Expanding the Physiological Role of Aryl-Alcohol Flavooxidases as Quinone Reductases

Aryl-alcohol oxidases (AAOs) are members of the glucose-methanol-choline oxidase/dehydrogenase (GMC) superfamily. These extracellular flavoproteins have been described as auxiliary enzymes in the degradation of lignin by several white-rot basidiomycetes. In this context, they oxidize fungal secondary metabolites and lignin-derived compounds using O2 as an electron acceptor, and supply H2O2 to ligninolytic peroxidases. Their substrate specificity, including mechanistic aspects of the oxidation reaction, has been characterized in Pleurotus eryngii AAO, taken as a model enzyme of this GMC superfamily. AAOs show broad reducing-substrate specificity in agreement with their role in lignin degradation, being able to oxidize both nonphenolic and phenolic aryl alcohols (and hydrated aldehydes). In the present work, the AAOs from Pleurotus ostreatus and Bjerkandera adusta were heterologously expressed in Escherichia coli, and their physicochemical properties and oxidizing abilities were compared with those of the well-known recombinant AAO from P. eryngii. In addition, electron acceptors different from O2, such as p-benzoquinone and the artificial redox dye 2,6-Dichlorophenolindophenol, were also studied. Differences in reducing-substrate specificity were found between the AAO enzymes from B. adusta and the two Pleurotus species. Moreover, the three AAOs oxidized aryl alcohols concomitantly with the reduction of p-benzoquinone, with similar or even higher efficiencies than when using their preferred oxidizing-substrate, O2. IMPORTANCE In this work, quinone reductase activity is analyzed in three AAO flavooxidases, whose preferred oxidizing-substrate is O2. The results presented, including reactions in the presence of both oxidizing substrates—benzoquinone and molecular oxygen—suggest that such aryl-alcohol dehydrogenase activity, although less important than its oxidase activity in terms of maximal turnover, may have a physiological role during fungal decay of lignocellulose by the reduction of quinones (and phenoxy radicals) from lignin degradation, preventing repolymerization. Moreover, the resulting hydroquinones would participate in redox-cycling reactions for the production of hydroxyl free radical involved in the oxidative attack of the plant cell-wall. Hydroquinones can also act as mediators for laccases and peroxidases in lignin degradation in the form of semiquinone radicals, as well as activators of lytic polysaccharide monooxygenases in the attack of crystalline cellulose. Moreover, reduction of these, and other phenoxy radicals produced by laccases and peroxidases, promotes lignin degradation by limiting repolymerization reactions. These findings expand the role of AAO in lignin biodegradation

Protein dynamics promote hydride tunnelling in substrate oxidation by aryl-alcohol oxidase

The temperature dependence of hydride transfer from the substrate to the N5 of the FAD cofactor during the reductive half-reaction of Pleurotus eryngii aryl-alcohol oxidase (AAO) is assessed here. Kinetic isotope effects on both the pre-steady state reduction of the enzyme and its steady-state kinetics, with differently deuterated substrates, suggest an environmentally-coupled quantum-mechanical tunnelling process. Moreover, those kinetic data, along with the crystallographic structure of the enzyme in complex with a substrate analogue, indicate that AAO shows a pre-organized active site that would only require the approaching of the hydride donor and acceptor for the tunnelled transfer to take place. Modification of the enzyme's active-site architecture by replacement of Tyr92, a residue establishing hydrophobic interactions with the substrate analogue in the crystal structure, in the Y92F, Y92L and Y92W variants resulted in different temperature dependence patterns that indicated a role of this residue in modulating the transfer reaction. [imagen en resumen

Stepwise Hydrogen Atom and Proton Transfers in Dioxygen Reduction by Aryl-Alcohol Oxidase

The mechanism of dioxygen reduction by the flavoenzyme aryl-alcohol oxidase was investigated with kinetic isotope, viscosity, and pL (pH/pD) effects in rapid kinetics experiments by stopped-flow spectrophotometry of the oxidative half-reaction of the enzyme. Double mixing of the enzyme in a stopped-flow spectrophotometer with [a-2H2]-p-methoxybenzyl alcohol and oxygen at varying aging times established a slow rate constant of 0.0023 s-1 for the wash-out of the D atom from the N5 atom of the reduced flavin. Thus, the deuterated substrate could be used to probe the cleavage of the N-H bond of the reduced flavin in the oxidative half-reaction. A significant and pH-independent substrate kinetic isotope effect (KIE) of 1.5 between pH 5.0 and 8.0 demonstrated that H transfer is partially limiting the oxidative half-reaction of the enzyme; a negligible solvent KIE of 1.0 between pD 5.0 and 8.0 proved a fast H+ transfer reaction that does not contribute to determining the flavin oxidation rates. Thus, a mechanism for dioxygen reduction in which the H atom originating from the reduced flavin and a H+ from a solvent exchangeable site are transferred in separate kinetic steps is proposed. The spectroscopic and kinetic data presented also showed a lack of stabilization of transient flavin intermediates. The substantial differences in the mechanistic details of O2 reduction by aryl-alcohol oxidase with respect to other alcohol oxidases like choline oxidase, pyranose 2-oxidase, and glucose oxidase further demonstrate the high level of versatility of the flavin cofactor in flavoenzymes

Multiple implications of an active site phenylalanine in the catalysis of aryl-alcohol oxidase

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.This work was supported by the EnzOx2 project (H2020-BBI-PPP-2015-720297) of the European Joint Undertaking of Bio-based Industries (http://bbi-europe.eu), the INDOX project (KBBE-2013-7-613549) of the European Seventh Framework Programme, and the NOESIS (BIO2014-56388-R), vMutate (CTQ2016-79138-R) and FLADIMOTEC (BIO2016-75183-P) projects of the Spanish Ministry of Economy and Competitiveness. J.C. acknowledges a FPU fellowship (FPU2012-2041) from the Spanish Ministry of Education, Culture and Sports.Peer ReviewedPostprint (published version

Aportación al conocimiento del género Cheilanthes en la Península Ibérica

En esta nota se citan por primera vez en la Peninsula Ibérica Cheilanthes pulchella y Cheilanthes x insularis y se amplia la distribución de Cheilanthes guanchica y Cheilanthes x iberica. Se completa el catálogo de los táxones de este género en la Peninsula Ibérica, añadiéndose una clave de determinación para los mismos.Cheilanthes pulchella and Cheilanthes x insularis are mentioned as new from the Iberian Peninsula and distribution of Cheilanthes guanchica and Cheilanthes x iberica is amplied. The Cheilanthes taxa from the Iberian Peninsul1 are listed. Key of determination is proposed

Contribución al estudio de las comunidades rupícolas de la Cheilanthetalia Maranto-Maderensis y Androsacetali Vancellii en la Península Ibérica

Se lleva a cabo un estudio de las comunidades rupícolas ibéricas presididas por diversas especies de los géneros Cheilanthes y Notholaena, proponiéndose dos nuevas asociaciones de la alianza Phagnalo saxatile-Cheilanthion maderensis (Cheilanthetalia maranto-maderensis, Asplenietea trichomanis): Notholaeno marantae-Cheilanthetum guanchicae, propia del Subsector Bermejense (Sector Rondeño, Provincia Bética) y Cheilantho maderensis-Cosentinietum velleae, de distribución fundamentalmente bética. Con datos propios y otros bibliográficos, se hace un análisis crítico, a la vez que un estudio comparado, de las comunidades casmofíticas incluídas en los órdenes Cheilanthetalia maranto-maderensis (termomediterránea y silicíbasícola) y Androsacetalia vandellii (montana y supra-crioromediterránea, silicícola), analizando sus relaciones con las comunidades comofíticas de la Phagnalo-Rumicetea indurati. De esta última se describe la asociación Phalacrocarpo-Saxifragetum continentalis (Saxifragion continentalis).An study on the Iberian Peninsula rocks comunities it is going on with dominantes by several species of the genus Cheilanthes and Notholaena, from these the new associations of the Phagnalo saxatile-Cheilanthion maderensis alliance (Cheilanthetalia maranto-maderensis, Asplenietea trichomanis): Notholaeno marantae-Cheilanthetum guanchicae are proposed the first distinctive of the Bermejense Subsector (Rondeño Sector, Be- " tica chorological Province) and the second Cheilantho maderensis-Cosentinietum velleae with is basically of the Betic distribution. Based on the bibliography and on our own data, a comparative analysis of the casmofitic comunities on the Cheilanthetalia maranto-maderensis order and Androsacetalia vandellii order are presented, taking into account their relationships with Phagnalo-Rumicetea indurati. Forthermore from this last one the association Phalacrocarpo-Saxifragetum continentalis is also described

Switching the substrate preference of fungal aryl-alcohol oxidase: towards stereoselective oxidation of secondary benzyl alcohols

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.This work was supported by the INDOX (KBBE-2013-7-613549) EU project and by the BIO2017-86559-R (GenoBioref), CTQ2016-79138-R and BIO2016-79106-R projects of the Spanish Ministry of Economy, Industry and Competitiveness, cofinanced by FEDER funds. Pedro Merino (University of Zaragoza, Spain) is acknowledged for his suggestions on chiral HPLC analyses.Peer ReviewedAward-winningPostprint (published version

Multiple implications of an active site phenylalanine in the catalysis of aryl-alcohol oxidase

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 a nity 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 ndings 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

Memristor Emulator Circuit Design and Applications

This chapter introduces a design guide of memristor emulator circuits, from conceptual idea until experimental tests. Three topologies of memristor emulator circuits in their incremental and decremental versions are analysed and designed at low and high frequency. The behavioural model of each topology is derived and programmed at SIMULINK under the MATLAB environment. An offset compensation technique is also described in order to achieve the frequency-dependent pinched hysteresis loop that is on the origin and when the memristor emulator circuit is operating at high frequency. Furthermore, from these topologies, a technique to transform normal non-linear resistors to inverse non-linear resistors is also addressed. HSPICE numerical simulations for each topology are also shown. Finally, three real analogue applications based on memristors are analysed and explained at the behavioural level of abstraction

TreeDet: a web server to explore sequence space

The TreeDet (Tree Determinant) Server is the first release of a system designed to integrate results from methods that predict functional sites in protein families. These methods take into account the relation between sequence conservation and evolutionary importance. TreeDet fully analyses the space of protein sequences in either user-uploaded or automatically generated multiple sequence alignments. The methods implemented in the server represent three main classes of methods for the detection of family-dependent conserved positions, a tree-based method, a correlation based method and a method that employs a principal component analyses coupled to a cluster algorithm. An additional method is provided to highlight the reliability of the position in the alignments. The server is available at