Rational design of ligninolytic peroxidases

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Biológicas, leída el 14-12-2015Fac. de Ciencias BiológicasTRUEunpu

Role of surface tryptophan for peroxidase oxidation of nonphenolic lignin

Background: Despite claims as key enzymes in enzymatic delignification, very scarce information on the reaction rates between the ligninolytic versatile peroxidase (VP) and lignin peroxidase (LiP) and the lignin polymer is available, due to methodological difficulties related to lignin heterogeneity and low solubility.Results: Two water-soluble sulfonated lignins (from Picea abies and Eucalyptus grandis) were chemically characterized and used to estimate single electron-transfer rates to the H2O2-activated Pleurotus eryngii VP (native enzyme and mutated variant) transient states (compounds I and II bearing two- and one-electron deficiencies, respectively). When the rate-limiting reduction of compound II was quantified by stopped-flow rapid spectrophotometry, from fourfold (softwood lignin) to over 100-fold (hardwood lignin) lower electron-transfer efficiencies (k 3app values) were observed for the W164S variant at surface Trp164, compared with the native VP. These lignosulfonates have ~20–30 % phenolic units, which could be responsible for the observed residual activity. Therefore, methylated (and acetylated) samples were used in new stopped-flow experiments, where negligible electron transfer to the W164S compound II was found. This revealed that the residual reduction of W164S compound II by native lignin was due to its phenolic moiety. Since both native lignins have a relatively similar phenolic moiety, the higher W164S activity on the softwood lignin could be due to easier access of its mono-methoxylated units for direct oxidation at the heme channel in the absence of the catalytic tryptophan. Moreover, the lower electron transfer rates from the derivatized lignosulfonates to native VP suggest that peroxidase attack starts at the phenolic lignin moiety. In agreement with the transient-state kinetic data, very low structural modification of lignin, as revealed by size-exclusion chromatography and two-dimensional nuclear magnetic resonance, was obtained during steady-state treatment (up to 24 h) of native lignosulfonates with the W164S variant compared with native VP and, more importantly, this activity disappeared when nonphenolic lignosulfonates were used.Conclusions: We demonstrate for the first time that the surface tryptophan conserved in most LiPs and VPs (Trp164 of P. eryngii VPL) is strictly required for oxidation of the nonphenolic moiety, which represents the major and more recalcitrant part of the lignin polymer

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Diseño racional de peroxidasas ligninolíticas Rational design of ligninolytic peroxidases

212 p.-36 fig.-10 tab.-anexo.[EN] Ligninolytic peroxidases are enzymes of biotechnological interest due to their ability to oxidize high redox potential aromatic compounds, including the recalcitrant lignin polymer. They are produced by white-rot fungi, the main organisms being able to mineralize lignin in nature. Lignin is the second most abundant polymer in nature. Due to its aromatic and heterogeneous nature, it is very recalcitrant and its degradation is difficult. White-rot fungi secrete a battery of oxidoreductases to the extracellular medium that, together with low molecular redox mediators, attack the lignin and achieve its degradation. Ligninolytic peroxidases play a key role in this process oxidizing the aromatic units of lignin. Ligninolytic peroxidases can be classified into three families: lignin peroxidases (LiP), manganese peroxidases (MnP) and versatile peroxidases (VP). VPs are characterized by the presence of three catalytic sites: i) the catalytic tryptophan, a solvent exposed residue that forms a protein radical trough a long range electron transfer from the heme, able to oxidize high redox potential compounds; ii) the manganese oxidation site, able to oxidize Mn2+ to Mn3+, which acts as a diffusible oxidizer, and iii) the main heme access channel where low redox potential compounds are oxidized. Among the different ligninolytic peroxidases, VP shows a special interest since it gathers the catalytic properties of LiP, MnP and generic peroxidases and, in addition, the enzyme does not need redox mediators to develop its activity. For these reasons, the VP from Pleurotus eryngii (isoenzyme VPL2) was the enzyme selected and studied in this thesis.The above catalytic properties allow VP to oxidize not only lignin but also other aromatic phenolic and non-phenolic (high or low redox potential) compounds, dyes and pesticides. The oxidative reactions VP and other ligninolytic peroxidases catalyze led to the break of lignin bonds or to the formation of new ones by radical condensation, or even indirectly to oxygenation reactions. All these reactions are of interest from a biotechnological point of view. On the other hand, ligninolytic peroxidases use hydrogen peroxide as substrate, which is cheap and of easy acquisition. Therefore, the use of these enzymes as biocatalysts in different industrial processes is very attractive as a sustainable and ecofriendly alternative to the conventional chemical methods. However, in spite of the high potential of ligninolytic peroxidases, they are not applicable as they are produced by natural organisms, since they do not present a proper selectivity and/or compatibility with the conditions under which the industrial processes are carried out. For these reasons, an optimization of the catalytic properties and stability of these enzymes is necessary previous to their biotechnological use. Among the drawbacks that limit the application of these peroxidases, the relative low stability towards pH, temperature and H2O2, or the insufficient production levels are found. The low stability of ligninolytic peroxidases towards H2O2 (their natural co-substrate) is one of the reasons delaying the development of applications based on these fungal enzymes. H2O2 is involved in enzyme inactivation by a mechanism-based process described as a suicide inactivation. This irreversible oxidative inactivation is produced in absence of reducing substrates or when the enzyme is exposed to a high excess of peroxide, and it is a consequence of multiple oxidization events affecting different components of the enzyme, including amino acids and the porphyrin ring. The relatively low pH stability of this and other fungal peroxidases is another drawback for their application. Under alkaline or even neutral conditions, ligninolytic peroxidases lose the structural calcium ions, which leads to a relaxation of the structure and the hexacoordination of the heme iron, resulting in an inactive enzyme. In a similar way, at acidic pH, the heme environment of these enzymes is affected being the interaction between the heme iron and the proximal histidine broken and the enzyme inactivated.On the other hand, although the catalytic properties of VP have been thoroughly studied, there are still catalytic aspects that are not clearly understood. Taking all this into account, studying the bases that regulate the stability and the catalytic properties of ligninolytic peroxidases is of interest for the future design of useful biocatalysts. 2. Aims The main aims of the present doctoral thesis were: i) the design of a VP with more adequate properties for its biotechnological application, and ii) the acquisition of an in-depth knowledge about the structural determinants that govern the catalysis and the stability of the enzyme. With this in mind, the following issues were addressed: i) The study of the oxidative inactivation of VP, and the factors that influence this process, as well as the design of a VP with improved H2O2-stability. ii) The design of a VP variant with optimized pH stability through the transfer of stabilizing structural motifs found in other peroxidases (Fernández-Fueyo et al. Biotechnology for Biofuels 2014, 7:2). iii) The analysis of the evolved variant 2-1B, obtained by directed evolution of VP (García-Ruiz et al. Biochem. J. (2012) 441, 487– 498), to determine which structural determinants generated by the mutations introduced improved its catalytic and stability properties. iv) To obtain an in-depth knowledge of the function of the catalytic tryptophan of VP, characterize the electron transfer between the peroxidase and the lignin, and study the ability of VP to oxidize technical lignins. Thanks to the structural-function information existing about VP and other ligninolytic peroxidases, and the availability of the crystal structure of VP, the optimization of VP using a rational approach has been possible. In this way, several VP variants were designed and produced and their properties were evaluated.The oxidative inactivation of VP by H2O2 was studied and different strategies were evaluated with the aim of improving its H2O2 stability. The studies performed demonstrated that VP is easily inactivated by H2O2 and that, during the inactivation process, the four methionine residues of VP are oxidized to methionine derivatives. Substitution of 3.1 Study and improvement of the oxidative stability of VP these residues, located in a sensitive region of the enzyme (near the heme cofactor and the catalytic tryptophan), rendered a variant with a 7.8-fold decreased oxidative inactivation rate. A second strategy was developed consisting in mutating two residues (Thr45 and Ile103) near the catalytic distal histidine (His47) with the aim of modifying the reactivity of the enzyme with H2O2 and consequently, slowing down the inactivation process. The T45A/I103T variant showed a 2.9-fold slower reaction rate with H2O2 and 2.8-fold enhanced oxidative stability. Finally, both strategies were combined in the T45A/I103T/M152F/M262F/M265L variant, whose stability in the presence of H2O2 was improved 11.7-fold. This variant showed an increased half-life, over 30 min compared with 3.4 min of the native enzyme, under an excess of 2000 equivalents of H2O2. Interestingly, the stability improvement achieved was related with the slower formation, subsequent stabilization and slower bleaching of the enzyme Compound III (FeIII-O2.-), a peroxidase intermediate that is not part of the catalytic cycle and leads to the inactivation of the enzyme.[ES] Las pLaeroxidasas ligninolíticas son enzimas que tienen una gran potencial biotecnológico debido a sus propiedades catalíticas que les permiten oxidar una gran variedad de sustratos recalcitrantes. Son producidas por los hongos de podredumbre blanca, los principales responsables de la degradación de la lignina en la naturaleza. La lignina es el material aromático renovable más abundante en la naturaleza y, junto con la celulosa y la hemicelulosa, compone la biomasa lignocelulósica. Estructuralmente, es un polímero aromático muy heterogéneo, y recalcitrante. Para degradar la lignina, los hongos de podredumbre blanca secretan un conjunto de oxidorreductasas al medio, entre las que se encuentran las peroxidasas ligninolíticas, que juegan un papel central en este proceso. Las peroxidasas ligninolíticas son hemo peroxidasas de alto potencial redox. Se clasifican en tres familias: lignina peroxidasas (LiP), manganeso peroxidasas (MnP) y peroxidasas versátiles (VP). Las VP se caracterizan por la presencia de tres sitios catalíticos: i) un triptófano catalítico expuesto en superficie, que puede formar un radical de proteína a través de una ruta de transferencia electrónica desde el hemo y es capaz de oxidar compuestos de alto potencial redox; ii) el sitio de oxidación de manganeso, que le permite oxidar Mn2+ a Mn3+; y iii) el canal principal de acceso al hemo en el que se oxidan compuestos de bajo potencial redox. Entre las distintas peroxidasas ligninolíticas, la VP presenta un especial interés debido a que combina las propiedades catalíticas propias de LiP, MnP y las peroxidasas genéricas y, además, no necesita de la presencia de mediadores para su actuación. Por ello, la VP, en concreto la variante alélica VPL2 de Pleurotus eryngii, fue la peroxidasa escogida para realizar los trabajos de la presente tesis doctoral.Las propiedades catalíticas de la VP y demás peroxidasas ligninolíticas les permiten oxidar compuestos aromáticos fenólicos y no fenólicos, así como diferentes tintes industriales y pesticidas. Por ello, las reacciones de oxidación que llevan a cabo estas enzimas son interesantes desde un punto de vista biotecnológico. Además, estas peroxidasas tienen la ventaja de ser autosuficientes, en el sentido de que son activadas por H2O2, un oxidante de fácil adquisición. Por tanto, la utilización de peroxidasas ligninolíticas como biocatalizadores en diferentes procesos industriales representa una alternativa sostenible y respetuosa con el medio ambiente frente al uso de métodos químicos convencionales. Sin embargo, muy pocas de ellas se comercializan y su aplicación industrial es modesta. Esto es debido, principalmente, a que no son aplicables tal cual se producen en la naturaleza, ya que no presentan una adecuada selectividad y compatibilidad con los rigurosos procesos industriales. Por ello, algunas de sus propiedades han de ser optimizadas antes de su aplicación. La estabilidad frente a H2O2 (estabilidad oxidativa) es uno de los principales obstáculos a superar para el uso biotecnológico de las peroxidasas ligninolíticas. El H2O2, necesario para el funcionamiento de las peroxidasas, es un fuerte oxidante que tiene efectos deletéreos sobre estas enzimas. La inactivación por H2O2 es un proceso irreversible, especialmente importante cuando existe un exceso de peróxido o no existe sustrato reductor. Su mecanismo no se conoce por completo, aunque se sabe que es consecuencia de diferentes reacciones de oxidación que afectan a distintos componentes de la enzima, incluyendo aminoácidos y el anillo de porfirina del cofactor. La estabilidad a pH es otro de los factores clave que a menudo necesita ser optimizado. La inactivación de las peroxidasas ligninolíticas debida al pH alcalino, o incluso neutro, se produce como consecuencia de la pérdida de los calcios estructurales responsables del mantenimiento de la correcta conformación del bolsillo del hemo. La ausencia de los calcios conduce al colapso de la estructura, lo que lleva a que el hierro del hemo quede hexacoordinado y la enzima inactiva. De igual forma, estas enzimas también se inactivan a pH ácido, aunque en este caso como consecuencia de la rotura del enlace entre el hierro del hemo y la histidina proximal. Por otra parte, aunque las propiedades de la VP y las otras peroxidasas ligninolíticas están ampliamente estudiadas, todavía hay aspectos catalíticos que no se conocen claramente.Por todo ello, es necesario comprender las bases estructurales que determinan y regulan la estabilidad y catálisis de estas enzimas para que su optimización y empleo sea una opción real. Los principales objetivos de la presente tesis fueron: i) el diseño de una VP que presente unas características más adecuadas para su uso biotecnológico, y ii) la profundización en el conocimiento de las bases estructurales que regulan la catálisis y estabilidad de la VP. Para ello, se abordaron las siguientes tareas: i) El estudio de la estabilidad oxidativa de la VP y de las bases estructurales que determinan esta propiedad, así como el diseño de una variante mutada que presente mayor resistencia oxidativa. ii) El diseño de una variante de VP con mayor estabilidad a pH mediante la transferencia de motivos estabilizantes encontrados en peroxidasas relacionadas (Fernández-Fueyo et al. Biotechnology for Biofuels 2014, 7:2). iii) El análisis de la variante VP 2-1B obtenida por evolución dirigida (García-Ruiz et al. Biochem. J. (2012) 441, 487–498) con objeto de averiguar qué determinantes estructurales generados por las mutaciones introducidas regulan sus propiedades catalíticas y estabilidad mejoradas. iv) Profundizar en el conocimiento del funcionamiento del triptófano catalítico, estudiar la transferencia electrónica entre la peroxidasa y la lignina durante la oxidación de ésta, y mostrar la capacidad de la VP para oxidar ligninas técnicas. Para realizar estas tareas, se utilizó un enfoque racional y se diseñaron diferentes variantes mutadas, que posteriormente fueron evaluadas. El estudio de la estabilidad oxidativa de la VP demostró que la enzima se inactiva fácilmente por H2O2. Durante la inactivación, las cuatro metioninas presentes en la VP se oxidaron a derivados de metionina. Teniendo en cuenta la proximidad de estos residuos a los sitios catalíticos de la enzima es probable que su oxidación provoque una alteración en la conformación de la enzima con un efecto directo sobre su actividad catalítica y estabilidad. De acuerdo con estas evidencias, la primera estrategia utilizada para mejorar la estabilidad oxidativa de la VP consistió en el diseño de variantes en las que las metioninas fueron sustituidas por residuos más resistentes a la oxidación. Asimismo, se diseñó una segunda estrategia consistente en la modificación del entorno de la histidina distal (His47) mediante la introducción de las mutaciones T45A e I103T. Los dos aminoácidos mutados se localizan por encima del área ocupada por la histidina 47, que está implicada, junto con la arginina 43, en la ruptura heterolítica del H2O2 durante la activación de la enzima. Las mutaciones se seleccionaron con la intención de provocar cambios sutiles en la posición de His47 y Arg43 que lleven a la ralentización de la velocidad de reacción con el H2O2 y, como consecuencia, a una menor inactivación. Finalmente, las dos estrategias se combinaron en la variante quíntuple T45A/I103T/M152F/M262F/M265L.Ambas estrategias dieron lugar a variantes mutadas con mayor estabilidad oxidativa. Los mejores resultados se obtuvieron con la variante quíntuple, ya que su velocidad de inactivación por H2O2 disminuyó 11 veces. Además, en presencia de un exceso de 2000 equivalentes de H2O2, la vida media de esta variante se incrementó hasta los 30 minutos, comparados con los 5 minutos de la enzima nativa. Finalmente, la mejora de la estabilidad del compuesto III (FeIII-O2.-), un intermediario catalítico relacionado con la inactivación por H2O2, parece ser la causa de las mejoras observadas asociadas a estas mutaciones. El trabajo llevado a cabo en esta tesis ha permitido profundizar en el conocimiento del mecanismo catalítico de la VP así como avanzar en el conocimiento de las bases estructurales que regulan algunas de sus propiedades como la estabilidad oxidativa, alcalina o ácida. Asimismo, se obtuvieron variantes mutadas que presentaron una mayor estabilidad frente a peróxido y pH, que son algunos de los principales factores que actualmente dificultan el uso biotecnológico de estas peroxidasas. La mejora en la estabilidad a pH ácido es especialmente importante teniendo en cuenta que la VP presenta un pH óptimo ácido para la oxidación de compuestos aromáticos de alto potencial redox. Además, el éxito de la estrategia consistente en transferir motivos estructurales estabilizadores de unas peroxidasas a otras sugiere que esta metodología se podría usar para el diseño de biocatalizadores de interés. Por otra parte, se confirmó la capacidad de la VP para degradar lignosulfonatos y se estudió el patrón de degradación de estas ligninas producido por la enzima. Además, se puso de manifiesto la transferencia electrónica que se produce entre el polímero de lignina y la peroxidasa, que fue caracterizada cinéticamente en condiciones de flujo detenido (stopped flow) y se asoció a la presencia del triptófano 164, el cual forma una radical catalíticamente activo.Beca predoctoral de Formación de Personal Investigador (FPI, Ref. BES-2009-014246). Proyectos europeos (PEROXICATS, KBBE-2010-4-265397) y (INDOX, KBBE- 2013-613549). Proyectos del plan nacional de I+D+i (HIPOP, BIO2011-26694) y (NOESIS, BIO2014-56388-R).Peer reviewe

Genomic analysis in the search for oxidoreductases of industrial interest

3 p.Peer reviewe

Improving the oxidative stability of a high redox potential fungal peroxidase by rational design

17 p.-2 tab.-8 fig.Ligninolytic peroxidases are enzymes of biotechnological interest due to their ability to oxidize high redox potential aromatic compounds, including the recalcitrant lignin polymer. However, different obstacles prevent their use in industrial and environmental applications, including low stability towards their natural oxidizing-substrate H2O2. In this work, versatile peroxidase was taken as a model ligninolytic peroxidase, its oxidative inactivation by H2O2 was studied and different strategies were evaluated with the aim of improving H2O2 stability. Oxidation of the methionine residues was produced during enzyme inactivation by H2O2 excess. Substitution of these residues, located near the heme cofactor and the catalytic tryptophan, rendered a variant with a 7.8-fold decreased oxidative inactivation rate. A second strategy consisted in mutating two residues (Thr45 and Ile103) near the catalytic distal histidine with the aim of modifying the reactivity of the enzyme with H2O2. The T45A/I103T variant showed a 2.9-fold slower reaction rate with H2O2 and 2.8-fold enhanced oxidative stability. Finally, both strategies were combined in the T45A/I103T/M152F/M262F/M265L variant, whose stability in the presence of H2O2 was improved 11.7-fold. This variant showed an increased half-life, over 30 min compared with 3.4 min of the native enzyme, under an excess of 2000 equivalents of H2O2. Interestingly, the stability improvement achieved was related with slower formation, subsequent stabilization and slower bleaching of the enzyme Compound III, a peroxidase intermediate that is not part of the catalytic cycle and leads to the inactivation of the enzyme.This work was funded by the Commission of the European Communities through the INDOX project (KBBE-2013-7-613549, "Optimized oxidoreductases for medium and large scale industrial biotransformations"), and by the Spanish Ministerio de Economía y Competitividad (MINECO) through the HIPOP project (BIO2011-26694, “Screening and engineering of new high-redox-potential peroxidases”).Peer reviewe

Demonstration of lignin-to-peroxidase direct electron transfer: A transient-state kinetics, directed mutagenesis, EPR, and NMR study

14 páginas.-- 10 figuras.-- 1 tabla.-- 46 referencias.-- Author's Choice—Final version free via Creative Commons CC-BY license.Versatile peroxidase (VP) is a high redox-potential peroxidase of biotechnological interest that is able to oxidize phenolic and non-phenolic aromatics, Mn2 , and different dyes. The ability of VP from Pleurotus eryngii to oxidize water-soluble lignins (softwood and hardwood lignosulfonates) is demonstrated here by a combination of directed mutagenesis and spectroscopic techniques, among others. In addition, direct electron transfer between the peroxidase and the lignin macromolecule was kinetically characterized using stopped-flow spectrophotometry. VP variants were used to show that this reaction strongly depends on the presence of a solvent-exposed tryptophan residue (Trp-164). Moreover, the tryptophanyl radical detected by EPR spectroscopy of H2O2-activated VP (being absent from the W164S variant) was identified as catalytically active because it was reduced during lignosulfonate oxidation, resulting in the appearance of a lignin radical. The decrease of lignin fluorescence (excitation at 355 nm/emission at 400 nm) during VP treatment under steady-state conditions was accompanied by a decrease of the lignin (aromatic nuclei and side chains) signals in one-dimensional and two-dimensional NMR spectra, confirming the ligninolytic capabilities of the enzyme. Simultaneously, size-exclusion chromatography showed an increase of the molecular mass of the modified residual lignin, especially for the (low molecular mass) hardwood lignosulfonate, revealing that the oxidation products tend to recondense during the VP treatment. Finally, mutagenesis of selected residues neighboring Trp-164 resulted in improved apparent second-order rate constants for lignosulfonate reactions, revealing that changes in its protein environment (modifying the net negative charge and/or substrate accessibility/binding) can modulate the reactivity of the catalytic tryptophanThis work was supported by the INDOX (KBBE-2013-613549) European Union project, the HIPOP (BIO2011-26694), NOESIS (BIO2014-56388-R), and BIORENZYMERY (AGL2014-53730-R) projects of the Spanish Ministry of Economy and Competitiveness (MINECO) co-financed by FEDER funds, and the PRIN 2009-STNWX3 project of the Italian Ministry of Education, Universities and Research (MIUR).Supported by an FPI Fellowship of the Spanish MINECO.-- Supported by a contract of the CSIC project 201440E097.-- Supported by a Ramón y Cajal contract of the Spanish MINECOPeer reviewe