 mM H2O2. CgaDyP1 decolorized five industrial dyes, and kinetics data confirmed that it belongs to the DyP class of enzymes. CgaDyP1 was shown to biotransform some of the 7 recalcitrant fluoroquinolone antibiotics tested here, including levofloxacin, moxifloxacin, and norfloxacin, and thus holds potential for biotechnological applications.

A dye-decolorizing peroxidase from Vibrio cholerae can demetallate heme

Iron is an essential element for bacterial survival. Bacterial pathogens have therefore developed methods to obtain iron. Vibrio cholerae, the intestinal pathogen that causes cholera, utilizes heme as an iron source. DyP from V. cholerae (VcDyP) is a dye-decolorizing peroxidase. When VcDyP was expressed in Escherichia coli and purified, it was found to contain protoporphyrin IX (PPIX) but not heme, indicating that the protein possesses deferrochelatase activity. Here, we examined the demetallation reaction of VcDyP using fluorescence spectroscopy. Treatment of heme-reconstituted VcDyP with sodium dithionite under anaerobic conditions led to an increase in the fluorescence intensity at 624 nm, suggesting the formation of PPIX. Although the same reaction was conducted using myoglobin, horseradish peroxidase and hemin, no increase in the fluorescence was observed. Therefore, demetallation of heme is specific to VcDyP. This reaction was faster at lower pH, but the amplitudes of the fluorescence increase were larger at pH 6.5–7.5, in clear contrast to the dye-decolorizing activity with the optimal pH of 4.5. In contrast to HutZ from V. cholerae, which is a heme-degrading enzyme that cleaves the heme macrocycle to release iron, VcDyP can remove iron from heme without degradation. To our knowledge, VcDyP is the first enzyme whose demetallation activity has been confirmed at neutral pH. Our results show that VcDyP is a bifunctional protein that degrades anthraquinone dyes and demetallates heme.

New application of a dye-decolorizing peroxidase immobilized on magnetic nanoparticles for efficient simultaneous degradation of two mycotoxins

Nowadays the enzymatic approaches are the most promising strategies for mycotoxins detoxification in food stuffs. Herein, the dye-decolorizing peroxidase RhDypB from Rhodococcus jostii was studied for its ability to degrade two mycotoxins in both free and the immobilized enzyme forms. This enzyme was recombinantly expressed and purified, while Fe3O4 nanoparticles were prepared and modified with chitosan as the immobilization carrier. The immobilized enzyme Fe3O4@CS@RhDypB demonstrated degradation rate of 85.61 % toward aflatoxin B1, while it was firstly found to be able to degrade zearalenone with the rate of 86.52 %, at pH 4.0 on 30 °C. The degradation products were identified as aflatoxin Q1 and 15-OH-ZEN respectively. After 5 cycles of reuse, Fe3O4@CS@RhDypB still exhibited degradation rates of 38.50 % and 49.76 % toward the mycotoxins, indicating its high reusability. Moreover, Fe3O4@CS@RhDypB exhibited excellent stability after 10 days of storage. This work identified potential applications of nanoparticle-immobilized enzyme for biodegradation of mycotoxins in food industry.

Heterologous expression and characterization of a dye-decolorizing peroxidase from Ganoderma lucidum, and its application in decolorization and detoxifization of different types of dyes.

Dye-decolorizing peroxidases (DyPs) belong to a novel superfamily of heme peroxidases that can oxidize recalcitrant compounds. In the current study, the GlDyP2 gene from Ganoderma lucidum was heterologously expressed in Escherichia coli, and the enzymatic properties of the recombinant GlDyP2 protein were investigated. The GlDyP2 protein could oxidize not only the typical peroxidase substrate ABTS but also two lignin substrates, namely guaiacol and 2,6-dimethoxy phenol (DMP). For the ABTS substrate, the optimum pH and temperature of GlDyP2 were 4.0 and 35°C, respectively. The pH stability and thermal stability of GlDyP2 were also measured; the results showed that GlDyP2 could function normally in the acidic environment, with a T50 value of 51°C. Moreover, compared to untreated controls, the activity of GlDyP2 was inhibited by 1.60 mM of Mg2+, Ni2+, Mn2+, and ethanol; 0.16 mM of Cu2+, Zn2+, methanol, isopropyl alcohol, and Na2EDTA·2H2O; and 0.016 mM of Fe2+ and SDS. The kinetic constants of recombinant GlDyP2 for oxidizing ABTS, Reactive Blue 19, guaiacol, and DMP were determined; the results showed that the recombination GlDyP2 exhibited the strongest affinity and the most remarkable catalytic efficiency towards guaiacol in the selected substrates. GlDyP2 also exhibited decolorization and detoxification capabilities towards several dyes, including Reactive Blue 19, Reactive Brilliant Blue X-BR, Reactive Black 5, Methyl Orange, Trypan Blue, and Malachite Green. In conclusion, GlDyP2 has good application potential for treating dye wastewater.

Oxidative degradation and detoxification of multiple mycotoxins using a dye-decolorizing peroxidase from the white-rot fungus Bjerkandera adusta

Mycotoxins are widely present from cereal crops to food products, posing a significant risk to food safety and causing serious health and economic losses. Therefore, it is imperative to develop a sustainable and efficient detoxification strategy to address mycotoxin contamination. Herein, a dye-decolorizing peroxidase, BaDyP, from the white-rot fungus Bjerkandera adusta was successfully expressed as a soluble form in Escherichia coli with the help of molecular chaperones. Purified recombinant BaDyP could efficiently degrade multiple mycotoxins including aflatoxin B1, zearalenone, and deoxynivalenol in the presence of mediators such as Mn2+ or 1-HBT. Meanwhile, their degradation products were identified using UPLC-MS/MS, including AFB1-diol, AFQ1, 15-OH-ZEN, HZEN, and C15H18O8. Furthermore, cell survival tests exhibited a significant reduction in the biological toxicity of these degradation products. These findings indicate that the use of dye-decolorizing peroxidase could be a promising approach for the effective removal of multiple mycotoxins in cereal crops and food products.

Effect of Electrical Stimulation of the External Circuit of Membraneless Bioelectrochemical Systems on Imidacloprid Degradation and Representation of the mtrB and DyP-Type Peroxidases Genes

Effect of Electrical Stimulation of the External Circuit of Membraneless Bioelectrochemical Systems on Imidacloprid Degradation and Representation of the mtrB and DyP-Type Peroxidases Genes

Kraft Lignin Decomposition by Forest Soil Bacterium Pseudomonas kribbensis CHA-19.

Identification of the biochemical metabolic pathway for lignin decomposition and the responsible degradative enzymes is needed for the effective biotechnological valorization of lignin to renewable chemical products. In this study, we investigated the decomposition of kraft lignin by the soil bacterium Pseudomonas kribbensis CHA-19, a strain that can utilize kraft lignin and its main degradation metabolite, vanillic acid, as growth substrates. Gel permeation chromatography revealed that CHA-19 decomposed polymeric lignin and degraded dehydrodivanillin (a representative lignin model compound); however, the degradative enzyme(s) and mechanism were not identified. Quantitative polymerase chain reaction with mRNAs from CHA-19 cells induced in the presence of lignin showed that the putative genes coding for two laccase-like multicopper oxidases (LMCOs) and three dye-decolorizing peroxidases (DyPs) were upregulated by 2.0- to 7.9-fold compared with glucose-induced cells, which indicates possible cooperation with multiple enzymes for lignin decomposition. Computational homology analysis of the protein sequences of LMCOs and DyPs also predicted their roles in lignin decomposition. Based on the above data, CHA-19 appears to initiate oxidative lignin decomposition using multifunctional LMCOs and DyPs, producing smaller metabolites such as vanillic acid, which is further degraded via ortho- and meta-ring cleavage pathways. This study not only helps to better understand the role of bacteria in lignin decomposition and thus in terrestrial ecosystems, but also expands the biocatalytic toolbox with new bacterial cells and their degradative enzymes for lignin valorization.

Response of soil fungi to textile dye contamination

This study examines the impact of textile dye contamination on the structure of soil fungal communities near a Shaoxing textile dye factory. We quantified the concentrations of various textile dyes, including anthraquinone azodye and phthalocyanine, which ranged from 20.20 to 140.62 mg kg^-1, 102.01–698.12 mg kg^-1, and 7.78–42.65 mg kg^-1, respectively, within a 1000 m radius of the factory. Our findings indicate that as dye concentration increases, the biodiversity of soil fungi, as measured by the Chao1 index, decreases significantly, highlighting the profound influence of dye contamination on fungal community structure. Additionally, microbial correlation network analysis revealed a reduction in fungal interactions correlating with increased dye concentrations. We also observed that textile dyes suppressed carbon and nitrogen metabolism in fungi while elevating the transcription levels of antioxidant-related genes. Enzymes such as lignin peroxidase (LiP), manganese peroxidase (MnP), laccase (Lac), dye-decolorizing peroxidases (DyPs), and versatile peroxidase (VP) were upregulated in contaminated soils, underscoring the critical role of fungi in dye degradation. These insights contribute to the foundational knowledge required for developing in situ bioremediation technologies for contaminated farmlands.

The oligomeric states of dye-decolorizing peroxidases from Streptomyces lividans and their implications for mechanism of substrate oxidation.

A common evolutionary mechanism in biology to drive function is protein oligomerization. In prokaryotes, the symmetrical assembly of repeating protein units to form homomers is widespread, yet consideration in vitro of whether such assemblies have functional or mechanistic consequences is often overlooked. Dye-decolorizing peroxidases (DyPs) are one such example, where their dimeric α + β barrel units can form various oligomeric states, but the oligomer influence, if any, on mechanism and function has received little attention. In this work, we have explored the oligomeric state of three DyPs found in Streptomyces lividans, each with very different mechanistic behaviors in their reactions with hydrogen peroxide and organic substrates. Using analytical ultracentrifugation, we reveal that except for one of the A-type DyPs where only a single sedimenting species is detected, oligomer states ranging from homodimers to dodecamers are prevalent in solution. Using cryo-EM on preparations of the B-type DyP, we determined a 3.02 Å resolution structure of a hexamer assembly that corresponds to the dominant oligomeric state in solution as determined by analytical ultracentrifugation. Furthermore, cryo-EM data detected sub-populations of higher-order oligomers, with one of these formed by an arrangement of two B-type DyP hexamers to give a dodecamer assembly. Our solution and structural insights of these oligomer states provide a new framework to consider previous mechanistic studies of these DyP members and are discussed in terms of long-range electron transfer for substrate oxidation and in the "storage" of oxidizable equivalents on the heme until a two-electron donor is available.

Brilliant green decolorization by dye decolorizing peroxidase producing bacterium, Achromobacter insolitus isolated from Forest soils of Similipal Biosphere Reserve, Odisha

Dye-decolorizing peroxidases (DyPs) are recently identified superfamily of heme-containing peroxidases which has received much attention for its potential application in dye decolourization. In the current research, six bacterial colonies (SDB1-6) showing lignolytic activity were isolated from forest soils of SBR using a minimal salt medium supplemented with alkali lignin and screened for DyP activity using brilliant green dye supplemented luria bertani agar medium. The highest DyP activity showing bacterium was identified using biochemical and 16SrRNA sequencing and was identified as Achromobacter insolitus. A. insolitus was found to be an efficient strain showing the highest DyP activity of 2.7 U/mL/ml at un-optimized condition. The RSM optimization was conducted to increase the DyP enzyme activity. A. insolitus demonstrated DyP activity of 6.20 U/mL/min with an increase of 2.29 times of enzyme activity in optimal condition of pH 7.0, temperature 25 °C, H2O2 concentration 0.1 mM, and time 24 h. The bacterium was further screened for dye decolorization taking crystal violet, methyl orange, methylene blue, brilliant green and methyl red dyes supplemented in LB medium. The bacterium showed maximum brilliant green degradation ability was analyzed using UV-Vis and FTIR analyses to characterize the degraded product. The highest brilliant green degradation activity of the A. insolitus in the present study could be exploited for bioremediation of brilliant green contaminated industrial effluents in a ecofriendly and cost effective way.

Characterization of Amycolatopsis 75iv2 dye-decolorizing peroxidase on O-glycosides.

Peroxidases require correct incorporation of the heme cofactor for activity. Heterologous overproduction of peroxidases often results in an inactive enzyme due to insufficient heme synthesis by the host organism. Therefore, peroxidases are incubated with excess heme during or after purification to reconstitute activity. S. lividans as a production host can produce fully active peroxidases both intracellularly and extracellularly without the need for heme supplementation. This reduces the number of downstream processing steps and is beneficial for more sustainable production of industrially relevant enzymes. Moreover, this research has extended the scope of dye-decolorizing peroxidase applications by studying naturally relevant plant secondary metabolites and analyzing the formed products. A previously overlooked artifact of radical polymerization leading to the release of the glycosyl moiety was revealed, shedding light on the mechanism of DyP peroxidases. The key aspect is the continuous addition, rather than the more common approach of a single addition, of the cosubstrate, hydrogen peroxide. This continuous addition allows the peroxidase to complete a high number of turnovers without self-oxidation.

Structural basis for peroxidase encapsulation inside the encapsulin from the Gram-negative pathogen Klebsiella pneumoniae

Encapsulins are self-assembling protein nanocompartments capable of selectively encapsulating dedicated cargo proteins, including enzymes involved in iron storage, sulfur metabolism, and stress resistance. They represent a unique compartmentalization strategy used by many pathogens to facilitate specialized metabolic capabilities. Encapsulation is mediated by specific cargo protein motifs known as targeting peptides (TPs), though the structural basis for encapsulation of the largest encapsulin cargo class, dye-decolorizing peroxidases (DyPs), is currently unknown. Here, we characterize a DyP-containing encapsulin from the enterobacterial pathogen Klebsiella pneumoniae. By combining cryo-electron microscopy with TP and TP-binding site mutagenesis, we elucidate the molecular basis for cargo encapsulation. TP binding is mediated by cooperative hydrophobic and ionic interactions as well as shape complementarity. Our results expand the molecular understanding of enzyme encapsulation inside protein nanocompartments and lay the foundation for rationally modulating encapsulin cargo loading for biomedical and biotechnological applications.

Engineering A-type Dye-Decolorizing Peroxidases by Modification of a Conserved Glutamate Residue.

Dye-decolorizing peroxidases (DyPs) are recently identified microbial enzymes that have been used in several Biotechnology applications from wastewater treatment to lignin valorization. However, their properties and mechanism of action still have many open questions. Their heme-containing active site is buried by three conserved flexible loops with a putative role in modulating substrate access and enzyme catalysis. Here, we investigated the role of a conserved glutamate residue in stabilizing interactions in loop 2 of A-type DyPs. First, we did site saturation mutagenesis of this residue, replacing it with all possible amino acids in bacterial DyPs from Bacillus subtilis (BsDyP) and from Kitasatospora aureofaciens (KaDyP1), the latter being characterized here for the first time. We screened the resulting libraries of variants for activity towards ABTS and identified variants with increased catalytic efficiency. The selected variants were purified and characterized for activity and stability. We furthermore used Molecular Dynamics simulations to rationalize the increased catalytic efficiency and found that the main reason is the electron channeling becoming easier from surface-exposed tryptophans. Based on our findings, we also propose that this glutamate could work as a pH switch in the wild-type enzyme, preventing intracellular damage.

Dye-decolorizing peroxidase of Thermobifida halotolerance displays complex kinetics with both substrate inhibition and apparent positive cooperativity

Dye-decolorizing peroxidases (DyPs) have been intensively investigated for the purpose of industrial dye decolourization and lignin degradation. Unfortunately, the characterization of these peroxidases is hampered by their non-Michaelis-Menten kinetics, exemplified by substrate inhibition and/or positive cooperativity. Although often observed, the underlying mechanisms behind the unusual kinetics of DyPs are poorly understood. Here we studied the kinetics of the oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), hydroquinones, and anthraquinone dyes by DyP from the bacterium Thermobifida halotolerans (ThDyP) and solved its crystal structure. We also provide rate equations for different kinetic mechanisms explaining the complex kinetics of heme peroxidases. Kinetic studies along with the analysis of the structure of ThDyP suggest that the substrate inhibition is caused by the non-productive binding of ABTS to the enzyme resting state. Strong irreversible inactivation of ThDyP by H2O2 in the absence of ABTS suggests that the substrate inhibition by H2O2 may be caused by the non-productive binding of H2O2 to compound I. Positive cooperativity was observed only with the oxidation of ABTS but not with the two electron-donating substrates. Although the conventional mechanism of cooperativity cannot be excluded, we propose that the oxidation of ABTS assumes the simultaneous binding of two ABTS molecules to reduce compound I to the enzyme resting state, and this causes the apparent positive cooperativity.

Biodegradation of malachite green by Pleurotus eryngii: a study on decolorization, mechanism, toxicity, and enzyme.

The purpose of this study was to investigate the biodegradation of malachite green (MG) by Pleurotus eryngii via decolorization. This study also explored the possible mechanisms and toxicity. The results indicated that this fungus exhibited strong decolorizing potential. MG degradation based on UPLC-TOF-Triple-MS analysis revealed the formation of intermediates such as 4-(dimethylamino)benzophenone, 4-(methylamino)benzophenone, and 4-(dimethylamino)phenol. Furthermore, a significant reduction in the toxicity of the degradation products was observed using the zebrafish animal model. A newly discovered dye-decolorizing peroxidase (DyP-PE) from P. eryngii was amplified, cloned, and expressed. The purified 56.4kDa DyP-PE strongly decolorized MG, suggesting potentially application in the bioremediation of MG pollution. Thus, the DyP-PE derived from P. eryngii may contribute to the degradation of MG.

Lignin bioconversion based on genome mining for ligninolytic genes in Erwinia billingiae QL-Z3

BackgroundBioconversion of plant biomass into biofuels and bio-products produces large amounts of lignin. The aromatic biopolymers need to be degraded before being converted into value-added bio-products. Microbes can be environment-friendly and efficiently degrade lignin. Compared to fungi, bacteria have some advantages in lignin degradation, including broad tolerance to pH, temperature, and oxygen and the toolkit for genetic manipulation.ResultsOur previous study isolated a novel ligninolytic bacterial strain Erwinia billingiae QL-Z3. Under optimized conditions, its rate of lignin degradation was 25.24% at 1.5 g/L lignin as the sole carbon source. Whole genome sequencing revealed 4556 genes in the genome of QL-Z3. Among 4428 protein-coding genes are 139 CAZyme genes, including 54 glycoside hydrolase (GH) and 16 auxiliary activity (AA) genes. In addition, 74 genes encoding extracellular enzymes are potentially involved in lignin degradation. Real-time PCR quantification demonstrated that the expression of potential ligninolytic genes were significantly induced by lignin. 8 knock-out mutants and complementary strains were constructed. Disruption of the gene for ELAC_205 (laccase) as well as EDYP_48 (Dyp-type peroxidase), ESOD_1236 (superoxide dismutase), EDIO_858 (dioxygenase), EMON_3330 (monooxygenase), or EMCAT_3587 (manganese catalase) significantly reduced the lignin-degrading activity of QL-Z3 by 47–69%. Heterologously expressed and purified enzymes further confirmed their role in lignin degradation. Fourier transform infrared spectroscopy (FTIR) results indicated that the lignin structure was damaged, the benzene ring structure and groups of macromolecules were opened, and the chemical bond was broken under the action of six enzymes encoded by genes. The abundant enzymatic metabolic products by EDYP_48, ELAC_205 and ESOD_1236 were systematically analyzed via liquid chromatography–mass spectrometry (LC–MS) analysis, and then provide a speculative pathway for lignin biodegradation. Finally, The activities of ligninolytic enzymes from fermentation supernatant, namely, LiP, MnP and Lac were 367.50 U/L, 839.50 U/L, and 219.00 U/L by orthogonal optimization.ConclusionsOur findings provide that QL-Z3 and its enzymes have the potential for industrial application and hold great promise for the bioconversion of lignin into bioproducts in lignin valorization.

PH-dependent and whole-cell catalytic decolorization of dyes using recombinant dye-decolorizing peroxidase from Rhodococcus jostii.

Dyes in wastewater have adverse effects on the environment and human health. Dye-decolorizing peroxidase (DyP) is a promising biocatalyst to dyes degradation, but the decolorization rates varied greatly which influencing factors and mechanisms remain to be fully disclosed. To explore an effective decolorizing approach, we have studied a DyP from Rhodococcus jostii (RhDyPB) which was overexpressed in Escherichia coli to decolorize four kinds of dyes, Reactive blue 19, Eosin Y, Indigo carmine, and Malachite green. We found the decolorization rates of the dyes by purified RhDyPB were all pH-dependent and the highest one was 94.4% of Malachite green at pH 6.0. ESI-MS analysis of intermediates in the decolorization process of Reactive blue 19 proved the degradation was due to peroxidase catalysis. Molecular docking predicated the interaction of RhDyPB with dyes, and a radical transfer reaction. In addition, we performed decolorization of dyes with whole E. coli cell with and without expressing RhDyPB. It was found that decolorization of dyes by E. coli cell wasdue to both cell absorption and degradation, and RhDyPB expression improved the degradation rates towards Reactive blue 19, Indigo carmine and Malachite green. The effective decolorization of Malachite green and the successful application of whole DyP-overexpressed cells in dye decolorization is conducive to the bioremediation of dye-containing wastewaters by DyPs.

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.