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.

Evolution and separation of actinobacterial pyranose and C-glycoside-3-oxidases.

FAD-dependent pyranose oxidase (POx) and C-glycoside-3-oxidase (CGOx) are both members of the glucose-methanol-choline superfamily of oxidoreductases and belong to the same sequence space. Pyranose oxidases had been studied for their oxidation of monosaccharides such as D-glucose, but recently, a bacterial C-glycoside-3-oxidase that is phylogenetically related to POx and that reacts with C-glycosides such as carminic acid, mangiferin or puerarin has been described. Since these actinobacterial CGOx enzymes belong to the same sequence space as bacterial POx, they must have evolved from the same ancestor. Here, we performed a phylogenetic analysis of actinobacterial sequences and resurrected seven ancestral enzymes of the POx/CGOx sequence space to study the evolutionary trajectory of substrate preferences for monosaccharides and C-glycosides. Clade I, with its dimeric member POx from Kitasatospora aureofaciens, shows strict preference for monosaccharides (D-glucose and D-xylose) and does not react with any of the glycosides tested. No extant member of clade II has been studied to date. The two extant members of clades III and IV, monomeric POx/CGOx from Pseudoarthrobacter siccitolerans and Streptomyces canus, oxidized both monosaccharides as well as various C-glycosides (homoorientin, isovitexin, mangiferin, and puerarin). Steady-state kinetic parameters of several clades III and IV ancestral enzymes indicate that the generalist ancestor N35 slowly evolved to present-day enzymes with a much higher preference for C-glycosides than monosaccharides. Based on structural predictions of ancestors, we hypothesize that the strict specificity of bacterial clade I POx (and also fungal POx) is the result of oligomerization, which in turn results from the evolution of protein segments that were shown to be important for oligomerization, the arm, and the head domain.IMPORTANCEC-Glycosides often form active compounds in various plants. Breakage of the C-C bond in these glycosides to release the aglycone is challenging and proceeds via a two-step reaction, the oxidation of the sugar and subsequent cleavage of the C-C bond. Recently, an enzyme from a soil bacterium, FAD-dependent C-glycoside-3-oxidase (CGOx), was shown to catalyze the initial oxidation reaction. Here, we show that CGOx belongs to the same sequence space as pyranose oxidase (POx), and that an actinobacterial ancestor of the POx/CGOx family evolved into four clades, two of which show a high preference for C-glycosides.

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.

Flexible active-site loops fine-tune substrate specificity of hyperthermophilic metallo-oxidases.

Hyperthermophilic ('superheat-loving') archaea found in high-temperature environments such as Pyrobaculum aerophilum contain multicopper oxidases (MCOs) with remarkable efficiency for oxidizing cuprous and ferrous ions. In this work, directed evolution was used to expand the substrate specificity of P. aerophilum McoP for organic substrates. Six rounds of error-prone PCR and DNA shuffling followed by high-throughput screening lead to the identification of a hit variant with a 220-fold increased efficiency (kcat/Km) than the wild-type for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) without compromising its intrinsic activity for metal ions. The analysis of the X-ray crystal structure reveals four proximal mutations close to the T1Cu active site. One of these mutations is within the 23-residues loop that occludes this site, a distinctive feature of prokaryotic MCOs. The increased flexibility of this loop results in an enlarged tunnel and one additional pocket that facilitates bulky substrate-enzyme interactions. These findings underscore the synergy between mutations that modulate the dynamics of the active-site loop enabling enhanced catalytic function. This study highlights the potential of targeting loops close to the T1Cu for engineering improvements suitable for biotechnological applications.

Environmentally Friendly Degradation and Detoxification of Rifampicin by a Bacterial Laccase and Hydrogen Peroxide.

Antibiotics are micropollutants accumulating in our rivers and wastewaters, potentially leading to bacterial antibiotic resistance, a worldwide problem to which there is no current solution. Here, we have developed an environmentally friendly two-step process to transform the antibiotic rifampicin (RIF) into non-antimicrobial compounds. The process involves an enzymatic oxidation step by the bacterial CotA-laccase and a hydrogen peroxide bleaching step. NMR identified rifampicin quinone as the main product of the enzymatic oxidation. Growth of Escherichia coli strains in the presence of final degradation products (FP) and minimum inhibitory concentration (MIC) measurements confirmed that FP are non-anti-microbial compounds, and bioassays suggest that FP is not toxic to eukaryotic organisms. Moreover, competitive fitness assays between susceptible and RIF-resistant bacteria show that susceptible bacteria is strongly favoured in the presence of FP. Our results show that we have developed a robust and environmentally friendly process to effectively remediate rifampicin from antibiotic contaminated environments.

The quest for new robust bacterial monoamine oxidases.

Microbial enzymes are versatile, cost-effective, and sustainable tools, making them a preferred choice for enzymatic processes. Santema et al. harnessed AlphaFold, a cutting-edge structure prediction tool, to discover new thermophilic monoamine oxidases (MAO) that could be relevant for drug development and use in biotechnology fields. The new enzyme displays thermal robustness, offering a unique structure-to-function profile compared to known MAOs. This bacterial enzyme, paired with recent advancements in enzyme engineering, has the potential to meet the biotech sector's need for customized enzymes.

Mechanistic insights into glycoside 3-oxidases involved in C-glycoside metabolism in soil microorganisms.

C-glycosides are natural products with important biological activities but are recalcitrant to degradation. Glycoside 3-oxidases (G3Oxs) are recently identified bacterial flavo-oxidases from the glucose-methanol-coline (GMC) superfamily that catalyze the oxidation of C-glycosides with the concomitant reduction of O2 to H2O2. This oxidation is followed by C-C acid/base-assisted bond cleavage in two-step C-deglycosylation pathways. Soil and gut microorganisms have different oxidative enzymes, but the details of their catalytic mechanisms are largely unknown. Here, we report that PsG3Ox oxidizes at 50,000-fold higher specificity (kcat/Km) the glucose moiety of mangiferin to 3-keto-mangiferin than free D-glucose to 2-keto-glucose. Analysis of PsG3Ox X-ray crystal structures and PsG3Ox in complex with glucose and mangiferin, combined with mutagenesis and molecular dynamics simulations, reveal distinctive features in the topology surrounding the active site that favor catalytically competent conformational states suitable for recognition, stabilization, and oxidation of the glucose moiety of mangiferin. Furthermore, their distinction to pyranose 2-oxidases (P2Oxs) involved in wood decay and recycling is discussed from an evolutionary, structural, and functional viewpoint.

Assessment of early harvest in the prevention of aflatoxins in peanuts during drought stress conditions.

The present study aimed to evaluate the effectiveness of early harvest in preventing aflatoxins in peanuts under drought-stress conditions. A field experiment was conducted on the 2018-2019 and 2019-2020 growing seasons in a greenhouse with an irrigation system to induce three drought stress conditions: no stress, mild, and severe stress. In addition, three harvest dates were proposed: two weeks earlier, one week earlier, and ideal harvest time. The mean peanut yield was 2634 kg/ha, considering the two growing seasons, and the drought stress conditions and harvest dates did not influence significantly. The shelling percentage was significantly higher in samples harvested at ideal harvest (77.7 %) than two weeks earlier (76.2 %) and was not influenced by drought stress conditions. Although a low mean percentage of grains with insect damage was identified, this percentage was statistically higher under severe stress (0.4 %) compared to no-stress conditions (0.2 %). The soil contamination ranged from 2.52 × 103 to 1.64 × 104 CFU/g of Aspergillus section Flavi, and the drought stress resulted in significantly higher concentrations in mild and severe stressed samples. A. section Flavi was found to infect all the peanut kernel samples. The drought stress resulted in higher percentages of A. section Flavi infections in samples from mild and severe stress conditions. The harvest date did not influence the soil and peanut kernel occurrence of A. section Flavi. A total of 435 and 796 strains of A. section Flavi were isolated from soil and peanut kernels, respectively. The potential of aflatoxin production by soil isolates was 31, 44, and 25 % for aflatoxin non-producers, aflatoxin B producers, and aflatoxin B and G producers, respectively, while in peanut kernel isolates were 44, 44, and 12 %. Three different A. section Flavi species were identified from peanut kernels: A. flavus, A. parasiticus, and A. pseudocaelatus. The mean aflatoxin concentration in peanut kernels was 42, 316, and 695.5 µg/kg in samples under no stress, mild stress, and severe stress conditions, respectively. Considering the harvest time, the mean aflatoxin concentration was 9.9, 334.3, and 614.2 µg/kg in samples harvested two weeks earlier, one week earlier, and in ideal harvest, respectively. In conclusion, the early harvest proved to be a viable, cost-free alternative for controlling aflatoxin in the peanut pre-harvest, resulting in a safer product and a better quality for sale and economic gain.

Acute Effects of Different Set Configurations on Neuromuscular, Metabolic, and Perceptual Responses in Young Women.

We compared neuromuscular, metabolic, and perceptual responses between different resistance training configurations in young women. In a counterbalanced randomized order, 13 young women performed the following protocols in separate sessions (sets x repetitions): traditional (TRAD): 5x10, 90-s of rest interval between sets; more frequent and shorter total rest (FSR): 10x5, 30-s of rest interval between sets. The sessions were composed of leg press exercise with the same intensity. Force (maximum voluntary isometric contraction [MVIC]) and metabolic (lactate concentration) responses were measured pre- and post-resistance training sessions. The rating of perceived exertion (RPE) was measured after each set. The internal training load was calculated using the session-RPE method. There was a significant reduction in the MVIC only after TRAD configuration (Effect size [ES] = 0.36). The lactate concentration increased in both conditions but was higher after TRAD (ES = 2.81) than FSR (ES = 1.23). The RPE has progressively increased in both configurations. On the other hand, the internal training load was lower in the FSR configuration. From our findings, we suggest that more frequent and shorter total rest is an effective strategy for maintaining the ability to produce force, generating less metabolic stress and lower perceived internal load in young women.

Retirada de projetil de arma de fogo alojado em assoalho infraorbitário com preservação do globo ocular: relato de caso / Removal of a firearm projectile lodged in the infraorbital floor with preservation of the eyeball: case report

Objetivo: relatar um caso de ferimento por arma de fogo (FAF), com projétil balístico alojado em região infraorbitária à esquerda, relatando tratamento cirúrgico de urgência para exérese do projetil por acesso subciliar. Relato do caso: Paciente do gênero masculino, 18 anos, foi encaminhado ao pronto socorro do Hospital Universitário por conta de ferimento por arma de fogo. Ao exame clínico, foi verificado discreto aumento de volume em região cervical e hemiface à esquerda, ausência de sangramento em face; presença de limitação para infraversão de olho esquerdo. O orifício de entrada do projetil foi identificado em região de tórax superior posterior à esquerda; após realização de tomografia de face, confirmou-se fratura de assoalho orbitário esquerdo, assim como projétil alojado abaixo do globo ocular. O ato cirúrgico foi realizado com caráter de urgência pela equipe de Cirurgia e Traumatologia Bucomaxilofacial. Discussão: estudos descrevem a importância da realização do adequado manejo de lesões traumáticas decorrentes de FAF seguido de remoção do projetil com urgência, visto que as complicações ao postergar o tempo cirúrgico só agravam o quadro clínico do paciente, além das afecções futuras que podem surgir com o decorrer da resolução do caso clínico. Conclusão: ratifica-se a importância da multidisciplinariedade entres as clínicas médicas, assim como, o correto manejo do paciente traumatizado, baseado nos critérios que conduzem o plano de tratamento.(AU)

Aim: to report a case of gunshot wound, with ballistic projectile lodged in the left infraorbital region, reporting emergency surgical treatment for projectile exeresis by subciliary access. Case Report: An 18-year-old male patient was referred to the emergency department of the University Hospital due to a gunshot wound. On clinical examination, it was found slight increase in volume in the cervical region and left hemiface, absence of bleeding in the face; presence of limitation to infraversion of the left eye. The entrance hole of the projectile was identified in the posterior left upper thorax region; after a tomography of the face, a fracture of the left orbital floor was confirmed, as well as the projectile lodged below the eyeball. The surgery was urgently performed by the Oral and Maxillofacial Surgery team. Discussion: Studies describe the importance of performing the proper management of traumatic injuries resulting from FAF followed by removal of the projectile with urgency, since the complications to postpone the surgical time only aggravate the clinical picture of the patient, in addition to future problems that may arise with the resolution of the clinical case. Conclusion: the importance of multidisciplinarity among medical clinics is ratified, as well as the correct management of the traumatized patient, based on the criteria that lead to the treatment plan.(AU)

Electrochemical Actuation of a DyP Peroxidase: A Facile Method for Drastic Improvement of the Catalytic Performance.

Dye decolorizing peroxidases (DyP) have attracted interest for applications such as dye-containing wastewater remediation and biomass processing. So far, efforts to improve operational pH ranges, activities, and stabilities have focused on site-directed mutagenesis and directed evolution strategies. Here, we show that the performance of the DyP from Bacillus subtilis can be drastically boosted without the need for complex molecular biology procedures by simply activating the enzyme electrochemically in the absence of externally added H2O2. Under these conditions, the enzyme shows specific activities toward a variety of chemically different substrates that are significantly higher than in its canonical operation. Moreover, it presents much broader pH activity profiles with the maxima shifted toward neutral to alkaline. We also show that the enzyme can be successfully immobilized on biocompatible electrodes. When actuated electrochemically, the enzymatic electrodes have two orders of magnitude higher turnover numbers than with the standard H2O2-dependent operation and preserve about 30% of the initial electrocatalytic activity after 5 days of operation-storage cycles.

Biocatalysis for biorefineries: The case of dye-decolorizing peroxidases.

Dye-decolorizing Peroxidases (DyPs) are heme-containing enzymes in fungi and bacteria that catalyze the reduction of hydrogen peroxide to water with concomitant oxidation of various substrates, including anthraquinone dyes, lignin-related phenolic and non-phenolic compounds, and metal ions. Investigation of DyPs has shed new light on peroxidases, one of the most extensively studied families of oxidoreductases; still, details of their microbial physiological role and catalytic mechanisms remain to be fully disclosed. They display a distinctive ferredoxin-like fold encompassing anti-parallel ß-sheets and α-helices, and long conserved loops surround the heme pocket with a role in catalysis and stability. A tunnel routes H2O2 to the heme pocket, whereas binding sites for the reducing substrates are in cavities near the heme or close to distal aromatic residues at the surface. Variations in reactions, the role of catalytic residues, and mechanisms were observed among different classes of DyP. They were hypothetically related to the presence or absence of distal H2O molecules in the heme pocket. The engineering of DyPs for improved properties directed their biotechnological applications, primarily centered on treating textile effluents and degradation of other hazardous pollutants, to fields such as biosensors and valorization of lignin, the most abundant renewable aromatic polymer. In this review, we track recent research contributions that furthered our understanding of the activity, stability, and structural properties of DyPs and their biotechnological applications. Overall, the study of DyP-type peroxidases has significant implications for environmental sustainability and the development of new bio-based products and materials with improved end-of-life options via biodegradation and chemical recyclability, fostering the transition to a sustainable bio-based industry in the circular economy realm.

Corrigendum to "Unveiling molecular details behind improved activity at neutral to alkaline pH of an engineered DyP-type peroxidase" Comput Struct Biotechnol J, 20 (2022), 3899-3910.

[This corrects the article DOI: 10.1016/j.csbj.2022.07.032.].

A wide array of lignin-related phenolics are oxidized by an evolved bacterial dye-decolourising peroxidase.

Lignin is the second most abundant natural polymer next to cellulose and by far the largest renewable source of aromatic compounds on the planet. Dye-decolourising peroxidases (DyPs) are biocatalysts with immense potential in lignocellulose biorefineries to valorize emerging lignin building blocks for environmentally friendly chemicals and materials. This work investigates the catalytic potential of the engineered PpDyP variant 6E10 for the oxidation of 24 syringyl, guaiacyl and hydroxybenzene lignin-phenolic derivatives. Variant 6E10 exhibited up to 100-fold higher oxidation rates at pH 8 for all the tested phenolic substrates compared to the wild-type enzyme and other acidic DyPs described in the literature. The main products of reactions were dimeric isomers with molecular weights of (2 × MWsubstrate - 2 H). Their structure depends on the substitution pattern of the aromatic ring of substrates, i.e., of the coupling possibilities of the primarily formed radicals upon enzymatic oxidation. Among the dimers identified were syringaresinol, divanillin and diapocynin, important sources of structural scaffolds exploitable in medicinal chemistry, food additives and polymers.

Rationally Guided Improvement of NOV1 Dioxygenase for the Conversion of Lignin-Derived Isoeugenol to Vanillin.

Biocatalysis is a key tool in both green chemistry and biorefinery fields. NOV1 is a dioxygenase that catalyzes the one-step, coenzyme-free oxidation of isoeugenol into vanillin and holds enormous biotechnological potential for the complete valorization of lignin as a sustainable starting material for biobased chemicals, polymers, and materials. This study integrates computational, kinetic, structural, and biophysical approaches to characterize a new NOV1 variant featuring improved activity and stability compared to those of the wild type. The S283F replacement results in a 2-fold increased turnover rate (kcat) for isoeugenol and a 4-fold higher catalytic efficiency (kcat/Km) for molecular oxygen compared to those of the wild type. Furthermore, the variant exhibits a half-life that is 20-fold higher than that of the wild type, which most likely relates to the enhanced stabilization of the iron cofactor in the active site. Molecular dynamics supports this view, revealing that the S283F replacement decreases the optimal pKa and favors conformations of the iron-coordinating histidines compatible with an increased level of binding to iron. Importantly, whole cells containing the S283F variant catalyze the conversion of ≤100 mM isoeugenol to vanillin, yielding >99% molar conversion yields within 24 h. This integrative strategy provided a new enzyme for biotechnological applications and mechanistic insights that will facilitate the future design of robust and efficient biocatalysts.

Distal Mutations Shape Substrate-Binding Sites during Evolution of a Metallo-Oxidase into a Laccase.

Laccases are in increasing demand as innovative solutions in the biorefinery fields. Here, we combine mutagenesis with structural, kinetic, and in silico analyses to characterize the molecular features that cause the evolution of a hyperthermostable metallo-oxidase from the multicopper oxidase family into a laccase (k cat 273 s-1 for a bulky aromatic substrate). We show that six mutations scattered across the enzyme collectively modulate dynamics to improve the binding and catalysis of a bulky aromatic substrate. The replacement of residues during the early stages of evolution is a stepping stone for altering the shape and size of substrate-binding sites. Binding sites are then fine-tuned through high-order epistasis interactions by inserting distal mutations during later stages of evolution. Allosterically coupled, long-range dynamic networks favor catalytically competent conformational states that are more suitable for recognizing and stabilizing the aromatic substrate. This work provides mechanistic insight into enzymatic and evolutionary molecular mechanisms and spots the importance of iterative experimental and computational analyses to understand local-to-global changes.

The gastrointestinal microbiome of browsing goats (Capra hircus).

Despite the growing interest in the ruminants' gastrointestinal tract (GIT) microbiomes' ability to degrade plant materials by animal husbandry and industrial sectors, only a few studies addressed browsing ruminants. The present work describes the taxonomic and functional profile of the bacterial and archaeal communities from five different gastrointestinal sections (rumen, omasum-abomasum, jejunum, cecum and colon) of browsing Capra hircus, by metabarcoding using 16S rRNA genes hypervariable regions. The bacterial communities across the GITs are mainly composed of Bacillota and Bacteroidota. Prevotella was the leading bacterial group found in the stomachs, Romboutsia in the jejuna, and Rikenellaceae_RC9_gut_group, Bacteroides, UCG-010_ge, UCG-005, and Alistipes in large intestines. The archaeal communities in the stomachs and jejuna revealed to be mainly composed of Methanobrevibacter, while in the large intestines its dominance is shared with Methanocorpusculum. Across the GITs, the main metabolic functions were related to carbohydrate, amino acid, and energy metabolisms. Significant differences in the composition and potential biological functions of the bacterial communities were observed among stomachs, jejuna and large intestines. In contrast, significant differences were observed among stomachs and jejuna verse large intestines for archaeal communities. Overall different regions of the GIT are occupied by different microbial communities performing distinct biological functions. A high variety of glycoside hydrolases (GHs) indispensable for degrading plant cell wall materials were predicted to be present in all the GIT sections.

Unveiling molecular details behind improved activity at neutral to alkaline pH of an engineered DyP-type peroxidase.

DyP-type peroxidases (DyPs) are microbial enzymes that catalyze the oxidation of a wide range of substrates, including synthetic dyes, lignin-derived compounds, and metals, such as Mn2+ and Fe2+, and have enormous biotechnological potential in biorefineries. However, many questions on the molecular basis of enzyme function and stability remain unanswered. In this work, high-resolution structures of PpDyP wild-type and two engineered variants (6E10 and 29E4) generated by directed evolution were obtained. The X-ray crystal structures revealed the typical ferredoxin-like folds, with three heme access pathways, two tunnels, and one cavity, limited by three long loops including catalytic residues. Variant 6E10 displays significantly increased loops' flexibility that favors function over stability: despite the considerably higher catalytic efficiency, this variant shows poorer protein stability compared to wild-type and 29E4 variants. Constant-pH MD simulations revealed a more positively charged microenvironment near the heme pocket of variant 6E10, particularly in the neutral to alkaline pH range. This microenvironment affects enzyme activity by modulating the pK a of essential residues in the heme vicinity and should account for variant 6E10 improved activity at pH 7-8 compared to the wild-type and 29E4 that show optimal enzymatic activity close to pH 4. Our findings shed light on the structure-function relationships of DyPs at the molecular level, including their pH-dependent conformational plasticity. These are essential for understanding and engineering the catalytic properties of DyPs for future biotechnological applications.

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.