Mecanismo catalítico de la aril-alcohol oxidasa de "Pleurotus eryngii"

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Biológicas, leída el 25-05-2012Durante la degradación de la lignina por Pleurotus eryngii la enzima extracelular aril-alcohol oxidasa (AAO) genera el H2O2 que actúa como sustrato oxidante de las peroxidasas ligninolíticas y para la formación del radical hidroxilo. El poder reductor para la activación del O2 a H2O2 proviene de la oxidación de alcoholes aromáticos a los correspondientes aldehídos, siendo el alcohol p-metoxibencílico el sustrato natural de la AAO de P. eryngii. Esta oxidasa es una flavoenzima que contiene como cofactor una molécula de FAD unida de forma no covalente. Durante el presente trabajo se han estudiado en profundidad el mecanismo catalítico y las relaciones estructura-función de la AAO. Todo ello ha sido posible gracias a la disponibilidad de la estructura cristalográfica de esta oxidasa y de un protocolo de expresión heteróloga en Escherichia coli y activación in vitro de la enzima. El mecanismo catalítico de la AAO se estudió empleando sustratos αdeuterados y agua deuterada como solvente de la reacción. Durante la semi-reacción de reducción tiene lugar un proceso concertado (es decir, sin formación un intermediario estable) de transferencia de hidruro, desde el carbono-α del alcohol al N5 de la flavina, y de protón, desde el hidroxilo del alcohol a la base catalítica. Este mecanismo es diferente del mecanismo secuencial (no-concertado) observado en otras oxidorreductasas de la superfamilia GMC. Empleando las formas α-monodeuteradas del alcohol p-metoxibencílico se encontró que la transferencia de hidruro es un proceso estereoselectivo, que viene determinado por la posición del sustrato en el centro activo de la AAO. Debido a que se había observado previamente que la AAO es también capaz de oxidar algunos aldehídos aromáticos, se estudió en detalle la oxidación de estos compuestos combinando diferentes técnicas experimentales. Se observó que la enzima únicamente es capaz de oxidar aldehídos aromáticos en su forma gem-diol. Esta característica explicó por qué la presencia de sustituyentes electrofílicos en el anillo aromático (que promueven la hidratación del carbonilo) favorecía la oxidación, y así mismo, explicó la diferente especificidad de la AAO por ambos tipos de sustratos (alcohol vs aldehído). Además, se postuló que el mecanismo de oxidación de la forma gem-diol de los aldehídos es similar al mecanismo de oxidación de los alcoholes, donde participa una base catalítica, activando el sustrato a través de la abstracción del protón de uno de los grupos hidroxilo de la forma gem-diol. Posteriormente, se combinaron técnicas experimentales y computacionales para investigar diferentes aspectos del mecanismo y las relaciones estructura-función en la AAO. En primer lugar se estudió la migración del alcohol al centro activo, escasamente accesible desde el solvente, identificando los residuos que actúan como puerta de entrada del canal hidrofóbico de la enzima y aquellos que participan en el proceso de migración (Tyr92/Phe397/Phe501). Una vez en el centro activo, el sustrato adoptó una posición catalítica coincidente con la identificada experimentalmente usando isómeros α-monodeuterados del alcohol, es decir, con el hidrógeno que se transfiere (pro-R) próximo al N5 de la flavina. A través de los estudios de mutagénesis dirigida, estado de protonación y perfiles energéticos teóricos, se concluyó que la His502 actúa como la base catalítica responsable de la abstracción del protón del grupo hidroxilo del alcohol (facilitando la posterior transferencia de hidruro a la flavina) mientras que la His546 facilita el correcto posicionamiento del sustrato en el centro activo. Los perfiles energéticos de la reacción obtenidos mediante simulaciones de mecánica quántica/mecánica molecular (QM/MM) mostraron que, a pesar de que la transferencia de protón e hidruro son procesos altamente acoplados (concertados), la abstracción del protón por la His502 es previa a la transferencia de hidruro (mecanismo concertado no-sincrónico). También, se estudió la segunda semi-reacción del ciclo catalítico de la AAO. La migración del O2 mostró que esta molécula accede al centro activo a través del mismo canal hidrofóbico empleado por los alcoholes, pero en este caso sin que se requieran reajustes significativos de las cadenas laterales de los residuos del canal. Durante esta semi-reacción el oxígeno es primero reducido a anión superoxido mediante la transferencia de un electrón desde la flavina reducida. Durante esta etapa, la His502H+ actúa como acido, facilitando la formación y posterior estabilización del anión superoxido, mientras que el residuo Phe501 facilita el correcto posicionamiento del O2 junto al locus C4a-N5 de la flavina. Esta semi-reacción incluye la transferencia de un segundo electrón desde la forma semiquinona de la flavina y dos protones desde el N5H y la His502 al anión superoxido dando lugar al H2O2 (aunque sólo la primera transferencia es parcialmente limitante en la reoxidación). Finalmente se encontró que la estereoselectividad de la AAO en la transferencia de hidruro desde la posición pro-R en el carbono-α del alcohol (mostrada utilizando los enantiómeros (R) y (S) del alcohol pmetoxibencílico α-deuterado) se mantiene durante la oxidación de alcoholes secundarios por la enzima nativa, y especialmente por la variante F501A con el centro activo ampliado. Este resultado (confirmado mediante cromatografía quiral) revela el interés biotecnológico de esta oxidasa no solo por su contribución en la degradación de la lignina y la síntesis de aromatizantes, sino también por su mecanismo de oxidación.Fac. de Ciencias BiológicasTRUEunpu

Substrate stacking interactions in aryl-alcohol oxidase

This is the peer reviewed version of the following article: [Ferreira, P., Hernández-Ortega, A., Lucas, F., Carro, J., Herguedas, B., Borrelli, K. W., Guallar, V., Martínez, 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).Aryl-alcohol oxidase (AAO, EC 1.1.3.7) generates H2O2 for lignin degradation at the expense of benzylic and other π system-containing primary alcohols, which are oxidized to the corresponding aldehydes. Ligand diffusion studies on Pleurotus eryngii AAO showed a T-shaped stacking interaction between the Tyr92 side chain and the alcohol substrate at the catalytically competent position for concerted hydride and proton transfers. Bi-substrate kinetics analysis revealed that reactions with 3-chloro- or 3-fluorobenzyl alcohols (halogen substituents) proceed via a ping–pong mechanism. However, mono- and dimethoxylated substituents (in 4-methoxybenzyl and 3,4-dimethoxybenzyl alcohols) altered the mechanism and a ternary complex was formed. Electron-withdrawing substituents resulted in lower quantum mechanics stacking energies between aldehyde and the tyrosine side chain, contributing to product release, in agreement with the ping–pong mechanism observed in 3-chloro- and 3-fluorobenzyl alcohol kinetics analysis. In contrast, the higher stacking energies when electron donor substituents are present result in reaction of O2 with the flavin through a ternary complex, in agreement with the kinetics of methoxylated alcohols. The contribution of Tyr92 to the AAO reaction mechanism was investigated by calculation of stacking interaction energies and site-directed mutagenesis. Replacement of Tyr92 by phenylalanine does not alter the AAO kinetic constants (on 4-methoxybenzyl alcohol), most probably because the stacking interaction is still possible. However, introduction of a tryptophan residue at this position strongly reduced the affinity for the substrate (i.e. the pre-steady state Kd and steady-state Km increase by 150-fold and 75-fold, respectively), and therefore the steady-state catalytic efficiency, suggesting that proper stacking is impossible with this bulky residue. The above results confirm the role of Tyr92 in substrate binding, thus governing the kinetic mechanism in AAO.This work was supported by the BIO2013-42978-P (to MM), BIO2011-26694 (to ATM), “Juan de la Cierva” (to FL) and CTQ2010-18123 (to VG) Grants of the Spanish Ministry of Economy and Competitiveness (MINECO) and by the INDOX (KBBE-2013-7-613549, to ATM) and PELE (ERC-2009-Adg 25027, to VG) European projects.Peer ReviewedPostprint (author's final draft

Progress and Research Needs of Plant Biomass Degradation by Basidiomycete Fungi

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Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, individuals with clonal mosaic events (clonal mosaicism for chromosome alterations and/or loss of chromosome Y) showed an increased risk of COVID-19 lethality

Association Between Preexisting Versus Newly Identified Atrial Fibrillation and Outcomes of Patients With Acute Pulmonary Embolism

Background Atrial fibrillation (AF) may exist before or occur early in the course of pulmonary embolism (PE). We determined the PE outcomes based on the presence and timing of AF. Methods and Results Using the data from a multicenter PE registry, we identified 3 groups: (1) those with preexisting AF, (2) patients with new AF within 2 days from acute PE (incident AF), and (3) patients without AF. We assessed the 90-day and 1-year risk of mortality and stroke in patients with AF, compared with those without AF (reference group). Among 16 497 patients with PE, 792 had preexisting AF. These patients had increased odds of 90-day all-cause (odds ratio [OR], 2.81; 95% CI, 2.33-3.38) and PE-related mortality (OR, 2.38; 95% CI, 1.37-4.14) and increased 1-year hazard for ischemic stroke (hazard ratio, 5.48; 95% CI, 3.10-9.69) compared with those without AF. After multivariable adjustment, preexisting AF was associated with significantly increased odds of all-cause mortality (OR, 1.91; 95% CI, 1.57-2.32) but not PE-related mortality (OR, 1.50; 95% CI, 0.85-2.66). Among 16 497 patients with PE, 445 developed new incident AF within 2 days of acute PE. Incident AF was associated with increased odds of 90-day all-cause (OR, 2.28; 95% CI, 1.75-2.97) and PE-related (OR, 3.64; 95% CI, 2.01-6.59) mortality but not stroke. Findings were similar in multivariable analyses. Conclusions In patients with acute symptomatic PE, both preexisting AF and incident AF predict adverse clinical outcomes. The type of adverse outcomes may differ depending on the timing of AF onset.info:eu-repo/semantics/publishedVersio

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Fungal aryl-alcohol oxidase: a peroxide-producing flavoenzyme involved in lignin degradation

Aryl-alcohol oxidase (AAO) is an extracellular flavoprotein providing the H2O2 required by ligninolytic peroxidases for fungal degradation of lignin, the key step for carbon recycling in land ecosystems. O2 activation by Pleurotus eryngii AAO takes place during the redox-cycling of p-methoxylated benzylic metabolites secreted by the fungus. Only Pleurotus AAO sequences were available for years, but the number strongly increased recently due to sequencing of different basidiomycete genomes, and a comparison of 112 GMC (glucose–methanol–choline oxidase) superfamily sequences including 40 AAOs is presented. As shown by kinetic isotope effects, alcohol oxidation by AAO is produced by hydride transfer to the flavin, and hydroxyl proton transfer to a base. Moreover, site-directed mutagenesis studies showed that His502 activates the alcohol substrate by proton abstraction, and this result was extended to other GMC oxidoreductases where the nature of the base was under discussion. However, in contrast with that proposed for GMC oxidoreductases, the two transfers are not stepwise but concerted. Alcohol docking at the buried AAO active site resulted in only one catalytically relevant position for concerted transfer, with the pro-R α-hydrogen at distance for hydride abstraction. The expected hydride-transfer stereoselectivity was demonstrated, for the first time in a GMC oxidoreductase, by using the (R) and (S) enantiomers of α-deuterated p-methoxybenzyl alcohol. Other largely unexplained aspects of AAO catalysis (such as the unexpected specificity on substituted aldehydes) can also be explained in the light of the recent results. Finally, the biotechnological interest of AAO in flavor production is extended by its potential in production of chiral compounds taking advantage from the above-described stereoselectivityThis work was supported by the Spanish projects BIO2008-01533 and BIO2011-26694, and by the PEROXICATS (KBBE-2010-4-265397) European project. The cited SAP (Saprotrophic Agaricomycotina project), C. subvermispora, and P. ostreatus JGI genome projectsPeer reviewe

Modulating O2 reactivity in a fungal flavoenzyme: Involvement of aryl-alcohol oxidase Phe-501 contiguous to catalytic histidine

11 p.-6 fig.-2 tab.3 fig. supl.-1 tab. supl.Aryl-alcohol oxidase (AAO) is a flavoenzyme responsible for activation of O2 to H2O2 in fungal degradation of lignin. The AAO crystal structure shows a buried active site connected to the solvent by a hydrophobic funnel-shaped channel, with Phe-501 and two other aromatic residues forming a narrow bottleneck that prevents the direct access of alcohol substrates. However, ligand diffusion simulations show O2 access to the active site following this channel. Site-directed mutagenesis of Phe-501 yielded a F501A variant with strongly reduced O2 reactivity. However, a variant with increased reactivity, as shown by kinetic constants and steady-state oxidation degree, was obtained by substitution of Phe-501 with tryptophan. The high oxygen catalytic efficiency of F501W, ∼2-fold that of native AAO and ∼120-fold that of F501A, seems related to a higher O2 availability because the turnover number was slightly decreased with respect to the native enzyme. Free diffusion simulations of O2 inside the active-site cavity of AAO (and several in silico Phe-501 variants) yielded >60% O2 population at 3–4 Å from flavin C4a in F501W compared with 44% in AAO and only 14% in F501A. Paradoxically, the O2 reactivity of AAO decreased when the access channel was enlarged and increased when it was constricted by introducing a tryptophan residue. This is because the side chain of Phe-501, contiguous to the catalytic histidine (His-502 in AAO), helps to position O2 at an adequate distance from flavin C4a (and His-502 Nϵ). Phe-501 substitution with a bulkier tryptophan residue resulted in an increase in the O2 reactivity of this flavoenzymeThis work was supported in part by Spanish Projects BIO2008-01533 and BIO2010-1493 (to A. T. M. and M. M., respectively) and by the PEROXICATS (KBBE-2010-4-265397) and PELE (ERC-2009-Adg 25027) European Projects (to A. T. M. and V. G., respectively)Peer reviewe

Substrate diffusion and oxidation in GMC oxidoreductases: an experimental and computational study on fungal aryl-alcohol oxidase

10 páginas, 7 figuras, 2 tablas -- PAGS nros. 341-350AAO (aryl-alcohol oxidase) provides H2O2 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 Tyr92. 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 His502 (and CHO His466), 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 AAOPeer reviewe

Role of active site histidines in the two half-reactions of the aryl-alcohol oxidase catalytic cycle

The crystal structure of aryl-alcohol oxidase (AAO), a flavoenzyme involved in lignin degradation, reveals two active-site histidines, whose role in the two enzyme half-reactions was investigated. The redox state of flavin during turnover of the variants obtained show a stronger histidine involvement in the reductive than in the oxidative half-reaction. This was confirmed by the k cat/K m(Al) and reduction constants that are 2-3 orders of magnitude decreased for the His546 variants and up to 5 orders for the His502 variants, while the corresponding O 2 constants only decreased up to 1 order of magnitude. These results confirm His502 as the catalytic base in the AAO reductive half-reaction. The solvent kinetic isotope effect (KIE) revealed that hydroxyl proton abstraction is partially limiting the reaction, while the α-deuterated alcohol KIE showed a stereoselective hydride transfer. Concerning the oxidative half-reaction, directed mutagenesis and computational simulations indicate that only His502 is involved. Quantum mechanical/molecular mechanical (QM/MM) reveals an initial partial electron transfer from the reduced FADH - to O 2, without formation of a flavin-hydroperoxide intermediate. Reaction follows with a nearly barrierless His502H + proton transfer that decreases the triplet/singlet gap. Spin inversion and second electron transfer, concomitant with a slower proton transfer from flavin N5, yields H 2O 2. No solvent KIE was found for O 2 reduction confirming that the His502 proton transfer does not limit the oxidative half-reaction. However, the small KIE on k cat/K m(Ox), during steady-state oxidation of α-deuterated alcohol, suggests that the second proton transfer from N5H is partially limiting, as predicted by the QM/MM simulations. © 2012 American Chemical Society.This work was supported by projects BIO2008–01533, BIO2011–26694 (to A.T.M. and co-workers), BIO2010–1493 (to M.M.) and CTQ2010-18123 (to V.G.) of the Spanish Ministry of Economy and Competitiveness (MINECO), and by PEROXICATS (KBBE-2010-4-265397, to A.T.M.) and PELE (ERC-2009-Adg 25027, to V.G.) European projectsPeer Reviewe