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

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

Cyber-Physical Vulnerability Assessment in Smart Grids Based on Multilayer Complex Networks

This article belongs to the Special Issue Cybersecurity and Privacy-Preserving in Modern Smart GridIn the last decade, the main attacks against smart grids have occurred in communication networks (ITs) causing the disconnection of physical equipment from power networks (OTs) and leading to electricity supply interruptions. To deal with the deficiencies presented in past studies, this paper addresses smart grids vulnerability assessment considering the smart grid as a cyber-physical heterogeneous interconnected system. The model of the cyber-physical system is composed of a physical power network model and the information and communication technology network model (ICT) both are interconnected and are interrelated by means of the communication and control equipment installed in the smart grid. This model highlights the hidden interdependencies between power and ICT networks and contains the interaction between both systems. To mimic the real nature of smart grids, the interconnected heterogeneous model is based on multilayer complex network theory and scale-free graph, where there is a one-to-many relationship between cyber and physical assets. Multilayer complex network theory centrality indexes are used to determine the interconnected heterogeneous system set of nodes criticality. The proposed methodology, which includes measurement, communication, and control equipment, has been tested on a standardized power network that is interconnected to the ICT network. Results demonstrate the model’s effectiveness in detecting vulnerabilities in the interdependent cyber-physical system compared to traditional vulnerability assessments applied to power networks (OT).This research was funded by Fundación Iberdrola España, within the 2020 research support scholarship program

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

Phase Transitions in Two-Dimensional Traffic Flow Models

We introduce two simple two-dimensional lattice models to study traffic flow in cities. We have found that a few basic elements give rise to the characteristic phase diagram of a first-order phase transition from a freely moving phase to a jammed state, with a critical point. The jammed phase presents new transitions corresponding to structural transformations of the jam. We discuss their relevance in the infinite size limit.Comment: RevTeX 3.0 file. Figures available upon request to e-address [email protected] (or 'dopico' or 'molera' or 'anxo', same node

Differential proteomic analysis of the secretome of Irpex lacteus and other white-rot fungi during wheat straw pretreatment

BACKGROUND: Identifying new high-performance enzymes or enzyme complexes to enhance biomass degradation is the key for the development of cost-effective processes for ethanol production. Irpex lacteus is an efficient microorganism for wheat straw pretreatment, yielding easily hydrolysable products with high sugar content. Thus, this fungus was selected to investigate the enzymatic system involved in lignocellulose decay, and its secretome was compared to those from Phanerochaete chrysosporium and Pleurotus ostreatus which produced different degradation patterns when growing on wheat straw. Extracellular enzymes were analyzed through 2D-PAGE, nanoLC/MS-MS, and homology searches against public databases. RESULTS: In wheat straw, I. lacteus secreted proteases, dye-decolorizing and manganese-oxidizing peroxidases, and H(2)O(2) producing-enzymes but also a battery of cellulases and xylanases, excluding those implicated in cellulose and hemicellulose degradation to their monosaccharides, making these sugars poorly available for fungal consumption. In contrast, a significant increase of β-glucosidase production was observed when I. lacteus grew in liquid cultures. P. chrysosporium secreted more enzymes implicated in the total hydrolysis of the polysaccharides and P. ostreatus produced, in proportion, more oxidoreductases. CONCLUSION: The protein pattern secreted during I. lacteus growth in wheat straw plus the differences observed among the different secretomes, justify the fitness of I. lacteus for biopretreatment processes in 2G-ethanol production. Furthermore, all these data give insight into the biological degradation of lignocellulose and suggest new enzyme mixtures interesting for its efficient hydrolysis

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

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

Deciphering the unique structure and acylation pattern of Posidonia oceanica lignin

Copyright © 2020 American Chemical Society. Lignins from different parts of the seagrass Posidonia oceanica-namely sheaths, rhizome, and roots-as well as from fibrous balls from P. oceanica detritus were isolated and thoroughly characterized by pyrolysis coupled with gas chromatography/mass spectrometry, derivatization followed by reductive cleavage, two-dimensional nuclear magnetic resonance spectroscopy, and gel permeation chromatography. The lignins of P. oceanica were enriched in guaiacyl (G) over syringyl (S) units, with S/G ratios ranging from 0.1 (fibrous balls) to 0.5 (rhizome). β-O-4′ ethers and phenylcoumarans were the most abundant lignin substructures, followed by resinols, and minor amounts of dibenzodioxocins and spirodienones. Moreover, all lignins were found to be highly I-Acylated (up to 44% of total units), mainly with p-hydroxybenzoates but also, to a lesser extent, with acetates. The data indicated that this acylation extensively occurred in both G-and S-lignin units, contrary to what happens in palms, poplar, and willow, where p-hydroxybenzoates overwhelmingly appear at the I-position of S-units

Lipomatosis epidural lumbosacra idiopática: revisión de la bibliografía y caso clínico

La lipomatosis epidural es una afección que aparece generalmente en relación con hipercortisolismos endógenos o exógenos, siendo más raras las formas idiopáticas, de las que se han descrito únicamente 17 casos. Se presenta un nuevo caso con sintomatología compatible con estenosis de canal, y que tras estudios de imagen se demostró que correspondía con una lipomatosis epidural lumbosacra. Se le encomendó al paciente una reducción de peso, con lo que se alivió parcialmente la sintomatología, permaneciendo estable en la actualidad. Se revisa la bibliografía de este raro proceso, exponiendo los distintos hallazgos encontrados. Algunos autores proponen una reducción de peso, lo cual puede aliviar la sintomatología. Para casos rebeldes está indicada la laminectomía asociada al despegamiento de la grasa sin realizar exploración intradural.Epidural lipomatosis is an affection that is usually related to endogenic or exogenous hipercortisolism. Less frequently, it can be idiopathic. To date only 17 cases have been documented. A new case is presented in a patient suffering from symptoms resembling lumbar spinal stenosis. The radiological studies revealed a compression of the dural sac at lumbosacral levéis, caused by tissue similar in density to fat. Weight reduction, was recommended to the patients and a partial relief of symptoms was achieved. The authors reviewed the pathogeny of epidural lipomatosis and its various different treatments. In the idiopathic form, weight reduction is recomended first, reserving surgery for those cases that do not respond to this initial treatment. Surgical treatment should consist of laminectomy and fat debulking, and should not include intradural exploration