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

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

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

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

Cristalización de proteínas redox y predicción estructural de la FAD sintetasa humana

Este trabajo ha abordado el estudio estructural de proteínas redox de interés biotecnológico y biomédico tales como el factor de inducción de la apoptosis humano, implicado en procesos de muerte celular (hAIF), la NADPH-flavodoxina reductasa del microorganismo fitopatógeno Xanthomonas axonopodis pv citri (XacFPR) y las proteínas riboflavin quinasa humana (hRFK) y FAD sintetasa humana (hFADS_2), implicadas en la síntesis del cofactor FAD necesario para múltiples rutas metabólicas así como para la actividad de las flavoproteinas. Con este fin se han utilizado varias técnicas: la cristalografía de rayos X, la predicción estructural de proteínas y la microscopia de fuerzas atómicas. La técnica de cristalografía de rayos X aplicada a proteínas está basada en la obtención de cristales proteicos que difractan los rayos X proporcionando datos que conducen a la resolución de la estructura tridimensional de la proteína. Esta técnica se puede llevar a cabo en presencia de ligandos de manera que se consigue información de la interacción proteína - ligando. Durante el desarrollo de este trabajo se han identificado condiciones iniciales de cristalización de las proteínas riboflavin quinasa humana (hRFK) y de las especies mutantes del factor de inducción de la apoptosis humano (hAIF), así como la resolución de la estructura de la proteína XacFPR cocristalizada con NADP+ y/o un inhibidor de su actividad reductasa en la que no se observa densidad electrónica extra debida al ligando. Con el fin de avanzar en el conocimiento de la estructura de la enzima hFADS_2, y a falta de un modelo cristalográfico, se realizó una predicción de su estructura tridimensional así como un estudio morfológico del dominio C terminal mediante microscopia de fuerzas atómicas. Se observó que el dominio C terminal aislado dimeriza y que los monómeros presentan una altura promedio de 3,5 ± 0,5 nm, que se ajusta a las dimensiones encontradas en las predicciones

Evaluación del efecto de potenciales inhibidores de la actividad ferredoxina reductasa de Xanthomonas axonopodis pv citri, que actúa como patógeno en cultivos de cítricos

Este trabajo se incluye dentro de un proyecto de búsqueda de compuestos que presenten acción bactericida contra el microorganismo patógeno Xanthomonas axonopodis pv citri (Xac), una bacteria que causa la enfermedad del cáncer de cítricos. Xanthomonas es una bacteria Gram negativa que posee una enzima, la NADPH-flavodoxina (ferredoxina) reductasa (XacFPR), que se ha utilizado en este proyecto como diana terapéutica, debido a sus diferencias estructurales y funcionales con sus homólogas en las células eucariotas vegetales, las FNRs plastídicas. De este modo se busca inhibir la enzima del fitopatógeno sin ocasionar daño a la planta hospedadora mediante el descubrimiento de un bactericida eficaz. El objetivo supone todo un reto, ya que existe un método efectivo para evitar los efectos de Xanthomonas, que incluyen graves daños en los cítricos de diferentes países del mundo, incluyendo España, reduciendo enormemente la producción, lo que conlleva grandes pérdidas económicas sin que existan métodos efectivos para frenar su avance. Los potenciales inhibidores de la XacFPR estudiados en esta memoria, 43 compuestos, fueron seleccionados con anterioridad mediante cribado masivo a partir de una quimioteca formada por 11.120 compuestos. En este trabajo se ha medido el valor IC50 de estos compuestos mediante la actividad diaforasa de la XacFPR y se han realizado ajustes globales de los datos cinéticos obtenidos en presencia de un compuesto con alta eficacia de inhibición, a las funciones de Michaelis-Menten y sus inversos modificadas por el factor de inhibición para determinar el tipo de inhibición. En este trabajo se ha concluido que dicho compuesto produce una inhibición efectiva de un 94,6 % de la actividad reductasa de la XacFPR y con un IC50 de 9.7 µM mediante un posible mecanismo de inhibición no competitiva, con un valor de KI de 5 µM. Los experimentos preliminares de acoplamiento molecular, "docking", muestran que dicho compuesto se une a XacFPR en el sitio donde se encuentra el cofactor FAD. Con este compuesto seleccionado se podrán realizar pruebas de inhibición in vivo donde se comprobará la efectividad del bactericida y su inocuidad en las plantas hospedadoras

Structural Insights into the Mechanism of Protein O-Fucosylation

Protein O-fucosylation is an essential post-translational modification, involved in the folding of target proteins and in the role of these target proteins during embryonic development and adult tissue homeostasis, among other things. Two different enzymes are responsible for this modification, Protein O-fucosyltransferase 1 and 2 (POFUT1 and POFUT2, respectively). Both proteins have been characterised biologically and enzymatically but nothing is known at the molecular or structural level. Here we describe the first crystal structure of a catalytically functional POFUT1 in an apo-form and in complex with GDP-fucose and GDP. The enzyme belongs to the GT-B family and is not dependent on manganese for activity. GDP-fucose/GDP is localised in a conserved cavity connected to a large solvent exposed pocket, which we show is the binding site of epidermal growth factor (EGF) repeats in the extracellular domain of the Notch Receptor. Through both mutational and kinetic studies we have identified which residues are involved in binding and catalysis and have determined that the Arg240 residue is a key catalytic residue. We also propose a novel SN1-like catalytic mechanism with formation of an intimate ion pair, in which the glycosidic bond is cleaved before the nucleophilic attack; and theoretical calculations at a DFT (B3LYP/6-31+G(d,p) support this mechanism. Thus, the crystal structure together with our mutagenesis studies explain the molecular mechanism of POFUT1 and provide a new starting point for the design of functional inhibitors to this critical enzyme in the future

PT-112 Induces Mitochondrial Stress and Immunogenic Cell Death, Targeting Tumor Cells with Mitochondrial Deficiencies

PT-112 is a novel pyrophosphate–platinum conjugate, with clinical activity reported in advanced pretreated solid tumors. While PT-112 has been shown to induce robust immunogenic cell death (ICD) in vivo but only minimally bind DNA, the molecular mechanism underlying PT-112 target disruption in cancer cells is still under elucidation. The murine L929 in vitro system was used to test whether differential metabolic status alters PT-112’s effects, including cell cytotoxicity. The results showed that tumor cells presenting mutations in mitochondrial DNA (mtDNA) (L929dt and L929dt cybrid cells) and reliant on glycolysis for survival were more sensitive to cell death induced by PT-112 compared to the parental and cybrid cells with an intact oxidative phosphorylation (OXPHOS) pathway (L929 and dtL929 cybrid cells). The type of cell death induced by PT-112 did not follow the classical apoptotic pathway: the general caspase inhibitor Z-VAD-fmk did not inhibit PT-112-induced cell death, alone or in combination with the necroptosis inhibitor necrostatin-1. Interestingly, PT-112 initiated autophagy in all cell lines, though this process was not complete. Autophagy is known to be associated with an integrated stress response in cancer cells and with subsequent ICD. PT-112 also induced a massive accumulation of mitochondrial reactive oxygen species, as well as changes in mitochondrial polarization—only in the sensitive cells harboring mitochondrial dysfunction—along with calreticulin cell-surface exposure consistent with ICD. PT-112 substantially reduced the amount of mitochondrial CoQ10 in L929 cells, while the basal CoQ10 levels were below our detection limits in L929dt cells, suggesting a potential relationship between a low basal level of CoQ10 and PT-112 sensitivity. Finally, the expression of HIF-1α was much higher in cells sensitive to PT-112 compared to cells with an intact OXPHOS pathway, suggesting potential clinical applications

External loops at the ferredoxin-NADP+ reductase protein-partner binding cavity contribute to substrates allocation

Ferredoxin-NADP+ reductase (FNR) is the structural prototype of a family of FAD-containing reductases that catalyze electron transfer between low potential proteins and NAD(P)+/H, and that display a two-domain arrangement with an open cavity at their interface. The inner part of this cavity accommodates the reacting atoms during catalysis. Loops at its edge are highly conserved among plastidic FNRs, suggesting that they might contribute to both flavin stabilization and competent disposition of substrates. Here we pay attention to two of these loops in Anabaena FNR. The first is a sheet-loop-sheet motif, loop102-114, that allocates the FAD adenosine. It was thought to determine the extended FAD conformation, and, indirectly, to modulate isoalloxazine electronic properties, partners binding, catalytic efficiency and even coenzyme specificity. The second, loop261-269, contains key residues for the allocation of partners and coenzyme, including two glutamates, Glu267 and Glu268, proposed as candidates to facilitate the key displacement of the C-terminal tyrosine (Tyr303) from its stacking against the isoalloxazine ring during the catalytic cycle. Our data indicate that the main function of loop102-114 is to provide the inter-domain cavity with flexibility to accommodate protein partners and to guide the coenzyme to the catalytic site, while the extended conformation of FAD must be induced by other protein determinants. Glu267 and Glu268 appear to assist the conformational changes that occur in the loop261-269 during productive coenzyme binding, but their contribution to Tyr303 displacement is minor than expected. Additionally, loop261-269 appears a determinant to ensure reversibility in photosynthetic FNRsPeer reviewe