The Experts below are selected from a list of 1413 Experts worldwide ranked by ideXlab platform
Kenneth L Sale - One of the best experts on this subject based on the ideXlab platform.
-
understanding factors controlling depolymerization and polymerization in catalytic degradation of β ether linked model lignin compounds by Versatile Peroxidase
Green Chemistry, 2017Co-Authors: Jijiao Zeng, Matthew J L Mills, Blake A. Simmons, Kenneth L Sale, Michael S. KentAbstract:Lignin is a major component of lignocellulosic biomass and is responsible for much of its recalcitrant nature. Enzymatic breakdown of lignin into valuable products potentially represents an additional revenue stream in biofuels production. Many enzymes have been characterized which perform oxidative catalysis of lignin decomposition. However, the nature of the decomposition products from a given enzyme-catalyzed reaction depends on competition between depolymerization of lignin and repolymerization of the resulting depolymerization products, resulting in either polymeric products or small, aromatic species. The latter have greater value, as aromatic monomers can be used as precursors in the production of fuels and specialty chemicals via chemical or synthetic biological routes. An understanding of the factors that control the equilibrium between depolymerization and polymerization remains elusive. In this study we investigated this equilibrium for a Versatile Peroxidase from B. adusta using several lignin model compounds containing β-ether bonds as substrates and characterized the effects of reaction conditions (pH, addition of H2O2 and mediators) on catalysis. In tandem, quantum chemistry calculations of free energy changes of relevant chemical reactions and of electron spin density distributions of radical species were performed. Due to the low oxidation potential of the neutral radical, this enzyme is unable to oxidize non-phenolic lignin subunits. The results indicate that for phenolic lignin dimers the Versatile Peroxidase first produces a neutral radical via oxidation of the 4-OH position, followed by polymerization and depolymerization reactions. Selection between polymerization and depolymerization reaction pathways was found to be dependent on the functional group at the 5 position of the guaiacyl group (G5). In the case of a hydrogen atom at the G5 position (guaiacylglycerol-β-ether), the unpaired electron is distributed between the 4-OH and G5 positions, resulting in polymerization. However, substitution of G5 with a methoxy group (S-O-4) results in roughly equal distribution of the unpaired electron at G1 and 4-OH, leading to extensive side chain cleavage. The degradation pathway of phenolic β-O-4 was identified as Cα-aryl cleavage rather than Cα–Cβ.
-
understanding factors controlling depolymerization and polymerization in catalytic degradation of β ether linked model lignin compounds by Versatile Peroxidase
Green Chemistry, 2017Co-Authors: Jijiao Zeng, Matthew J L Mills, Blake A. Simmons, Kenneth L Sale, Michael S. KentAbstract:Lignin is a major component of lignocellulosic biomass and is responsible for much of its recalcitrant nature. Enzymatic breakdown of lignin into valuable products potentially represents an additional revenue stream in biofuels production. Many enzymes have been characterized which perform oxidative catalysis of lignin decomposition. However, the nature of the decomposition products from a given enzyme-catalyzed reaction depends on competition between depolymerization of lignin and repolymerization of the resulting depolymerization products, resulting in either polymeric products or small, aromatic species. The latter have greater value, as aromatic monomers can be used as precursors in the production of fuels and specialty chemicals via chemical or synthetic biological routes. An understanding of the factors that control the equilibrium between depolymerization and polymerization remains elusive. In this study we investigated this equilibrium for a Versatile Peroxidase from B. adusta using several lignin model compounds containing β-ether bonds as substrates and characterized the effects of reaction conditions (pH, addition of H2O2 and mediators) on catalysis. In tandem, quantum chemistry calculations of free energy changes of relevant chemical reactions and of electron spin density distributions of radical species were performed. Due to the low oxidation potential of the neutral radical, this enzyme is unable to oxidize non-phenolic lignin subunits. The results indicate that for phenolic lignin dimers the Versatile Peroxidase first produces a neutral radical via oxidation of the 4-OH position, followed by polymerization and depolymerization reactions. Selection between polymerization and depolymerization reaction pathways was found to be dependent on the functional group at the 5 position of the guaiacyl group (G5). In the case of a hydrogen atom at the G5 position (guaiacylglycerol-β-ether), the unpaired electron is distributed between the 4-OH and G5 positions, resulting in polymerization. However, substitution of G5 with a methoxy group (S-O-4) results in roughly equal distribution of the unpaired electron at G1 and 4-OH, leading to extensive side chain cleavage. The degradation pathway of phenolic β-O-4 was identified as Cα-aryl cleavage rather than Cα–Cβ.
Ángel T. Martínez - One of the best experts on this subject based on the ideXlab platform.
-
Unveiling the basis of alkaline stability of an evolved Versatile Peroxidase.
Biochemical Journal, 2016Co-Authors: Verónica Sáez-jiménez, Eva Garcia-ruiz, Ángel T. Martínez, Miguel Alcalde, Antonio A. Romero, Francisco J. Medrano, Sandra Acebes, Victor Guallar, Francisco J. Ruiz-dueñasAbstract:A variant of high biotechnological interest (called 2-1B) was obtained by directed evolution of the Pleurotus eryngii VP expressed in Saccharomyces cerevisiae (Garcia-Ruiz et al. Biochem. J. 441, 487, 2012). 2-1B shows seven mutations in the mature protein that resulted in improved functional expression, activity and thermostability, along with a remarkable stronger alkaline stability (it retains 60% of the initial activity after 120 h incubation at pH 9 vs complete inactivation of the native enzyme after only 1 h). The latter is highly demanded for biorefinery applications. Here we investigate the structural basis behind the enhanced alkaline stabilization of this evolved enzyme. In order to do this, several VP variants containing one or several of the mutations present in 2-1B were designed, and their alkaline stability and biochemical properties determined. In addition, the crystal structures of 2-1B and one of the intermediate variants (both expressed in Escherichia coli ) were solved and carefully analyzed, and molecular dynamics simulations were carried out. We concluded that the introduction of three basic residues in VP (Lys-37, Arg-39 and Arg-330) led to new connections at the heme-helix B (where the distal histidine is located) interface, and formation of new electrostatic interactions that avoided the hexacoordination of the heme iron. These new structural determinants stabilized the heme and its environment, helping to maintain the structural enzyme integrity (with pentacoordinated heme iron) under alkaline conditions. Moreover, the reinforcement of the solvent-exposed area around Gln-305 in the proximal side, prompted by the Q202L mutation, further enhanced the stability.
-
Correction: Alkaline Versatile Peroxidase by directed evolution
Catalysis Science & Technology, 2016Co-Authors: David Gonzalez-perez, Ivan Mateljak, Eva Garcia-ruiz, Francisco J. Ruiz-dueñas, Ángel T. Martínez, Miguel AlcaldeAbstract:Correction for ‘Alkaline Versatile Peroxidase by directed evolution’ by David Gonzalez-Perez et al., Catal. Sci. Technol., 2016, 6, 6625–6636.
-
Alkaline Versatile Peroxidase by directed evolution
Catalysis Science & Technology, 2016Co-Authors: David Gonzalez-perez, Ivan Mateljak, Eva Garcia-ruiz, Francisco J. Ruiz-dueñas, Ángel T. Martínez, Miguel AlcaldeAbstract:Ligninolytic Peroxidases are involved in natural wood decay in strict acid environments. Despite their biotechnological interest, these high-redox potential enzymes are not functional at basic pH due to the loss of calcium ions that affects their structural integrity. In this study, we have built catalytic activity at basic pH in a Versatile Peroxidase (VP) previously engineered for thermostability. By using laboratory evolution and hybrid approaches, we designed an active and highly stable alkaline VP while the catalytic bases behind the alkaline activation were unveiled. A stabilizing mutational backbone allowed the pentacoordinated heme state to be maintained, and the new alkaline mutations hyperactivated the enzyme after incubation at basic pHs. The final mutant oxidises substrates at alkaline pHs both at the heme channel and at the Mn2+ site, while the catalytic tryptophan was not operational under these conditions. Mutations identified in this work could be transferred to other ligninolytic Peroxidases for alkaline activation.
-
Improving the Ph-Stability of Versatile Peroxidase by Comparative Structural Analysis with a Naturally-Stable Manganese Peroxidase.
PLOS ONE, 2015Co-Authors: Verónica Sáez-jiménez, Ángel T. Martínez, Antonio A. Romero, Elena Fernández-fueyo, Francisco J. Medrano, Francisco J. Ruiz-dueñasAbstract:Versatile Peroxidase (VP) from the white-rot fungus Pleurotus eryngii is a high redox potential Peroxidase of biotechnological interest able to oxidize a wide range of recalcitrant substrates including lignin, phenolic and non-phenolic aromatic compounds and dyes. However, the relatively low stability towards pH of this and other fungal Peroxidases is a drawback for their industrial application. A strategy based on the comparative analysis of the crystal structures of VP and the highly pH-stable manganese Peroxidase (MnP4) from Pleurotus ostreatus was followed to improve the VP pH stability. Several interactions, including hydrogen bonds and salt bridges, and charged residues exposed to the solvent were identified as putatively contributing to the pH stability of MnP4. The eight amino acid residues responsible for these interactions and seven surface basic residues were introduced into VP by directed mutagenesis. Furthermore, two cysteines were also included to explore the effect of an extra disulfide bond stabilizing the distal Ca2+ region. Three of the four designed variants were crystallized and new interactions were confirmed, being correlated with the observed improvement in pH stability. The extra hydrogen bonds and salt bridges stabilized the heme pocket at acidic and neutral pH as revealed by UV-visible spectroscopy. They led to a VP variant that retained a significant percentage of the initial activity at both pH 3.5 (61% after 24 h) and pH 7 (55% after 120 h) compared with the native enzyme, which was almost completely inactivated. The introduction of extra solvent-exposed basic residues and an additional disulfide bond into the above variant further improved the stability at acidic pH (85% residual activity at pH 3.5 after 24 h when introduced separately, and 64% at pH 3 when introduced together). The analysis of the results provides a rational explanation to the pH stability improvement achieved.
-
Structural Determinants of Oxidative Stabilization in an Evolved Versatile Peroxidase
ACS Catalysis, 2014Co-Authors: David Gonzalez-perez, Eva Garcia-ruiz, Francisco J. Ruiz-dueñas, Ángel T. Martínez, Miguel AlcaldeAbstract:Versatile Peroxidases (VP) are promiscuous biocatalysts with the highest fragility to hydroperoxides yet reported due to a complex molecular architecture, with three catalytic sites and several oxidation pathways. To improve the VP resistance to H2O2, an evolved version of this enzyme was subjected to a range of directed evolution and hybrid strategies in Saccharomyces cerevisiae. After five generations of random, saturation, and domain mutagenesis, together with in vivo DNA recombination, several structural determinants behind the oxidative destabilization of the enzyme were unmasked. To establish a balance between activity and stability, selected beneficial mutations were introduced into novel mutational environments by the in vivo exchange of sequence blocks, promoting epistatic interactions. The best variant of this process accumulated 8 mutations that increased the half-life of the protein from 3 (parental type) to 35 min in the presence of 3000 equiv of H2O2 and with a 6 °C upward shift in thermosta...
Jijiao Zeng - One of the best experts on this subject based on the ideXlab platform.
-
understanding factors controlling depolymerization and polymerization in catalytic degradation of β ether linked model lignin compounds by Versatile Peroxidase
Green Chemistry, 2017Co-Authors: Jijiao Zeng, Matthew J L Mills, Blake A. Simmons, Kenneth L Sale, Michael S. KentAbstract:Lignin is a major component of lignocellulosic biomass and is responsible for much of its recalcitrant nature. Enzymatic breakdown of lignin into valuable products potentially represents an additional revenue stream in biofuels production. Many enzymes have been characterized which perform oxidative catalysis of lignin decomposition. However, the nature of the decomposition products from a given enzyme-catalyzed reaction depends on competition between depolymerization of lignin and repolymerization of the resulting depolymerization products, resulting in either polymeric products or small, aromatic species. The latter have greater value, as aromatic monomers can be used as precursors in the production of fuels and specialty chemicals via chemical or synthetic biological routes. An understanding of the factors that control the equilibrium between depolymerization and polymerization remains elusive. In this study we investigated this equilibrium for a Versatile Peroxidase from B. adusta using several lignin model compounds containing β-ether bonds as substrates and characterized the effects of reaction conditions (pH, addition of H2O2 and mediators) on catalysis. In tandem, quantum chemistry calculations of free energy changes of relevant chemical reactions and of electron spin density distributions of radical species were performed. Due to the low oxidation potential of the neutral radical, this enzyme is unable to oxidize non-phenolic lignin subunits. The results indicate that for phenolic lignin dimers the Versatile Peroxidase first produces a neutral radical via oxidation of the 4-OH position, followed by polymerization and depolymerization reactions. Selection between polymerization and depolymerization reaction pathways was found to be dependent on the functional group at the 5 position of the guaiacyl group (G5). In the case of a hydrogen atom at the G5 position (guaiacylglycerol-β-ether), the unpaired electron is distributed between the 4-OH and G5 positions, resulting in polymerization. However, substitution of G5 with a methoxy group (S-O-4) results in roughly equal distribution of the unpaired electron at G1 and 4-OH, leading to extensive side chain cleavage. The degradation pathway of phenolic β-O-4 was identified as Cα-aryl cleavage rather than Cα–Cβ.
-
understanding factors controlling depolymerization and polymerization in catalytic degradation of β ether linked model lignin compounds by Versatile Peroxidase
Green Chemistry, 2017Co-Authors: Jijiao Zeng, Matthew J L Mills, Blake A. Simmons, Kenneth L Sale, Michael S. KentAbstract:Lignin is a major component of lignocellulosic biomass and is responsible for much of its recalcitrant nature. Enzymatic breakdown of lignin into valuable products potentially represents an additional revenue stream in biofuels production. Many enzymes have been characterized which perform oxidative catalysis of lignin decomposition. However, the nature of the decomposition products from a given enzyme-catalyzed reaction depends on competition between depolymerization of lignin and repolymerization of the resulting depolymerization products, resulting in either polymeric products or small, aromatic species. The latter have greater value, as aromatic monomers can be used as precursors in the production of fuels and specialty chemicals via chemical or synthetic biological routes. An understanding of the factors that control the equilibrium between depolymerization and polymerization remains elusive. In this study we investigated this equilibrium for a Versatile Peroxidase from B. adusta using several lignin model compounds containing β-ether bonds as substrates and characterized the effects of reaction conditions (pH, addition of H2O2 and mediators) on catalysis. In tandem, quantum chemistry calculations of free energy changes of relevant chemical reactions and of electron spin density distributions of radical species were performed. Due to the low oxidation potential of the neutral radical, this enzyme is unable to oxidize non-phenolic lignin subunits. The results indicate that for phenolic lignin dimers the Versatile Peroxidase first produces a neutral radical via oxidation of the 4-OH position, followed by polymerization and depolymerization reactions. Selection between polymerization and depolymerization reaction pathways was found to be dependent on the functional group at the 5 position of the guaiacyl group (G5). In the case of a hydrogen atom at the G5 position (guaiacylglycerol-β-ether), the unpaired electron is distributed between the 4-OH and G5 positions, resulting in polymerization. However, substitution of G5 with a methoxy group (S-O-4) results in roughly equal distribution of the unpaired electron at G1 and 4-OH, leading to extensive side chain cleavage. The degradation pathway of phenolic β-O-4 was identified as Cα-aryl cleavage rather than Cα–Cβ.
María Jesús Martínez - One of the best experts on this subject based on the ideXlab platform.
-
Versatile Peroxidase as a valuable tool for generating new biomolecules by homogeneous and heterogeneous cross-linking.
Enzyme and Microbial Technology, 2013Co-Authors: Davinia Salvachúa, Ángel T. Martínez, María Jesús Martínez, Alicia Prieto, Maija-liisa Mattinen, Tarja Tamminen, Tiina Liitiä, Martina Lille, Stefan Willför, Craig B. FauldsAbstract:The modification and generation of new biomolecules intended to give higher molecular-mass species for biotechnological purposes, can be achieved by enzymatic cross-linking. The Versatile Peroxidase (VP) from Pleurotus eryngii is a high redox-potential enzyme with oxidative activity on a wide variety of substrates. In this study, VP was successfully used to catalyze the polymerization of low molecular mass compounds, such as lignans and peptides, as well as larger macromolecules, such as protein and complex polysaccharides. Different analytical, spectroscopic, and rheological techniques were used to determine structural changes and/or variations of the physicochemical properties of the reaction products. The lignans secoisolariciresinol and hydroxymatairesinol were condensed by VP forming up to 8 unit polymers in the presence of organic co-solvents and Mn(2+). Moreover, 11 unit of the peptides YIGSR and VYV were homogeneously cross-linked. The heterogeneous cross-linking of one unit of the peptide YIGSR and several lignan units was also achieved. VP could also induce gelation of feruloylated arabinoxylan and the polymerization of β-casein. These results demonstrate the efficacy of VP to catalyze homo- and hetero-condensation reactions, and reveal its potential exploitation for polymerizing different types of compounds.
-
two oxidation sites for low redox potential substrates a directed mutagenesis kinetic and crystallographic study on pleurotus eryngii Versatile Peroxidase
Journal of Biological Chemistry, 2012Co-Authors: M P Morales, Ángel T. Martínez, María Jesús Martínez, Maria J. Maté, Antonio A. Romero, Francisco J RuizduenasAbstract:Abstract Versatile Peroxidase shares with manganese Peroxidase and lignin Peroxidase the ability to oxidize Mn2+ and high redox potential aromatic compounds, respectively. Moreover, it is also able to oxidize phenols (and low redox potential dyes) at two catalytic sites, as shown by biphasic kinetics. A high efficiency site (with 2,6-dimethoxyphenol and p-hydroquinone catalytic efficiencies of ∼70 and ∼700 s−1 mm−1, respectively) was localized at the same exposed Trp-164 responsible for high redox potential substrate oxidation (as shown by activity loss in the W164S variant). The second site, characterized by low catalytic efficiency (∼3 and ∼50 s−1 mm−1 for 2,6-dimethoxyphenol and p-hydroquinone, respectively) was localized at the main heme access channel. Steady-state and transient-state kinetics for oxidation of phenols and dyes at the latter site were improved when side chains of residues forming the heme channel edge were removed in single and multiple variants. Among them, the E140G/K176G, E140G/P141G/K176G, and E140G/W164S/K176G variants attained catalytic efficiencies for oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) at the heme channel similar to those of the exposed tryptophan site. The heme channel enlargement shown by x-ray diffraction of the E140G, P141G, K176G, and E140G/K176G variants would allow a better substrate accommodation near the heme, as revealed by the up to 26-fold lower Km values (compared with native VP). The resulting interactions were shown by the x-ray structure of the E140G-guaiacol complex, which includes two H-bonds of the substrate with Arg-43 and Pro-139 in the distal heme pocket (at the end of the heme channel) and several hydrophobic interactions with other residues and the heme cofactor.
-
A new strain of Bjerkandera sp. production, purification and characterization of Versatile Peroxidase
World Journal of Microbiology and Biotechnology, 2010Co-Authors: Roberto Taboada-puig, Thelmo A. Lu-chau, Gumersindo Feijoo, María Jesús Martínez, Maria Teresa Moreira, Juan M. LemaAbstract:The lignin modifying enzymes (LMEs) secreted by a new white rot fungus isolated from Chile were studied in this work. This fungus has been identified as a new anamorph of Bjerkandera sp. based on the sequences of the ribosomal DNA and morphological analysis at light microscopy showing hyaline hyphae without clamp connection, cylindrical conidia and lack of sexual forms, similar to those reported in other Bjerkandera anamorphs. The characterization of the culture medium for the highest LMEs production was performed in flask cultures, with a formulation of the culture medium containing high levels of glucose and peptone. The highest Mn-oxidizing Peroxidase activity (1,400 U/L) was achieved on day 6 in Erlenmeyer flasks. Four Peroxidases (named R1B1, R1B2, R1B3 and R1B4), have been purified by using ion-exchange and exclusion molar chromatographies. All of them showed typical activity on Mn2+ and exhibited Mn-independent activity against 2,6-dimethoxyphenol. R1B4 showed also activity on veratryl alcohol (pH 3) indicating that this enzyme belongs to the Versatile Peroxidase family. The high VP production capacities of this strain, as well as the enzymatic characteristics of the LMEs suggest that it may be successfully used in the degradation of recalcitrant compounds.
-
Protein Radicals in Fungal Versatile Peroxidase CATALYTIC TRYPTOPHAN RADICAL IN BOTH COMPOUND I AND COMPOUND II AND STUDIES ON W164Y, W164H, AND W164S VARIANTS
Journal of Biological Chemistry, 2009Co-Authors: Francisco J. Ruiz-dueñas, Rebecca Pogni, Riccardo Basosi, María Jesús Martínez, Stefania Giansanti, María Del Puerto Morales, Maria J. Maté, Antonio A. Romero, Ángel T. MartínezAbstract:Abstract Lignin-degrading Peroxidases, a group of biotechnologically interesting enzymes, oxidize high redox potential aromatics via an exposed protein radical. Low temperature EPR of Pleurotus eryngii Versatile Peroxidase (VP) revealed, for the first time in a fungal Peroxidase, the presence of a tryptophanyl radical in both the two-electron (VPI) and the one-electron (VPII) activated forms of the enzyme. Site-directed mutagenesis was used to substitute this tryptophan (Trp-164) by tyrosine and histidine residues. No changes in the crystal structure were observed, indicating that the modified behavior was due exclusively to the mutations introduced. EPR revealed the formation of tyrosyl radicals in both VPI and VPII of the W164Y variant. However, no protein radical was detected in the W164H variant, whose VPI spectrum indicated a porphyrin radical identical to that of the inactive W164S variant. Stopped-flow spectrophotometry showed that the W164Y mutation reduced 10-fold the apparent second-order rate constant for VPI reduction (k2app) by veratryl alcohol (VA), when compared with over 50-fold reduction in W164S, revealing some catalytic activity of the tyrosine radical. Its first-order rate constant (k2) was more affected than the dissociation constant (KD2). Moreover, VPII reduction by VA was impaired by the above mutations, revealing that the Trp-164 radical was involved in catalysis by both VPI and VPII. The low first-order rate constant (k3) values were similar for the W164Y, W164H, and W164S variants, indicating that the tyrosyl radical in VPII was not able to oxidize VA (in contrast with that observed for VPI). VPII self-reduction was also suppressed, revealing that Trp-164 is involved in this autocatalytic process.
-
Substrate oxidation sites in Versatile Peroxidase and other basidiomycete Peroxidases
Journal of Experimental Botany, 2009Co-Authors: Francisco J. Ruiz-dueñas, María Jesús Martínez, María Del Puerto Morales, Eva García, Yuta Miki, Ángel T. MartínezAbstract:Versatile Peroxidase (VP) is defined by its capabilities to oxidize the typical substrates of other basidiomycete Peroxidases: (i) Mn 2+ , the manganese Peroxidase (MnP) substrate (Mn 3+ being able to oxidize phenols and initiate lipid peroxidation reactions); (ii) veratryl alcohol (VA), the typical lignin Peroxidase (LiP) substrate; and (iii) simple phenols, which are the substrates of Coprinopsis cinerea Peroxidase (CIP). Crystallographic, spectroscopic, directed mutagenesis, and kinetic studies showed that these ‘hybrid’ properties are due to the coexistence in a single protein of different catalytic sites reminiscent of those present in the other basidiomycete Peroxidase families. Crystal structures of wild and recombinant VP, and kinetics of mutated variants, revealed certain differences in its Mnoxidation site compared with MnP. These result in efficient Mn 2+ oxidation in the presence of only two of the three acidic residues forming its binding site. On the other hand, a solvent-exposed tryptophan is the catalytically-active residue in VA oxidation, initiating an electron transfer pathway to haem (two other putative pathways were discarded by mutagenesis). Formation of a tryptophanyl radical after VP activation by peroxide was detected using electron paramagnetic resonance. This was the first time that a protein radical was directly demonstrated in a ligninolytic Peroxidase. In contrast with LiP, the VP catalytic tryptophan is not b-hydroxylated under hydrogen peroxide excess. It was also shown that the tryptophan environment affected catalysis, its modification introducing some LiP properties in VP. Moreover, some phenols and dyes are oxidized by VP at the edge of the main haem access channel, as found in CIP. Finally, the biotechnological interest of VP is discussed.
Francisco J. Ruiz-dueñas - One of the best experts on this subject based on the ideXlab platform.
-
Unveiling the basis of alkaline stability of an evolved Versatile Peroxidase.
Biochemical Journal, 2016Co-Authors: Verónica Sáez-jiménez, Eva Garcia-ruiz, Ángel T. Martínez, Miguel Alcalde, Antonio A. Romero, Francisco J. Medrano, Sandra Acebes, Victor Guallar, Francisco J. Ruiz-dueñasAbstract:A variant of high biotechnological interest (called 2-1B) was obtained by directed evolution of the Pleurotus eryngii VP expressed in Saccharomyces cerevisiae (Garcia-Ruiz et al. Biochem. J. 441, 487, 2012). 2-1B shows seven mutations in the mature protein that resulted in improved functional expression, activity and thermostability, along with a remarkable stronger alkaline stability (it retains 60% of the initial activity after 120 h incubation at pH 9 vs complete inactivation of the native enzyme after only 1 h). The latter is highly demanded for biorefinery applications. Here we investigate the structural basis behind the enhanced alkaline stabilization of this evolved enzyme. In order to do this, several VP variants containing one or several of the mutations present in 2-1B were designed, and their alkaline stability and biochemical properties determined. In addition, the crystal structures of 2-1B and one of the intermediate variants (both expressed in Escherichia coli ) were solved and carefully analyzed, and molecular dynamics simulations were carried out. We concluded that the introduction of three basic residues in VP (Lys-37, Arg-39 and Arg-330) led to new connections at the heme-helix B (where the distal histidine is located) interface, and formation of new electrostatic interactions that avoided the hexacoordination of the heme iron. These new structural determinants stabilized the heme and its environment, helping to maintain the structural enzyme integrity (with pentacoordinated heme iron) under alkaline conditions. Moreover, the reinforcement of the solvent-exposed area around Gln-305 in the proximal side, prompted by the Q202L mutation, further enhanced the stability.
-
Correction: Alkaline Versatile Peroxidase by directed evolution
Catalysis Science & Technology, 2016Co-Authors: David Gonzalez-perez, Ivan Mateljak, Eva Garcia-ruiz, Francisco J. Ruiz-dueñas, Ángel T. Martínez, Miguel AlcaldeAbstract:Correction for ‘Alkaline Versatile Peroxidase by directed evolution’ by David Gonzalez-Perez et al., Catal. Sci. Technol., 2016, 6, 6625–6636.
-
Alkaline Versatile Peroxidase by directed evolution
Catalysis Science & Technology, 2016Co-Authors: David Gonzalez-perez, Ivan Mateljak, Eva Garcia-ruiz, Francisco J. Ruiz-dueñas, Ángel T. Martínez, Miguel AlcaldeAbstract:Ligninolytic Peroxidases are involved in natural wood decay in strict acid environments. Despite their biotechnological interest, these high-redox potential enzymes are not functional at basic pH due to the loss of calcium ions that affects their structural integrity. In this study, we have built catalytic activity at basic pH in a Versatile Peroxidase (VP) previously engineered for thermostability. By using laboratory evolution and hybrid approaches, we designed an active and highly stable alkaline VP while the catalytic bases behind the alkaline activation were unveiled. A stabilizing mutational backbone allowed the pentacoordinated heme state to be maintained, and the new alkaline mutations hyperactivated the enzyme after incubation at basic pHs. The final mutant oxidises substrates at alkaline pHs both at the heme channel and at the Mn2+ site, while the catalytic tryptophan was not operational under these conditions. Mutations identified in this work could be transferred to other ligninolytic Peroxidases for alkaline activation.
-
Improving the Ph-Stability of Versatile Peroxidase by Comparative Structural Analysis with a Naturally-Stable Manganese Peroxidase.
PLOS ONE, 2015Co-Authors: Verónica Sáez-jiménez, Ángel T. Martínez, Antonio A. Romero, Elena Fernández-fueyo, Francisco J. Medrano, Francisco J. Ruiz-dueñasAbstract:Versatile Peroxidase (VP) from the white-rot fungus Pleurotus eryngii is a high redox potential Peroxidase of biotechnological interest able to oxidize a wide range of recalcitrant substrates including lignin, phenolic and non-phenolic aromatic compounds and dyes. However, the relatively low stability towards pH of this and other fungal Peroxidases is a drawback for their industrial application. A strategy based on the comparative analysis of the crystal structures of VP and the highly pH-stable manganese Peroxidase (MnP4) from Pleurotus ostreatus was followed to improve the VP pH stability. Several interactions, including hydrogen bonds and salt bridges, and charged residues exposed to the solvent were identified as putatively contributing to the pH stability of MnP4. The eight amino acid residues responsible for these interactions and seven surface basic residues were introduced into VP by directed mutagenesis. Furthermore, two cysteines were also included to explore the effect of an extra disulfide bond stabilizing the distal Ca2+ region. Three of the four designed variants were crystallized and new interactions were confirmed, being correlated with the observed improvement in pH stability. The extra hydrogen bonds and salt bridges stabilized the heme pocket at acidic and neutral pH as revealed by UV-visible spectroscopy. They led to a VP variant that retained a significant percentage of the initial activity at both pH 3.5 (61% after 24 h) and pH 7 (55% after 120 h) compared with the native enzyme, which was almost completely inactivated. The introduction of extra solvent-exposed basic residues and an additional disulfide bond into the above variant further improved the stability at acidic pH (85% residual activity at pH 3.5 after 24 h when introduced separately, and 64% at pH 3 when introduced together). The analysis of the results provides a rational explanation to the pH stability improvement achieved.
-
Structural Determinants of Oxidative Stabilization in an Evolved Versatile Peroxidase
ACS Catalysis, 2014Co-Authors: David Gonzalez-perez, Eva Garcia-ruiz, Francisco J. Ruiz-dueñas, Ángel T. Martínez, Miguel AlcaldeAbstract:Versatile Peroxidases (VP) are promiscuous biocatalysts with the highest fragility to hydroperoxides yet reported due to a complex molecular architecture, with three catalytic sites and several oxidation pathways. To improve the VP resistance to H2O2, an evolved version of this enzyme was subjected to a range of directed evolution and hybrid strategies in Saccharomyces cerevisiae. After five generations of random, saturation, and domain mutagenesis, together with in vivo DNA recombination, several structural determinants behind the oxidative destabilization of the enzyme were unmasked. To establish a balance between activity and stability, selected beneficial mutations were introduced into novel mutational environments by the in vivo exchange of sequence blocks, promoting epistatic interactions. The best variant of this process accumulated 8 mutations that increased the half-life of the protein from 3 (parental type) to 35 min in the presence of 3000 equiv of H2O2 and with a 6 °C upward shift in thermosta...
