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

Structural analysis of FAD synthetase from Corynebacterium ammoniagenes

Abstract Background The prokaryotic FAD synthetase family – a group of bifunctional enzymes that catalyse riboflavin phosphorylation and FMN adenylylation within a single polypeptide chain- was analysed in terms of sequence and structure. Results Sequences of nearly 800 prokaryotic species were aligned. Those related with bifunctional FAD synthetase activities showed conservation of several consensus regions and highly conserved residues. A 3D model for the FAD synthetase from Corynebacterium ammoniagenes (CaFADS) was generated. This model confirms that the N-terminal and C-terminal domains are related to nucleotydyltransferases and riboflavin kinases, respectively. Models for the interaction of CaFADS with its substrates were also produced, allowing location of all the protein substrates in their putative binding pockets. These include two independent flavin binding sites for each CaFADS activity. Conclusion For the first time, the putative presence of a flavin binding site for the adenylylation activity, independent from that related with the phosphorylation activity, is shown. Additionally, these models suggest the functional relevance of some residues putatively involved in the catalytic processes. Their relevant roles were analysed by site-directed mutagenesis. A role was confirmed for H28, H31, S164 and T165 in the stabilisation of the P groups and the adenine moiety of ATP and, the P of FMN for the adenylylation. Similarly, T208, N210 and E268 appear critical for accommodation of the P groups of ATP and the ribityl end of RF in the active site for the phosphorylation process. Finally, the C-terminal domain was shown to catalyse the phosphorylation process on its own, but no reaction at all was observed with the individually expressed N-terminal domain.</p

Mining the Flavoproteome of Brucella ovis, the Brucellosis causing agent in Ovis aries

Flavoproteins are a diverse class of proteins that are mostly enzymes and contain as cofactors flavin mononucleotide (FMN) and/or flavin adenine dinucleotide (FAD), which enable them to participate in a wide range of physiological reactions. We have compiled 78 potential proteins building the flavoproteome of Brucella ovis (B. ovis), the causative agent of ovine brucellosis. The curated list of flavoproteins here reported is based on (i) the analysis of sequence, structure and function of homologous proteins, and their classification according to their structural domains, clans, and expected enzymatic functions; (ii) the constructed phylogenetic trees of enzyme functional classes using 19 Brucella strains and 26 pathogenic and/or biotechnological relevant alphaproteobacteria together with B. ovis; and (iii) the evaluation of the genetic context for each entry. Candidates account for;2.7% of the B. ovis proteome, and 75% of them use FAD as cofactor. Only 55% of these flavoproteins belong to the core proteome of Brucella and contribute to B. ovis processes involved in maintenance activities, survival and response to stress, virulence, and/or infectivity. Several of the predicted flavoproteins are highly divergent in Brucella genus from revised proteins and for them it is difficult to envisage a clear function. This might indicate modified catalytic activities or even divergent processes and mechanisms still not identified. We have also detected the lack of some functional flavoenzymes in B. ovis, which might contribute to it being nonzoonotic. Finally, potentiality of B. ovis flavoproteome as the source of antimicrobial targets or biocatalyst is discussed. IMPORTANCE Some microorganisms depend heavily on flavin-dependent activities, but others maintain them at a minimum. Knowledge about flavoprotein content and functions in different microorganisms will help to identify their metabolic requirements, as well as to benefit either industry or health. Currently, most flavoproteins from the sheep pathogen Brucella ovis are only automatically annotated in databases, and only two have been experimentally studied. Indeed, certain homologues with unknown function are not characterized, and they might relate to still not identified mechanisms or processes. Our research has identified 78 members that comprise its flavoproteome, 76 of them flavoenzymes, which mainly relate to bacteria survival, virulence, and/or infectivity. The list of flavoproteins here presented allows us to better understand the peculiarities of Brucella ovis and can be applied as a tool to search for candidates as new biocatalyst or antimicrobial targets

Estudio del efecto inhibitorio de algunos compuestos químicos en la actividad reductasa de flavoenzimas de organismos patógenos

Este trabajo aborda la búsqueda de compuestos químicos con carácter inhibitorio de la actividad de flavoenzimas de microorganismos patógenos. Concretamente se centra en la búsqueda de inhibidores de la enzima NADPH-flavodoxina reductasa del fitopatógeno Xanthomonas axonopodis pv citri denominada XacFPR. Esta bacteria es conocida por provocar la enfermedad denominada cancrosis o cáncer de cítricos que afecta a múltiples plantaciones y cultivos alrededor del mundo provocando necrosis y la caída prematura de los frutos. Por tanto esta infección supone grandes pérdidas económicas a nivel global y resulta de vital importancia desarrollar un bactericida útil. Con el fin de lograr este objetivo se escogió como diana terapéutica la XacFPR, ya que interviene en procesos de fotosíntesis y respuesta a estrés oxidativo y por tanto resulta esencial para la supervivencia del patógeno. De entre una serie de compuestos derivados de un inhibidor ensayado con anterioridad (compuesto C12 actualmente descatalogado), se seleccionaron los más competentes realizando medidas de actividad enzimática. Estos fueron D2, D5 Y D10 con valores de IC50 de 25,38, 17,24 y 174,72 µM respectivamente y porcentajes de inhibición próximos al 100%. Posteriormente y debido a su homología estructural con XacFPR, se elaboraron ensayos con AnFNR, la enzima ferredoxina NADP+-reductasa presente en la cianobacteria de Anabaena. Con lo que se pudo concluir que solo D5 resultó prácticamente inocuo frente a la FNR (IC50 de 102,38 µM y 47,31% de inhibición). Un estudio posterior del tipo de inhibición reveló un posible mecanismo de inhibición no competitiva para los tres compuestos mencionados, con valores de kI de 21,3 µM en el caso de D2, kI de 8,3 µM para D5 y kI de 74,1 µM para D10

El videojuego como herramienta para la enseñanza de la Historia en el aula: El caso de la saga Europa Universalis

El videojuego es un elemento que desde hace tres décadas acompaña a los más jóvenes en su vida diaria y emplean gran parte de su tiempo en ellos. Los videojuegos pueden generan una gran motivación en el alumnado en comparación con las actividades de aprendizaje tradicionales, por esta razón, en el presente trabajo se pretende destacar el valor del videojuego de estrategia, concretamente los videojuegos que forman parte de la saga Europa Universalis, como un instrumento para la enseñanza de la historia en el aula teniendo en cuenta las ventajas e inconvenientes que puede tener el hecho de realizar actividades con videojuegos. <br /

Espectro de excitaciones de nanodiscos magnéticos

En los dispositivos superconductores de interferencia cuántica o SQUIDs, la combinación del efecto Josephson junto con la cuantización del flujo magnético nos permite utilizarlos como sensores de campo magnético de extremada sensibilidad. Estos dispositivos nos permiten transformar el flujo de campo magnético en señales medibles mediante la modulación de la corriente crítica que atraviesa el material superconductor.A lo largo de este trabajo vamos a utilizar estos sensores en el estudio de las configuraciones magnéticas de un nanodisco ferromagnético. Empezaremos explicando los fundamentos teóricos y realizando una caracterización de estos dispositivos superconductores que nos van a acompañar a lo largo de todo el estudio. A continuación hablaremos de la configuración de vórtice magnético que aparece en nanodiscos de Permalloy y estudiaremos sus ciclos de histéresis, tanto de forma experimental como numérica.El objetivo final del trabajo es estudiar el espectro de excitaciones de estas nanopartículas bajo la aplicación de pulsos de campo magnético, comparando de nuevo medidas experimentales con las obtenidas por simulación numérica.<br /

Structural insights into the synthesis of FMN in prokaryotic organisms

Riboflavin kinases (RFKs) catalyse the phosphorylation of riboflavin to produce FMN. In most bacteria this activity is catalysed by the C-terminal module of a bifunctional enzyme, FAD synthetase (FADS), which also catalyses the transformation of FMN into FAD through its N-terminal FMN adenylyl transferase (FMNAT) module. The RFK module of FADS is a homologue of eukaryotic monofunctional RFKs, while the FMNAT module lacks homologyto eukaryotic enzymes involved in FAD production. Previously, the crystal structure of Corynebacterium ammoniagenes FADS (CaFADS) was determined in its apo form. This structure predicted a dimer-of-trimers organization with the catalytic sites of two modules of neighbouring protomers approaching each other, leading to a hypothesis about the possibility of FMN channelling in the oligomeric protein. Here, two crystal structures of the individually expressed RFK module of CaFADS in complex with the products of the reaction, FMN and ADP, are presented. Structures are complemented with computational simulations, binding studies and kinetic characterization. Binding of ligands triggers dramatic structural changes in the RFK module, which affect large portions of the protein. Substrate inhibition and molecular-dynamics simulations allowed the conformational changes that take place along the RFK catalytic cycle to be established. The influence of these conformational changes in the FMNAT module is also discussed in the context of the full-length CaFADS protomer and the quaternary organization.This work has been supported by MINECO, Spain (BIO2013-42978-P to MM and BFU2014-59389-P to JAH), the Aragonian Government-FEDER (B18), Autonomous Community of Madrid (S2010/BMD-2457), Departamento Administrativo de Ciencia, Tecnología e Innovación (COLCIENCIAS) and Universidad Industrial de Santander (project 1818 to IL).Peer Reviewe

An efficient method for enzyme immobilization evidenced by atomic force microscopy

Immobilization of proteins in a functionally active form and proper orientation is fundamental for effective surface-based protein analysis. A new method is presented for the controlled and oriented immobilization of ordered monolayers of enzymes whose interaction site had been protected using the protein ligand. The utility of this method was demonstrated by analyzing the interactions between the enzyme ferredoxin-NADP+ reductase (FNR) and its redox partner ferredoxin (Fd). The quality of the procedure was deeply evaluated through enzymatic assays and atomic force microscopy. Single-molecule force spectroscopy revealed that site-specifically targeted FNR samples increased the ratio of recognition events 4-fold with regard to the standard randomly modified FNR samples. The results were corroborated using the cytochrome c reductase activity that gave an increase on surface between 6 and 12 times for the site-specifically targeted FNR samples. The activity in solution for the enzyme labeled from the complex was similar to that exhibited by wild-type FNR while FNR randomly tagged showed a 3-fold decrease. This indicates that random targeting protocols affect not only the efficiency of immobilized proteins to recognize their ligands but also their general functionality. The present methodology is expected to find wide applications in surface-based protein–protein interactions biosensors, single-molecule analysis, bioelectronics or drug screening

Crystal structure of the FAD-containing ferredoxin-NADP+ reductase from the plant pathogen Xanthomonas axonopodis pv. citri

We have solved the structure of ferredoxin-NADP(H) reductase, FPR, from the plant pathogen Xanthomonas axonopodis pv. citri, responsible for citrus canker, at a resolution of 1.5¿Å. This structure reveals differences in the mobility of specific loops when compared to other FPRs, probably unrelated to the hydride transfer process, which contributes to explaining the structural and functional divergence between the subclass I FPRs. Interactions of the C-terminus of the enzyme with the phosphoadenosine of the cofactor FAD limit its mobility, thus affecting the entrance of nicotinamide into the active site. This structure opens the possibility of rationally designing drugs against the X. axonopodis pv. citri phytopathogen

Cristalización y difracción de rayos X para la resolución de estructuras de proteínas y complejos proteicos

Estudio del papel de los residuos E413, R422 y R430 en el proceso de oligomerización de la enzima AIF mediante cristalografía de rayos X