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.

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.

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.

Laccases of prokaryotic origin: enzymes at the interface of protein science and protein technology.

The ubiquitous members of the multicopper oxidase family of enzymes oxidize a range of aromatic substrates such as polyphenols, methoxy-substituted phenols, amines and inorganic compounds, concomitantly with the reduction of molecular dioxygen to water. This family of enzymes can be broadly divided into two functional classes: metalloxidases and laccases. Several prokaryotic metalloxidases have been described in the last decade showing a robust activity towards metals, such as Cu(I), Fe(II) or Mn(II) and have been implicated in the metal metabolism of the corresponding microorganisms. Many laccases, with a superior efficiency for oxidation of organic compounds when compared with metals, have also been identified and characterized from prokaryotes, playing roles that more closely conform to those of intermediary metabolism. This review aims to present an update of current knowledge on prokaryotic multicopper oxidases, with a special emphasis on laccases, anticipating their enormous potential for industrial and environmental applications.

An integrated view of redox and catalytic properties of B-type PpDyP from Pseudomonas putida MET94 and its distal variants.

PpDyP from Pseudomonas putida MET94 is an extremely versatile B-type dye-decolourising peroxidase (DyP) capable of efficient oxidation of a wide range of anthraquinonic and azo dyes, phenolic substrates, the non-phenolic veratryl alcohol and even manganese and ferrous ions. In reaction with H2O2 it forms a stable Compound I at a rate of (1.4±0.3)×10(6)M(-1)s(-1), comparable to those of classical peroxidases and other DyPs. We provide the first report of standard redox potential (E(0')) of the Compound I/Native redox couple in a DyP-type peroxidase. The value of E(0')Cpd I/N=1.10±0.04 (V) is similar to those found in peroxidases from the mammalian superfamily but higher than in peroxidases from the plant superfamily. Site-directed mutagenesis has been used to investigate the role of conserved distal residues, i.e. to replace aspartate 132 by asparagine, and arginine 214 and asparagine 136 by leucine. The structural, redox and catalytic properties of variants are addressed by spectroscopic, electrochemical and kinetic measurements. Our data point to the importance of the distal arginine in the catalytic mechanism of PpDyP, as also observed in DyPB from Rhodococcus jostii RHA1 but not in DyPs from the A and D subfamilies. This work reinforces the idea of existence of mechanistic variations among members of the different sub-families of DyPs with direct implications for their enzymatic properties and potential for biotechnological applications.

New dye-decolorizing peroxidases from Bacillus subtilis and Pseudomonas putida MET94: towards biotechnological applications.

This work provides spectroscopic, catalytic, and stability fingerprints of two new bacterial dye-decolorizing peroxidases (DyPs) from Bacillus subtilis (BsDyP) and Pseudomonas putida MET94 (PpDyP). DyPs are a family of microbial heme-containing peroxidases with wide substrate specificity, including high redox potential aromatic compounds such as synthetic dyes or phenolic and nonphenolic lignin units. The genes encoding BsDyP and PpDyP, belonging to subfamilies A and B, respectively, were cloned and heterologously expressed in Escherichia coli. The recombinant PpDyP is a 120-kDa homotetramer while BsDyP enzyme consists of a single 48-kDa monomer. The optimal pH of both enzymes is in the acidic range (pH 4-5). BsDyP has a bell-shape profile with optimum between 20 and 30 °C whereas PpDyP shows a peculiar flat and broad (10-30 °C) temperature profile. Anthraquinonic or azo dyes, phenolics, methoxylated aromatics, and also manganese and ferrous ions are substrates used by the enzymes. In general, PpDyP exhibits higher activities and accepts a wider scope of substrates than BsDyP; the spectroscopic data suggest distinct heme microenvironments in the two enzymes that might account for the distinctive catalytic behavior. However, the Bs enzyme with activity lasting for up to 53 h at 40 °C is more stable towards temperature or chemical denaturation than the PpDyP. The results of this work will guide future optimization of the biocatalytis towards their utilization in the fields of environmental or industrial biotechnology.

Distal mutations enhance efficiency of free and immobilized NOV1 dioxygenase for vanillin synthesis.

Protein engineering is crucial to improve enzymes' efficiency and robustness for industrial biocatalysis. NOV1 is a bacterial dioxygenase that holds biotechnological potential by catalyzing the one-step oxidation of the lignin-derived isoeugenol into vanillin, a popular flavoring agent used in food, cleaning products, cosmetics and pharmaceuticals. This study aims to enhance NOV1 activity and operational stability through the identification of distal hotspots, located at more than 9 Šfrom the active site using Zymspot, a tool that predicts advantageous distant mutations, streamlining protein engineering. A total of 41 variants were constructed using site-directed mutagenesis and the six most active enzyme variants were then recombined. Two variants, with two and three mutations, showed nearly a 10-fold increase in activity and up to 40-fold higher operational stability than the wild-type. Furthermore, these variants show 90-100 % immobilization efficiency in metal affinity resins, compared to approximately 60 % for the wild-type. In bioconversions where 50 mM of isoeugenol was added stepwise over 24-h cycles, the 1D2 variant produced approximately 144 mM of vanillin after six reaction cycles, corresponding to around 22 mg, indicating a 35 % molar conversion yield. This output was around 2.5 times higher than that obtained using the wild-type. Our findings highlight the efficacy of distal protein engineering in enhancing enzyme functions like activity, stability, and metal binding selectivity, thereby fulfilling the criteria for industrial biocatalysts. This study provides a novel approach to enzyme optimization that could have significant implications for various biotechnological applications.

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.].

The role of Asp116 in the reductive cleavage of dioxygen to water in CotA laccase: assistance during the proton-transfer mechanism.

Multi-copper oxidases constitute a family of proteins that are capable of coupling the one-electron oxidation of four substrate equivalents to the four-electron reduction of dioxygen to two molecules of water. The main catalytic stages occurring during the process have already been identified, but several questions remain, including the nature of the protonation events that take place during the reductive cleavage of dioxygen to water. The presence of a structurally conserved acidic residue (Glu498 in CotA laccase from Bacillus subtilis) at the dioxygen-entrance channel has been reported to play a decisive role in the protonation mechanisms, channelling protons during the reduction process and stabilizing the site as a whole. A second acidic residue that is sequentially conserved in multi-copper oxidases and sited within the exit channel (Asp116 in CotA) has also been identified as being important in the protonation process. In this study, CotA laccase has been used as a model system to assess the role of Asp116 in the reduction process of dioxygen to water. The crystal structures of three distinct mutants, D116E, D116N and D116A, produced by site-saturation mutagenesis have been determined. In addition, theoretical calculations have provided further support for a role of this residue in the protonation events.

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.

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.

Immobilized dye-decolorizing peroxidase (DyP) and directed evolution variants for hydrogen peroxide biosensing.

Immobilized dye-decolorizing peroxidase from Pseudomonas putida MET94 (PpDyP) and three variants generated by directed evolution (DE) are studied aiming at the design of a biosensor for H2O2 detection. Structural properties of the enzymes in solution and immobilized state are addressed by resonance Raman (RR) and surface enhanced RR (SERR) spectroscopy, and the electrocatalytic properties are analyzed by electrochemistry. The wild-type (wt) and 29E4 variant (with E188K and H125Y mutations) represent excellent candidates for development of H2O2 biosensors, since they exhibit a good dynamic response range (1-200 µM H2O2), short response times (2 s) and a superior sensitivity (1.3-1.4 A⋅M-1⋅cm-2) for H2O2, as well as selectivity and long term stability. In contrast to the solution state, 6E10 (with E188K, A142V and H125Y mutations) and 25F6 (with E188K, A142V, H125Y and G129D mutations) variants display much lower activity and are inhibited by high concentrations of H2O2 upon adsorption on an electrode. In terms of sensitivity, the bioelectrodes employing wt PpDyP and 29E4 variant outperform HRP based counterparts reported in the literature by 1-4 orders of magnitude. We propose the development of wt or 29E4 PpDyP based biosensor as a valuable alternative to devices that rely on peroxidases.

Decolorization and detoxification of textile dyes using a versatile Streptomyces laccase-natural mediator system.

Currently, there is increasing interest in assessing the potential of bacterial laccases for industrial and environmental applications especially in harsh conditions. The environmental impact of the textile industry requires novel and effective technologies to mitigate the presence of dyes in wastewaters before discharging into the environment. Dyes usually remain stable in the presence of a variety of chemicals, light and are recalcitrant to microbial degradation. Among available technologies the biological treatments offer environmentally friendly strategies for decolorizing and detoxifying these compounds. The recent discovery of versatile laccases in streptomycetes opens up new opportunities for their commercial application. The aim of this study is to assess the potential of a novel bacterial laccase SilA produced by Streptomyces ipomoeae CECT 3341 active over wide temperature and pH ranges for use as an eco-friendly, biological treatment for the degradation of textile dyes. Insights into the enhancement of the oxidative action of this enzyme through the use of natural redox mediators are presented together with an assessment of the potential toxicity of the degradation products. Our results confirm that the combination of the laccase and natural mediators such as acetosyringone and methyl syringate enhanced the decolorization and detoxification of a variety of textile dyes up to sixfold and 20-fold, respectively. Mediator concentration was found to have a significant effect (p < 0.05) on dye decolorization at 60 °C; thus, the decolorization of Acid Orange 63 increased from 6 to 70-fold when the mediator concentration was increased from 0.1 to 0.5 mM. Further, the toxicity of tartrazine decreased 36-fold when the SilA-MeS system was used to decolorize the dye. The thermal properties of the SilA coupled with the stability of SilA at high pH suggest a potential commercial application for use in the decolorization of textile wastewaters which generally are performed at high temperature (>55 °C) and salinity and neutral pH, conditions which are unfavourable for conventional fungal laccases.

Improving kinetic or thermodynamic stability of an azoreductase by directed evolution.

Protein stability arises from a combination of factors which are often difficult to rationalise. Therefore its improvement is better addressed through directed evolution than by rational design approaches. In this study, five rounds of mutagenesis/recombination followed by high-throughput screening (≈10,000 clones) yielded the hit 1B6 showing a 300-fold higher half life at 50°C than that exhibited by the homodimeric wild type PpAzoR azoreductase from Pseudomonas putida MET94. The characterization using fluorescence, calorimetry and light scattering shows that 1B6 has a folded state slightly less stable than the wild type (with lower melting and optimal temperatures) but in contrast is more resistant to irreversible denaturation. The superior kinetic stability of 1B6 variant was therefore related to an increased resistance of the unfolded monomers to aggregation through the introduction of mutations that disturbed hydrophobic patches and increased the surface net charge of the protein. Variants 2A1 and 2A1-Y179H with increased thermodynamic stability (10 to 20°C higher melting temperature than wild type) were also examined showing the distinctive nature of mutations that lead to improved structural robustness: these occur in residues that are mostly involved in strengthening the solvent-exposed loops or the inter-dimer interactions of the folded state.

The kinetic role of carboxylate residues in the proximity of the trinuclear centre in the O2 reactivity of CotA-laccase.

Multicopper oxidases catalyze the four-electron reduction of dioxygen to water without the release of any reactive oxygen intermediate species. The role of carboxylate residue Asp116 located at the exit channel for water molecules of CotA-laccase has been investigated by site-saturation mutagenesis. A total of 300 clones was picked and screened for activity. Five variant enzymes, D116E, D116A, D116N, D116T and D116L, were selected for further characterisation. Spectroscopic analysis revealed only small perturbations in the geometry of the catalytic Cu sites of variants. However, a severe drop in turnover numbers (k(cat)) and downshifts by approximately 1-2 units of the optimal pH were observed for the oxidation of substrates, as compared with the wild type. The kinetics of formation and decay of peroxide intermediate (PI) was studied in type 1 depleted (T1D) CotA-laccase and in T1D-D116 or T1D-E498 mutants, previously shown to be involved in the mechanism of dioxygen reduction. It is noteworthy that CotA shows 10 times lower rates of PI formation and 10(3) higher PI decay rates as compared with other studied multicopper oxidases. The generation of PI is pH independent and mostly unaffected by the D116 or E498 mutations. In contrast, the decay of PI is markedly compromised by the replacement of D116 or E498 with non-carboxylate residues. The E498 residue appears to be the main protonable species for acceleration of PI decay at low pH. The D116 residue seems to be essential in the modulation of E498 protonation and in assisting protons to hydroxyl groups bound to the T2 Cu.

Expression system of CotA-laccase for directed evolution and high-throughput screenings for the oxidation of high-redox potential dyes.

Laccases are useful biocatalysts for many diverse biotechnological applications. In this study we have established efficient and reliable expression systems and high-throughput screenings for the recombinant CotA-laccase from Bacillus subtilis. The expression levels of cotA-laccase were compared in five different Escherichia coli host strains growing in 96-well microtiter plates under different culture conditions. Lower coefficients of variance (around 15%) were achieved using crude cell lysates of BL21 and KRX host strains growing under microaerobic conditions. Reproducible high-throughput screenings for the decolorization of high redox potential azo and anthraquinonic dyes were developed and optimized for identification of variants with increased redox potential. The enzymatic assays developed were tested for the screening of one mutant library from CotA-laccase created by error-prone PCR.