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Angel T Martinez - One of the best experts on this subject based on the ideXlab platform.
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Reaction mechanisms and applications of aryl-Alcohol Oxidase.
The Enzymes, 2020Co-Authors: Ana Serrano, Juan Carro, Angel T MartinezAbstract:Aryl-Alcohol Oxidases (AAO) constitute a family of FAD-containing enzymes, included in the glucose-methanol-choline Oxidase/dehydrogenase superfamily of proteins. They are commonly found in fungi, where their eco-physiological role is to produce hydrogen peroxide that activates ligninolytic perOxidases in white-rot (lignin-degrading) basidiomycetes or to trigger the Fenton reactions in brown-rot (carbohydrate-degrading) basidiomycetes. These enzymes catalyze the oxidation of a plethora of aromatic, and some aliphatic, polyunsaturated Alcohols bearing conjugated primary hydroxyl group. Besides, the enzymes show activity on the hydrated forms of the corresponding aldehydes. Some AAO features, such as the broad range of substrates that it can oxidize (with the only need of molecular oxygen as co-substrate) and its stereoselective mechanism, confer good properties to these enzymes as industrial biocatalysts. In fact, AAO can be used for different biotechnological applications, such as flavor synthesis, secondary Alcohol deracemization and oxidation of furfurals for the production of furandicarboxylic acid as a chemical building block. Also, AAO can participate in processes of interest in the wood biorefinery and textile industries as an auxiliary enzyme providing hydrogen peroxide to ligninolytic or dye-decolorizing perOxidases. Both rational design and directed molecular evolution have been employed to engineer AAO for some of the above biotechnological applications.
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Switching the substrate preference of fungal aryl-Alcohol Oxidase: towards stereoselective oxidation of secondary benzyl Alcohols
Catalysis Science & Technology, 2019Co-Authors: Ana Serrano, Juan Carro, Miguel Alcalde, Victor Guallar, Javier Viña-gonzalez, Ferran Sancho, Angel T MartinezAbstract:Oxidation of primary Alcohols by aryl-Alcohol Oxidase (AAO), a flavoenzyme that provides H2O2 to fungal perOxidases for lignin degradation in nature, is achieved by concerted hydroxyl proton transfer and stereoselective hydride abstraction from the pro-R benzylic position. In racemic secondary Alcohols, the R-hydrogen abstraction would result in the selective oxidation of the S-enantiomer to the corresponding ketone. This stereoselectivity of AAO may be exploited for enzymatic deracemization of chiral mixtures and isolation of R-enantiomers of industrial interest by switching the enzyme activity from primary to secondary Alcohols. A combination of computational simulations and mutagenesis has been used to produce AAO variants with increased activity on secondary Alcohols, using the already available F501A variant of Pleurotus eryngii AAO as a starting point. Adaptive-PELE simulations for the diffusion of (S)-1-(p-methoxyphenyl)-ethanol in this variant allowed Ile500 to be identified as one of the key residues with a higher number of contacts with the substrate during its transition from the solvent to the active site. Substitution of Ile500 produced more efficient variants for the oxidation of several secondary Alcohols, and the I500M/F501W double variant was able to fully oxidize (after 75 min) with high selectivity (ee >99%) the S-enantiomer of the model secondary aryl-Alcohol (±)-1-(p-methoxyphenyl)-ethanol, while the R-enantiomer remained unreacted.
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switching the substrate preference of fungal aryl Alcohol Oxidase towards stereoselective oxidation of secondary benzyl Alcohols
Catalysis Science & Technology, 2019Co-Authors: Ana Serrano, Juan Carro, Miguel Alcalde, Victor Guallar, Ferran Sancho, Javier Vinagonzalez, Angel T MartinezAbstract:Oxidation of primary Alcohols by aryl-Alcohol Oxidase (AAO), a flavoenzyme that provides H2O2 to fungal perOxidases for lignin degradation in nature, is achieved by concerted hydroxyl proton transfer and stereoselective hydride abstraction from the pro-R benzylic position. In racemic secondary Alcohols, the R-hydrogen abstraction would result in the selective oxidation of the S-enantiomer to the corresponding ketone. This stereoselectivity of AAO may be exploited for enzymatic deracemization of chiral mixtures and isolation of R-enantiomers of industrial interest by switching the enzyme activity from primary to secondary Alcohols. A combination of computational simulations and mutagenesis has been used to produce AAO variants with increased activity on secondary Alcohols, using the already available F501A variant of Pleurotus eryngii AAO as a starting point. Adaptive-PELE simulations for the diffusion of (S)-1-(p-methoxyphenyl)-ethanol in this variant allowed Ile500 to be identified as one of the key residues with a higher number of contacts with the substrate during its transition from the solvent to the active site. Substitution of Ile500 produced more efficient variants for the oxidation of several secondary Alcohols, and the I500M/F501W double variant was able to fully oxidize (after 75 min) with high selectivity (ee >99%) the S-enantiomer of the model secondary aryl-Alcohol (±)-1-(p-methoxyphenyl)-ethanol, while the R-enantiomer remained unreacted.
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Multiple implications of an active site phenylalanine in the catalysis of aryl-Alcohol Oxidase
Scientific reports, 2018Co-Authors: Juan Carro, Patricia Ferreira, Milagros Medina, Victor Guallar, Pep Amengual-rigo, Ferran Sancho, Angel T MartinezAbstract:Aryl-Alcohol Oxidase (AAO) has demonstrated to be an enzyme with a bright future ahead due to its biotechnological potential in deracemisation of chiral compounds, production of bioplastic precursors and other reactions of interest. Expanding our understanding on the AAO reaction mechanisms, through the investigation of its structure-function relationships, is crucial for its exploitation as an industrial biocatalyst. In this regard, previous computational studies suggested an active role for AAO Phe397 at the active-site entrance. This residue is located in a loop that partially covers the access to the cofactor forming a bottleneck together with two other aromatic residues. Kinetic and affinity spectroscopic studies, complemented with computational simulations using the recently developed adaptive-PELE technology, reveal that the Phe397 residue is important for product release and to help the substrates attain a catalytically relevant position within the active-site cavity. Moreover, removal of aromaticity at the 397 position impairs the oxygen-reduction activity of the enzyme. Experimental and computational findings agree very well in the timing of product release from AAO, and the simulations help to understand the experimental results. This highlights the potential of adaptive-PELE to provide answers to the questions raised by the empirical results in the study of enzyme mechanisms.
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aromatic stacking interactions govern catalysis in aryl Alcohol Oxidase
FEBS Journal, 2015Co-Authors: Patricia Ferreira, Juan Carro, Angel T Martinez, Kenneth W. Borrelli, Victor Guallar, Aitor Hernandezortega, Fatima Lucas, Beatriz Herguedas, Milagros MedinaAbstract:This is the peer reviewed version of the following article: [Ferreira, P., Hernandez-Ortega, A., Lucas, F., Carro, J., Herguedas, B., Borrelli, K. W., Guallar, V., Martinez, A. T. and Medina, M. (2015), Aromatic stacking interactions govern catalysis in aryl-Alcohol Oxidase. FEBS J, 282: 3091–3106. doi:10.1111/febs.13221], which has been published in final form at [10.1111/febs.13221]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving." http://olabout.wiley.com/WileyCDA/Section/id-820227.html The version posted may not be updated or replaced with the final published version (the Version of Record).
Juan Carro - One of the best experts on this subject based on the ideXlab platform.
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Reaction mechanisms and applications of aryl-Alcohol Oxidase.
The Enzymes, 2020Co-Authors: Ana Serrano, Juan Carro, Angel T MartinezAbstract:Aryl-Alcohol Oxidases (AAO) constitute a family of FAD-containing enzymes, included in the glucose-methanol-choline Oxidase/dehydrogenase superfamily of proteins. They are commonly found in fungi, where their eco-physiological role is to produce hydrogen peroxide that activates ligninolytic perOxidases in white-rot (lignin-degrading) basidiomycetes or to trigger the Fenton reactions in brown-rot (carbohydrate-degrading) basidiomycetes. These enzymes catalyze the oxidation of a plethora of aromatic, and some aliphatic, polyunsaturated Alcohols bearing conjugated primary hydroxyl group. Besides, the enzymes show activity on the hydrated forms of the corresponding aldehydes. Some AAO features, such as the broad range of substrates that it can oxidize (with the only need of molecular oxygen as co-substrate) and its stereoselective mechanism, confer good properties to these enzymes as industrial biocatalysts. In fact, AAO can be used for different biotechnological applications, such as flavor synthesis, secondary Alcohol deracemization and oxidation of furfurals for the production of furandicarboxylic acid as a chemical building block. Also, AAO can participate in processes of interest in the wood biorefinery and textile industries as an auxiliary enzyme providing hydrogen peroxide to ligninolytic or dye-decolorizing perOxidases. Both rational design and directed molecular evolution have been employed to engineer AAO for some of the above biotechnological applications.
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Switching the substrate preference of fungal aryl-Alcohol Oxidase: towards stereoselective oxidation of secondary benzyl Alcohols
Catalysis Science & Technology, 2019Co-Authors: Ana Serrano, Juan Carro, Miguel Alcalde, Victor Guallar, Javier Viña-gonzalez, Ferran Sancho, Angel T MartinezAbstract:Oxidation of primary Alcohols by aryl-Alcohol Oxidase (AAO), a flavoenzyme that provides H2O2 to fungal perOxidases for lignin degradation in nature, is achieved by concerted hydroxyl proton transfer and stereoselective hydride abstraction from the pro-R benzylic position. In racemic secondary Alcohols, the R-hydrogen abstraction would result in the selective oxidation of the S-enantiomer to the corresponding ketone. This stereoselectivity of AAO may be exploited for enzymatic deracemization of chiral mixtures and isolation of R-enantiomers of industrial interest by switching the enzyme activity from primary to secondary Alcohols. A combination of computational simulations and mutagenesis has been used to produce AAO variants with increased activity on secondary Alcohols, using the already available F501A variant of Pleurotus eryngii AAO as a starting point. Adaptive-PELE simulations for the diffusion of (S)-1-(p-methoxyphenyl)-ethanol in this variant allowed Ile500 to be identified as one of the key residues with a higher number of contacts with the substrate during its transition from the solvent to the active site. Substitution of Ile500 produced more efficient variants for the oxidation of several secondary Alcohols, and the I500M/F501W double variant was able to fully oxidize (after 75 min) with high selectivity (ee >99%) the S-enantiomer of the model secondary aryl-Alcohol (±)-1-(p-methoxyphenyl)-ethanol, while the R-enantiomer remained unreacted.
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switching the substrate preference of fungal aryl Alcohol Oxidase towards stereoselective oxidation of secondary benzyl Alcohols
Catalysis Science & Technology, 2019Co-Authors: Ana Serrano, Juan Carro, Miguel Alcalde, Victor Guallar, Ferran Sancho, Javier Vinagonzalez, Angel T MartinezAbstract:Oxidation of primary Alcohols by aryl-Alcohol Oxidase (AAO), a flavoenzyme that provides H2O2 to fungal perOxidases for lignin degradation in nature, is achieved by concerted hydroxyl proton transfer and stereoselective hydride abstraction from the pro-R benzylic position. In racemic secondary Alcohols, the R-hydrogen abstraction would result in the selective oxidation of the S-enantiomer to the corresponding ketone. This stereoselectivity of AAO may be exploited for enzymatic deracemization of chiral mixtures and isolation of R-enantiomers of industrial interest by switching the enzyme activity from primary to secondary Alcohols. A combination of computational simulations and mutagenesis has been used to produce AAO variants with increased activity on secondary Alcohols, using the already available F501A variant of Pleurotus eryngii AAO as a starting point. Adaptive-PELE simulations for the diffusion of (S)-1-(p-methoxyphenyl)-ethanol in this variant allowed Ile500 to be identified as one of the key residues with a higher number of contacts with the substrate during its transition from the solvent to the active site. Substitution of Ile500 produced more efficient variants for the oxidation of several secondary Alcohols, and the I500M/F501W double variant was able to fully oxidize (after 75 min) with high selectivity (ee >99%) the S-enantiomer of the model secondary aryl-Alcohol (±)-1-(p-methoxyphenyl)-ethanol, while the R-enantiomer remained unreacted.
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Multiple implications of an active site phenylalanine in the catalysis of aryl-Alcohol Oxidase
Scientific reports, 2018Co-Authors: Juan Carro, Patricia Ferreira, Milagros Medina, Victor Guallar, Pep Amengual-rigo, Ferran Sancho, Angel T MartinezAbstract:Aryl-Alcohol Oxidase (AAO) has demonstrated to be an enzyme with a bright future ahead due to its biotechnological potential in deracemisation of chiral compounds, production of bioplastic precursors and other reactions of interest. Expanding our understanding on the AAO reaction mechanisms, through the investigation of its structure-function relationships, is crucial for its exploitation as an industrial biocatalyst. In this regard, previous computational studies suggested an active role for AAO Phe397 at the active-site entrance. This residue is located in a loop that partially covers the access to the cofactor forming a bottleneck together with two other aromatic residues. Kinetic and affinity spectroscopic studies, complemented with computational simulations using the recently developed adaptive-PELE technology, reveal that the Phe397 residue is important for product release and to help the substrates attain a catalytically relevant position within the active-site cavity. Moreover, removal of aromaticity at the 397 position impairs the oxygen-reduction activity of the enzyme. Experimental and computational findings agree very well in the timing of product release from AAO, and the simulations help to understand the experimental results. This highlights the potential of adaptive-PELE to provide answers to the questions raised by the empirical results in the study of enzyme mechanisms.
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aromatic stacking interactions govern catalysis in aryl Alcohol Oxidase
FEBS Journal, 2015Co-Authors: Patricia Ferreira, Juan Carro, Angel T Martinez, Kenneth W. Borrelli, Victor Guallar, Aitor Hernandezortega, Fatima Lucas, Beatriz Herguedas, Milagros MedinaAbstract:This is the peer reviewed version of the following article: [Ferreira, P., Hernandez-Ortega, A., Lucas, F., Carro, J., Herguedas, B., Borrelli, K. W., Guallar, V., Martinez, A. T. and Medina, M. (2015), Aromatic stacking interactions govern catalysis in aryl-Alcohol Oxidase. FEBS J, 282: 3091–3106. doi:10.1111/febs.13221], which has been published in final form at [10.1111/febs.13221]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving." http://olabout.wiley.com/WileyCDA/Section/id-820227.html The version posted may not be updated or replaced with the final published version (the Version of Record).
Patricia Ferreira - One of the best experts on this subject based on the ideXlab platform.
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Multiple implications of an active site phenylalanine in the catalysis of aryl-Alcohol Oxidase
Scientific reports, 2018Co-Authors: Juan Carro, Patricia Ferreira, Milagros Medina, Victor Guallar, Pep Amengual-rigo, Ferran Sancho, Angel T MartinezAbstract:Aryl-Alcohol Oxidase (AAO) has demonstrated to be an enzyme with a bright future ahead due to its biotechnological potential in deracemisation of chiral compounds, production of bioplastic precursors and other reactions of interest. Expanding our understanding on the AAO reaction mechanisms, through the investigation of its structure-function relationships, is crucial for its exploitation as an industrial biocatalyst. In this regard, previous computational studies suggested an active role for AAO Phe397 at the active-site entrance. This residue is located in a loop that partially covers the access to the cofactor forming a bottleneck together with two other aromatic residues. Kinetic and affinity spectroscopic studies, complemented with computational simulations using the recently developed adaptive-PELE technology, reveal that the Phe397 residue is important for product release and to help the substrates attain a catalytically relevant position within the active-site cavity. Moreover, removal of aromaticity at the 397 position impairs the oxygen-reduction activity of the enzyme. Experimental and computational findings agree very well in the timing of product release from AAO, and the simulations help to understand the experimental results. This highlights the potential of adaptive-PELE to provide answers to the questions raised by the empirical results in the study of enzyme mechanisms.
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aromatic stacking interactions govern catalysis in aryl Alcohol Oxidase
FEBS Journal, 2015Co-Authors: Patricia Ferreira, Juan Carro, Angel T Martinez, Kenneth W. Borrelli, Victor Guallar, Aitor Hernandezortega, Fatima Lucas, Beatriz Herguedas, Milagros MedinaAbstract:This is the peer reviewed version of the following article: [Ferreira, P., Hernandez-Ortega, A., Lucas, F., Carro, J., Herguedas, B., Borrelli, K. W., Guallar, V., Martinez, A. T. and Medina, M. (2015), Aromatic stacking interactions govern catalysis in aryl-Alcohol Oxidase. FEBS J, 282: 3091–3106. doi:10.1111/febs.13221], which has been published in final form at [10.1111/febs.13221]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving." http://olabout.wiley.com/WileyCDA/Section/id-820227.html The version posted may not be updated or replaced with the final published version (the Version of Record).
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5-hydroxymethylfurfural conversion by fungal aryl-Alcohol Oxidase and unspecific peroxygenase.
The FEBS journal, 2015Co-Authors: Juan Carro, Patricia Ferreira, Alicia Prieto, Ana Gutierrez, Jesús Jiménez-barbero, Ana Serrano, Leonor Rodríguez, Beatriz Balcells, Ana Ardá, Rene UllrichAbstract:Oxidative conversion of 5-hydroxymethylfurfural (HMF) is of biotechnological interest for the production of renewable (lignocellulose-based) platform chemicals, such as 2,5-furandicarboxylic acid (FDCA). To the best of our knowledge, the ability of fungal aryl-Alcohol Oxidase (AAO) to oxidize HMF is reported here for the first time, resulting in almost complete conversion into 2,5-formylfurancarboxylic acid (FFCA) in a few hours. The reaction starts with Alcohol oxidation, yielding 2,5-diformylfuran (DFF), which is rapidly converted into FFCA by carbonyl oxidation, most probably without leaving the enzyme active site. This agrees with the similar catalytic efficiencies of the enzyme with respect to oxidization of HMF and DFF, and its very low activity on 2,5-hydroxymethylfurancarboxylic acid (which was not detected by GC-MS). However, AAO was found to be unable to directly oxidize the carbonyl group in FFCA, and only modest amounts of FDCA are formed from HMF (most probably by chemical oxidation of FFCA by the H2O2 previously generated by AAO). As aldehyde oxidation by AAO proceeds via the corresponding geminal diols (aldehyde hydrates), the various carbonyl oxidation rates may be related to the low degree of hydration of FFCA compared with DFF. The conversion of HMF was completed by introducing a fungal unspecific heme peroxygenase that uses the H2O2 generated by AAO to transform FFCA into FDCA, albeit more slowly than the previous AAO reactions. By adding this peroxygenase when FFCA production by AAO has been completed, transformation of HMF into FDCA may be achieved in a reaction cascade in which O2 is the only co-substrate required, and water is the only by-product formed.
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Substrate diffusion and oxidation in GMC oxidoreductases: an experimental and computational study on fungal aryl-Alcohol Oxidase
The Biochemical journal, 2011Co-Authors: Aitor Hernández-ortega, Patricia Ferreira, Angel T Martinez, Milagros Medina, Kenneth W. Borrelli, Victor GuallarAbstract:AAO (aryl-Alcohol Oxidase) provides H 2 O 2 in fungal degradation of lignin, a process of high biotechnological interest. The crystal structure of AAO does not show open access to the active site, where different aromatic Alcohols are oxidized. In the present study we investigated substrate diffusion and oxidation in AAO compared with the structurally related CHO (choline Oxidase). Cavity finder and ligand diffusion simulations indicate the substrate-entrance channel, requiring side-chain displacements and involving a stacking interaction with Tyr 92 . Mixed QM (quantum mechanics)/MM (molecular mechanics) studies combined with site-directed mutagenesis showed two active-site catalytic histidine residues, whose substitution strongly decreased both catalytic and transient-state reduction constants for p -anisyl Alcohol in the H502A (over 1800-fold) and H546A (over 35-fold) variants. Combination of QM/MM energy profiles, protonation predictors, molecular dynamics, mutagenesis and pH profiles provide a robust answer regarding the nature of the catalytic base. The histidine residue in front of the FAD ring, AAO His 502 (and CHO His 466 ), acts as a base. For the two substrates assayed, it was shown that proton transfer preceded hydride transfer, although both processes are highly coupled. No stable intermediate was observed in the energy profiles, in contrast with that observed for CHO. QM/MM, together with solvent KIE (kinetic isotope effect) results, suggest a non-synchronous concerted mechanism for Alcohol oxidation by AAO.
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Novel structural features in the GMC family of oxidoreductases revealed by the crystal structure of fungal aryl-Alcohol Oxidase
Acta Crystallographica Section D Biological Crystallography, 2009Co-Authors: Israel S. Fernández, Patricia Ferreira, Angel T Martinez, Francisco J. Ruiz-dueñas, Elena Santillana, Antonio A. RomeroAbstract:Lignin biodegradation, a key step in carbon recycling in land ecosystems, is carried out by white-rot fungi through an H(2)O(2)-dependent process defined as enzymatic combustion. Pleurotus eryngii is a selective lignin-degrading fungus that produces H(2)O(2) during redox cycling of p-anisylic compounds involving the secreted flavoenzyme aryl-Alcohol Oxidase (AAO). Here, the 2.4 A resolution X-ray crystal structure of this oxidoreductase, which catalyzes dehydrogenation reactions on various primary polyunsaturated Alcohols, yielding the corresponding aldehydes, is reported. The AAO crystal structure was solved by single-wavelength anomalous diffraction of a selenomethionine derivative obtained by Escherichia coli expression and in vitro folding. This monomeric enzyme is composed of two domains, the overall folding of which places it into the GMC (glucose-methanol-choline Oxidase) oxidoreductase family, and a noncovalently bound FAD cofactor. However, two additional structural elements exist in the surroundings of its active site that modulate the access of substrates; these are absent in the structure of the model GMC oxidoreductase glucose Oxidase. The folding of these novel elements gives rise to a funnel-like hydrophobic channel that connects the solvent region to the buried active-site cavity of AAO. This putative active-site cavity is located in front of the re side of the FAD isoalloxazine ring and near two histidines (His502 and His546) that could contribute to Alcohol activation as catalytic bases. Moreover, three aromatic side chains from two phenylalanines (Phe397 and Phe502) and one tyrosine (Tyr92) at the inner region of the channel form an aromatic gate that may regulate the access of the enzyme substrates to the active site as well as contribute to the recognition of the Alcohols that can effectively be oxidized by AAO.
Martínez, Ángel T. - One of the best experts on this subject based on the ideXlab platform.
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Sequential oxidation of 5-hydroxymethylfurfural to furan-2,5-dicarboxylic acid by an evolved aryl-Alcohol Oxidase
'Elsevier BV', 2021Co-Authors: Viña-gonzález Javier, Martínez, Ángel T., Guallar Víctor, Alcalde Galeote MiguelAbstract:[EN] Furan-2,5-dicarboxylic acid (FDCA) is a building block of biodegradable plastics that can be used to replace those derived from fossil carbon sources. In recent years, much interest has focused on the synthesis of FDCA from the bio-based 5-hydroxymethylfurfural (HMF) through a cascade of enzyme reactions. Aryl-Alcohol Oxidase (AAO) and 5-hydroxymethylfurfural Oxidase (HMFO) are glucose-methanol-choline flavoenzymes that may be used to produce FDCA from HMF through three sequential oxidations, and without the assistance of auxiliary enzymes. Such a challenging process is dependent on the degree of hydration of the original aldehyde groups and of those formed, the rate-limiting step lying in the final oxidation of the intermediate 5-formyl-furancarboxylic acid (FFCA) to FDCA. While HMFO accepts FFCA as a final substrate in the HMF reaction pathway, AAO is virtually incapable of oxidizing it. Here, we have engineered AAO to perform the stepwise oxidation of HMF to FDCA through its structural alignment with HMFO and directed evolution. With a 3-fold enhanced catalytic efficiency for HMF and a 6-fold improvement in overall conversion, this evolved AAO is a promising point of departure for further engineering aimed at generating an efficient biocatalyst to synthesize FDCA from HMF.This research was supported by the EU project H2020-BBI-PPP-2015-2-720297-ENZOX2, by the Spanish Government projects BIO2016-79106-R-Lignolution, and by the Comunidad de Madrid project Y2018/BIO4738-EVOCHIMERA
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Sequential oxidation of 5-hydroxymethylfurfural to furan-2,5-dicarboxylic acid by an evolved aryl-Alcohol Oxidase
'Elsevier BV', 2020Co-Authors: Viña-gonzález Javier, Martínez, Ángel T., Guallar Víctor, Alcalde MiguelAbstract:Furan-2,5-dicarboxylic acid (FDCA) is a building block of biodegradable plastics that can be used to replace those derived from fossil carbon sources. In recent years, much interest has focused on the synthesis of FDCA from the bio-based 5-hydroxymethylfurfural (HMF) through a cascade of enzyme reactions. Aryl-Alcohol Oxidase (AAO) and 5-hydroxymethylfurfural Oxidase (HMFO) are glucose-methanol-choline flavoenzymes that may be used to produce FDCA from HMF through three sequential oxidations, and without the assistance of auxiliary enzymes. Such a challenging process is dependent on the degree of hydration of the original aldehyde groups and of those formed, the rate-limiting step lying in the final oxidation of the intermediate 5-formyl-furancarboxylic acid (FFCA) to FDCA. While HMFO accepts FFCA as a final substrate in the HMF reaction pathway, AAO is virtually incapable of oxidizing it. Here, we have engineered AAO to perform the stepwise oxidation of HMF to FDCA through its structural alignment with HMFO and directed evolution. With a 3-fold enhanced catalytic efficiency for HMF and a 6-fold improvement in overall conversion, this evolved AAO is a promising point of departure for further engineering aimed at generating an efficient biocatalyst to synthesize FDCA from HMF.This research was supported by the EU project H2020-BBI-PPP-2015-2-720297-ENZOX2, by the Spanish Government projects BIO2016-79106-R-Lignolution, and by the Comunidad de Madrid project Y2018/BIO4738-EVOCHIMERA.Peer ReviewedPostprint (author's final draft
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Reaction mechanisms and applications of aryl-Alcohol Oxidase
'Elsevier BV', 2020Co-Authors: Serrano Ana, Carro Juan, Martínez, Ángel T.Abstract:26 p.-7 fig.-2 tab. The Enzymes (2020)Aryl-Alcohol Oxidases (AAO) constitute a family of FAD-containing enzymes, included in the glucose-methanol-choline Oxidase/dehydrogenase superfamily of proteins. They are commonly found in fungi, where their eco-physiological role is to produce hydrogen peroxide that activates ligninolytic perOxidases in white-rot (lignin-degrading) basidiomycetes or to trigger the Fenton reactions in brown-rot (carbohydrate-degrading) basidiomycetes. These enzymes catalyze the oxidation of a plethora of aromatic, and some aliphatic, polyunsaturated Alcohols bearing conjugated primary hydroxyl group. Besides, the enzymes show activity on the hydrated forms of the corresponding aldehydes. Some AAO features, such as the broad range of substrates that it can oxidize (with the only need of molecular oxygen as co-substrate) and its stereoselective mechanism, confer good properties to these enzymes as industrial biocatalysts. In fact, AAO can be used for different biotechnological applications, such as flavor synthesis, secondary Alcohol deracemization and oxidation of furfurals for the production of furandicarboxylic acid as a chemical building block. Also, AAO can participate in processes of interest in the wood biorefinery and textile industries as an auxiliary enzyme providing hydrogen peroxide to ligninolytic or dye-decolorizing perOxidases. Both rational design and directed molecular evolution have been employed to engineer AAO for some of the above biotechnological applications.This work has been supported by the EnzOx2 project (H2020-BBI-PPP-2015-2-720297,https://www.enzox2.eu) of the European BBI-JU (https://www.bbi-europe.eu), the GenoBioref project (BIO2017-86559-R) of the Spanish Ministry of Economy, Industry and Competitiveness, co-financed by FEDER funds, and the PIE201620E081 CSIC project.Peer reviewe
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Laboratory evolution platform for aryl Alcohol Oxidase in Saccharomyces cerevisiae
2019Co-Authors: Viña-gonzález Javier, Alcalde Galeote Miguel, González-pérez David, Martínez, Ángel T.Abstract:Trabajo presentado en el 3rd Multistep Enzyme Catalyzed Processes Congress, celebrado en Madrid (España) del 07 al 10 de abril de 2014.Among the ligninolytic oxidoreductases secreted by white-rot fungi, the aryl Alcohol Oxidase (AAO) plays an outstanding role as H2O2 supplying enzyme during lignin decay1. With high enantioselectivity and broad substrate specificity, this flavo-enzyme is also a promising departure point for directed evolution studies towards different biotechnological fates, but the lack of heterologous functional expression levels precludes further advances in the field. In this study, the native signal peptide of AAO from Pleurotus eryngii was replaced by those of the mating α-factor, the toxin K1 Killer, as well as combinations of pre- and pro-regions from both leaders to achieve secretion inSaccharomyces cerevisiae2,3. AAO expression in yeast was measured with the help of an ad-hoc colorimetric-dual HTS-protocol based on the detection of H2O2 with a chemical (FOX) and an enzymatic assay (ABTS-HRP). All constructs were successfully processed and secreted by yeast showing extracellular AAO activities with several aromatic Alcohols, which opens new paths for future developments.Peer Reviewe
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Complete oxidation of hydroxymethylfurfural to furandicarboxylic acid by aryl-Alcohol Oxidase
'Springer Science and Business Media LLC', 2019Co-Authors: Serrano-lotina, Ana M., Calviño Eva, Carro Juan, Sánchez-ruiz María, Cañada F. Javier, Martínez, Ángel T.Abstract:[Background] 5-Hydroxymethylfurfural (HMF) is a highly valuable platform chemical that can be obtained from plant biomass carbohydrates. HMF can be oxidized to 2,5-furandicarboxylic acid (FDCA), which is used as a renewable substitute for the petroleum-based terephthalic acid in polymer production.[Results] Aryl-Alcohol Oxidase (AAO) from the white-rot fungus Pleurotus eryngii is able to oxidize HMF and its derivative 2,5-diformylfuran (DFF) producing formylfurancarboxylic acid (FFCA) thanks to its activity on benzylic Alcohols and hydrated aldehydes. Here, we report the ability of AAO to produce FDCA from FFCA, opening up the possibility of full oxidation of HMF by this model enzyme. During HMF reactions, an inhibitory effect of the H2O2 produced in the first two oxidation steps was found to be the cause of the lack of AAO activity on FFCA. In situ monitoring of the whole reaction by 1H-NMR confirmed the absence of any unstable dead-end products, undetected in the HPLC analyses, that could be responsible for the incomplete conversion. The deleterious effect of H2O2 was confirmed by successful HMF conversion into FDCA when the AAO reaction was carried out in the presence of catalase. On the other hand, no H2O2 formation was detected during the slow FFCA conversion by AAO in the absence of catalase, in contrast to typical Oxidase reaction with HMF and DFF, suggesting an alternative mechanism as reported in some reactions of related flavo-Oxidases. Moreover, several active-site AAO variants that yield nearly complete conversion in shorter reaction times than the wild-type enzyme have been identified.[Conclusions] The use of catalase to remove H2O2 from the reaction mixture leads to 99% conversion of HMF into FDCA by AAO and several improved variants, although the mechanism of peroxide inhibition of the AAO action on the aldehyde group of FFCA is not fully understood.We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI). This work has been funded by the H2020 BBI-JU (https://www.bbi-europe.eu) project EnzOx2 (H2020-BBI-PPP-2015-2-720297; https://www.enzox2.eu) and the GENOBIOREF (BIO2017-86559-R) and CTQ2015-64597-C2-2-P projects of the Spanish Ministry of Economy, Industry and Competitiveness, co-financed by FEDER funds.Peer reviewe
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Sequential oxidation of 5-hydroxymethylfurfural to furan-2,5-dicarboxylic acid by an evolved aryl-Alcohol Oxidase
'Elsevier BV', 2021Co-Authors: Viña-gonzález Javier, Martínez, Ángel T., Guallar Víctor, Alcalde Galeote MiguelAbstract:[EN] Furan-2,5-dicarboxylic acid (FDCA) is a building block of biodegradable plastics that can be used to replace those derived from fossil carbon sources. In recent years, much interest has focused on the synthesis of FDCA from the bio-based 5-hydroxymethylfurfural (HMF) through a cascade of enzyme reactions. Aryl-Alcohol Oxidase (AAO) and 5-hydroxymethylfurfural Oxidase (HMFO) are glucose-methanol-choline flavoenzymes that may be used to produce FDCA from HMF through three sequential oxidations, and without the assistance of auxiliary enzymes. Such a challenging process is dependent on the degree of hydration of the original aldehyde groups and of those formed, the rate-limiting step lying in the final oxidation of the intermediate 5-formyl-furancarboxylic acid (FFCA) to FDCA. While HMFO accepts FFCA as a final substrate in the HMF reaction pathway, AAO is virtually incapable of oxidizing it. Here, we have engineered AAO to perform the stepwise oxidation of HMF to FDCA through its structural alignment with HMFO and directed evolution. With a 3-fold enhanced catalytic efficiency for HMF and a 6-fold improvement in overall conversion, this evolved AAO is a promising point of departure for further engineering aimed at generating an efficient biocatalyst to synthesize FDCA from HMF.This research was supported by the EU project H2020-BBI-PPP-2015-2-720297-ENZOX2, by the Spanish Government projects BIO2016-79106-R-Lignolution, and by the Comunidad de Madrid project Y2018/BIO4738-EVOCHIMERA
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Evolved Peroxygenase-Aryl Alcohol Oxidase Fusions for Self-Sufficient Oxyfunctionalization Reactions
'American Chemical Society (ACS)', 2021Co-Authors: Gómez De Santos, Patricia, Lázaro Sofia, Viña-gonzález Javier, Hoang, Manh Dat, Sánchez-moreno Israel, Glieder Anton, Hollmann Frank, Alcalde Galeote MiguelAbstract:[EN] Fungal peroxygenases are deemed emergent biocatalysts for selective C¿H bond oxyfunctionalization reactions. In this study, we have engineered a functional and stable self-sufficient chimeric peroxygenase-Oxidase fusion. The bifunctional biocatalyst carried a laboratory-evolved version of the fungal peroxygenase fused to an evolved fungal aryl-Alcohol Oxidase that supplies H2O2in situ. Enzyme fusion libraries with peptide linkers of different sizes and amino acid compositions were designed, while attached leader sequences favored secretion in yeast. The most promising functional enzyme fusions were characterized biochemically and further tested for the synthesis of dextrorphan, a metabolite of the antitussive drug dextromethorphan. This reaction system was optimized to control the aromatic Alcohol transformation rate, and therefore the H2O2 supply, to achieve total turnover numbers of 62,000, the highest value reported for the biocatalytic synthesis of dextrorphan to date. Accordingly, our study opens an avenue for the use of peroxygenase-aryl Alcohol Oxidase fusions in the pharmaceutical and chemical sectors.This work was supported by the Comunidad de Madrid Synergy CAM Project Y2018/BIO-4738-EVOCHIMERA-CM, the Spanish Government Projects BIO2016-79106-R-Lignolution, PID2019-106166RB-100-OXYWAVE and the CSIC Project PIE-201580E042. P. Gomez de Santos is grateful to the Ministry of Science, Innovation and Universities (Spain) for her FPI contract (BES-2017- 080040)
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Sequential oxidation of 5-hydroxymethylfurfural to furan-2,5-dicarboxylic acid by an evolved aryl-Alcohol Oxidase
'Elsevier BV', 2020Co-Authors: Viña-gonzález Javier, Martínez, Ángel T., Guallar Víctor, Alcalde MiguelAbstract:Furan-2,5-dicarboxylic acid (FDCA) is a building block of biodegradable plastics that can be used to replace those derived from fossil carbon sources. In recent years, much interest has focused on the synthesis of FDCA from the bio-based 5-hydroxymethylfurfural (HMF) through a cascade of enzyme reactions. Aryl-Alcohol Oxidase (AAO) and 5-hydroxymethylfurfural Oxidase (HMFO) are glucose-methanol-choline flavoenzymes that may be used to produce FDCA from HMF through three sequential oxidations, and without the assistance of auxiliary enzymes. Such a challenging process is dependent on the degree of hydration of the original aldehyde groups and of those formed, the rate-limiting step lying in the final oxidation of the intermediate 5-formyl-furancarboxylic acid (FFCA) to FDCA. While HMFO accepts FFCA as a final substrate in the HMF reaction pathway, AAO is virtually incapable of oxidizing it. Here, we have engineered AAO to perform the stepwise oxidation of HMF to FDCA through its structural alignment with HMFO and directed evolution. With a 3-fold enhanced catalytic efficiency for HMF and a 6-fold improvement in overall conversion, this evolved AAO is a promising point of departure for further engineering aimed at generating an efficient biocatalyst to synthesize FDCA from HMF.This research was supported by the EU project H2020-BBI-PPP-2015-2-720297-ENZOX2, by the Spanish Government projects BIO2016-79106-R-Lignolution, and by the Comunidad de Madrid project Y2018/BIO4738-EVOCHIMERA.Peer ReviewedPostprint (author's final draft
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Evolved Peroxygenase-Aryl Alcohol Oxidase Fusions for Self-Sufficient Oxyfunctionalization Reactions
'American Chemical Society (ACS)', 2020Co-Authors: Gómez De Santos, Patricia, Lázaro Sofia, Viña-gonzález Javier, Hoang, Manh Dat, Sánchez-moreno Israel, Glieder Anton, Hollmann F., Alcalde MiguelAbstract:Fungal peroxygenases are deemed emergent biocatalysts for selective C-H bond oxyfunctionalization reactions. In this study, we have engineered a functional and stable self-sufficient chimeric peroxygenase-Oxidase fusion. The bifunctional biocatalyst carried a laboratory-evolved version of the fungal peroxygenase fused to an evolved fungal aryl-Alcohol Oxidase that supplies H2O2 in situ. Enzyme fusion libraries with peptide linkers of different sizes and amino acid compositions were designed, while attached leader sequences favored secretion in yeast. The most promising functional enzyme fusions were characterized biochemically and further tested for the synthesis of dextrorphan, a metabolite of the antitussive drug dextromethorphan. This reaction system was optimized to control the aromatic Alcohol transformation rate, and therefore the H2O2 supply, to achieve total turnover numbers of 62,000, the highest value reported for the biocatalytic synthesis of dextrorphan to date. Accordingly, our study opens an avenue for the use of peroxygenase-aryl Alcohol Oxidase fusions in the pharmaceutical and chemical sectors. Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work publicBT/Biocatalysi
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Laboratory evolution platform for aryl Alcohol Oxidase in Saccharomyces cerevisiae
2019Co-Authors: Viña-gonzález Javier, Alcalde Galeote Miguel, González-pérez David, Martínez, Ángel T.Abstract:Trabajo presentado en el 3rd Multistep Enzyme Catalyzed Processes Congress, celebrado en Madrid (España) del 07 al 10 de abril de 2014.Among the ligninolytic oxidoreductases secreted by white-rot fungi, the aryl Alcohol Oxidase (AAO) plays an outstanding role as H2O2 supplying enzyme during lignin decay1. With high enantioselectivity and broad substrate specificity, this flavo-enzyme is also a promising departure point for directed evolution studies towards different biotechnological fates, but the lack of heterologous functional expression levels precludes further advances in the field. In this study, the native signal peptide of AAO from Pleurotus eryngii was replaced by those of the mating α-factor, the toxin K1 Killer, as well as combinations of pre- and pro-regions from both leaders to achieve secretion inSaccharomyces cerevisiae2,3. AAO expression in yeast was measured with the help of an ad-hoc colorimetric-dual HTS-protocol based on the detection of H2O2 with a chemical (FOX) and an enzymatic assay (ABTS-HRP). All constructs were successfully processed and secreted by yeast showing extracellular AAO activities with several aromatic Alcohols, which opens new paths for future developments.Peer Reviewe