RESUMEN

In plants, salicylic acid (SA) hydroxylation regulates SA homoeostasis, playing an essential role during plant development and response to pathogens. This reaction is catalysed by SA hydroxylase enzymes, which hydroxylate SA producing 2,3-dihydroxybenzoic acid (2,3-DHBA) and/or 2,5-dihydroxybenzoic acid (2,5-DHBA). Several SA hydroxylases have recently been identified and characterised from different plant species, but no such activity has yet been reported in maize. In this work, we describe the identification and characterisation of a new SA hydroxylase in maize plants. This enzyme, with high sequence similarity to previously described SA hydroxylases from Arabidopsis and rice, converts SA into 2,5-DHBA; however, it has different kinetic properties to those of previously characterised enzymes, and it also catalysers the conversion of the flavonoid dihydroquercetin into quercetin in in vitro activity assays, suggesting that the maize enzyme may have different roles in vivo to those previously reported from other species. Despite this, ZmS5H can complement the pathogen resistance and the early senescence phenotypes of Arabidopsis s3h mutant plants. Finally, we characterised a maize mutant in the S5H gene (s5hMu) that has altered growth, senescence and increased resistance against Colletotrichum graminicola infection, showing not only alterations in SA and 2,5-DHBA but also in flavonol levels. Together, the results presented here provide evidence that SA hydroxylases in different plant species have evolved to show differences in catalytic properties that may be important to fine tune SA levels and other phenolic compounds such as flavonols, to regulate different aspects of plant development and pathogen defence.

Asunto(s)

Colletotrichum , Resistencia a la Enfermedad , Enfermedades de las Plantas , Proteínas de Plantas , Ácido Salicílico , Zea mays , Zea mays/genética , Zea mays/enzimología , Zea mays/microbiología , Ácido Salicílico/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Resistencia a la Enfermedad/genética , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/inmunología , Colletotrichum/fisiología , Cinética , Ácidos Cetoglutáricos/metabolismo , Oxigenasas de Función Mixta/metabolismo , Oxigenasas de Función Mixta/genética , Regulación de la Expresión Génica de las Plantas , Arabidopsis/genética , Arabidopsis/enzimología , Arabidopsis/microbiología , Gentisatos/metabolismo , Filogenia , Quercetina/metabolismo , Hidroxibenzoatos

RESUMEN

Tissue accumulation and high urinary excretion of ethylmalonic acid (EMA) are found in ethylmalonic encephalopathy (EE), an inherited disorder associated with cerebral and cerebellar atrophy whose pathogenesis is poorly established. The in vitro and in vivo effects of EMA on bioenergetics and redox homeostasis were investigated in rat cerebellum. For the in vitro studies, cerebellum preparations were exposed to EMA, whereas intracerebellar injection of EMA was used for the in vivo evaluation. EMA reduced state 3 and uncoupled respiration in vitro in succinate-, glutamate-, and malate-supported mitochondria, whereas decreased state 4 respiration was observed using glutamate and malate. Furthermore, mitochondria permeabilization and succinate supplementation diminished the decrease in state 3 with succinate. EMA also inhibited the activity of KGDH, an enzyme necessary for glutamate oxidation, in a mixed manner and augmented mitochondrial efflux of α-ketoglutarate. ATP levels were markedly reduced by EMA, reflecting a severe bioenergetic disruption. Docking simulations also indicated interactions between EMA and KGDH and a competition with glutamate and succinate for their mitochondrial transporters. In vitro findings also showed that EMA decreased mitochondrial membrane potential and Ca2+ retention capacity, and induced swelling in the presence of Ca2+ , which were prevented by cyclosporine A and ADP and ruthenium red, indicating mitochondrial permeability transition (MPT). Moreover, EMA, at high concentrations, mildly increased ROS levels and altered antioxidant defenses in vitro and in vivo. Our data indicate that EMA-induced impairment of glutamate and succinate oxidation and MPT may contribute to the pathogenesis of the cerebellum abnormalities in EE.

Asunto(s)

Cerebelo/efectos de los fármacos , Cerebelo/metabolismo , Metabolismo Energético/efectos de los fármacos , Glutamatos/metabolismo , Malonatos/toxicidad , Poro de Transición de la Permeabilidad Mitocondrial , Succinatos/metabolismo , Animales , Ácidos Cetoglutáricos/metabolismo , Malatos/metabolismo , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Proteínas Mitocondriales/efectos de los fármacos , Proteínas Mitocondriales/metabolismo , Simulación del Acoplamiento Molecular , Oxidación-Reducción , Consumo de Oxígeno/efectos de los fármacos , Ratas , Ratas Wistar , Succinatos/farmacología

RESUMEN

PURPOSE: Glutamine plays an important role in tumor metabolism and progression. This research aimed to find out how Gln exert their effects on laryngeal squamous cell carcinoma (LSCC). METHODS: Cell proliferation was measured by CCK8 and EdU assay, mitochondrial bioenergetic activity was measured by mitochondrial stress tests. Gene expression profiling was revealed by RNA sequencing and validated by RT-qPCR. In LSCC patients, protein expression in tumor and adjacent tissues was examined and scored by IHC staining. RNAi was performed by stably expressed shRNA in TU177 cells. In vivo tumor growth analysis was performed using a nude mouse tumorigenicity model. RESULTS: Gln deprivation suppressed TU177 cell proliferation, which was restored by αKG supplementation. By transcriptomic analysis, we identified CECR2, which encodes a histone acetyl-lysine reader, as the downstream target gene for Gln and αKG. In LSCC patients, the expression of CECR2 in tumors was lower than adjacent tissues. Furthermore, deficiency of CECR2 promoted tumor cell growth both in vitro and in vivo, suggesting it has tumor suppressor effects. Besides, cell proliferation inhibited by Gln withdrawal could be restored by CECR2 depletion, and the proliferation boosted by αKG supplementation could be magnified either, suggested that CECR2 feedback suppressed Gln and αKG's effect on tumor growth. Transcriptomic profiling revealed CECR2 regulated the expression of a series of genes involved in tumor progression. CONCLUSION: We confirmed the Gln-αKG-CECR2 axis contributes to tumor growth in LSCC. This finding provided a potential therapeutic opportunity for the use of associated metabolites as a potential treatment for LSCC.

Asunto(s)

Genes Supresores de Tumor , Glutamina/metabolismo , Neoplasias Laríngeas/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Factores de Transcripción/genética , Anciano , Anciano de 80 o más Años , Animales , Recuento de Células , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Progresión de la Enfermedad , Regulación hacia Abajo , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glutamina/farmacología , Humanos , Ácidos Cetoglutáricos/metabolismo , Ácidos Cetoglutáricos/farmacología , Neoplasias Laríngeas/metabolismo , Neoplasias Laríngeas/patología , Masculino , Ratones , Ratones Desnudos , Persona de Mediana Edad , Mitocondrias/metabolismo , Proteínas de Neoplasias/metabolismo , Consumo de Oxígeno , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Factores de Transcripción/deficiencia , Factores de Transcripción/metabolismo

RESUMEN

XanA is an FeII- and α-ketoglutarate-dependent enzyme responsible for the conversion of xanthine to uric acid. It is unique to fungi and it was first described in Aspergillus nidulans. In this work, we present the preliminary characterization of the XanA enzyme from Aspergillus oryzae, a relevant fungus in food production in Japan. The XanA protein (GenBank BAE56701.1) was expressed as a recombinant protein in Escherichia coli BL21 (DE3) Arctic cells. Initial purification assays showed low protein solubility; therefore, the buffer composition was optimized using a fluorescence-based thermal shift assay. The protein was stabilized in solution in the presence of either 600 µM xanthine, 1 M NaCl, 600 µM α-ketoglutarate or 20% glycerol, which increases the melting temperature (Tm) by 2, 4, 5 and 6 °C respectively. The XanA protein was purified by following a three-step purification protocol. The nickel affinity purified protein was subjected to ion-exchange chromatography once the N-terminal 6XHis-tag had been successfully removed, followed by size-exclusion purification. Dynamic light scattering experiments showed that the purified protein was monodisperse and behaved as a monomer in solution. Preliminary activity assays in the presence of xanthine, α-ketoglutarate, and iron suggest that the enzyme is an iron- and α-ketoglutarate-dependent xanthine dioxygenase. Furthermore, the enzyme's optimum activity conditions were determined to be 25 °C, pH of 7.2, HEPES buffer, and 1% of glycerol. In conclusion, we established the conditions to purify the XanA enzyme from A. oryzae in its active form from E. coli bacteria and determined the optimal activity conditions.

Asunto(s)

Aspergillus oryzae , Dioxigenasas , Proteínas Fúngicas , Aspergillus oryzae/enzimología , Aspergillus oryzae/genética , Dioxigenasas/biosíntesis , Dioxigenasas/química , Dioxigenasas/genética , Dioxigenasas/aislamiento & purificación , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/aislamiento & purificación , Hierro/química , Hierro/metabolismo , Ácidos Cetoglutáricos/química , Ácidos Cetoglutáricos/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación

RESUMEN

In many cell types, epigenetic changes are partially regulated by the availability of metabolites involved in the activity of chromatin-modifying enzymes. Even so, the association between metabolism and the typical epigenetic reprogramming that occurs during preimplantation embryo development remains poorly understood. In this work, we explore the link between energy metabolism, more specifically the tricarboxylic acid cycle (TCA), and epigenetic regulation in bovine preimplantation embryos. Using a morphokinetics model of embryonic development (fast- and slow-developing embryos), we show that DNA methylation (5mC) and hydroxymethylation (5hmC) are dynamically regulated and altered by the speed of the first cleavages. More specifically, slow-developing embryos fail to perform the typical reprogramming that is necessary to ensure the generation of blastocysts with higher ability to establish specific cell lineages. Transcriptome analysis revealed that such differences were mainly associated with enzymes involved in the TCA cycle rather than specific writers/erasers of DNA methylation marks. This relationship was later confirmed by disturbing the embryonic metabolism through changes in α-ketoglutarate or succinate availability in culture media. This was sufficient to interfere with the DNA methylation dynamics despite the fact that blastocyst rates and total cell number were not quite affected. These results provide the first evidence of a relationship between epigenetic reprogramming and energy metabolism in bovine embryos. Likewise, levels of metabolites in culture media may be crucial for precise epigenetic reprogramming, with possible further consequences in the molecular control and differentiation of cells.

Asunto(s)

Blastocisto/enzimología , Blastocisto/metabolismo , Ciclo del Ácido Cítrico , Metilación de ADN , Animales , Blastocisto/citología , Bovinos , Medios de Cultivo/metabolismo , Desarrollo Embrionario/genética , Metabolismo Energético , Epigénesis Genética , Femenino , Perfilación de la Expresión Génica , Ontología de Genes , Ácidos Cetoglutáricos/metabolismo , Embarazo , Ácido Succínico/metabolismo

RESUMEN

Nitazoxanide (NTZ) is a broad-spectrum drug used in intestinal infections, but still poorly explored in the treatment of parasitic tissular infections. This study aimed to evaluate the in vitro responses of the energetic metabolism of T. crassiceps cysticerci induced by NTZ. The organic acids of the tricarboxylic acid cycle, products derived from fatty acids oxidation and protein catabolism were analyzed. These acids were quantified after 24 h of in vitro exposure to different NTZ concentrations. A positive control group was performed with albendazole sulfoxide (ABZSO). The significant alterations in citrate, fumarate and malate concentrations showed the NTZ influence in the tricarboxylic acid (TCA) cycle. The non-detection of acetate confirmed that the main mode of action of NTZ is effective against T. crassiceps cysticerci. The statistical differences in fumarate, urea and beta-hydroxybutyrate concentrations showed the NTZ effect on protein catabolism and fatty acid oxidation. Therefore, the main energetic pathways such as the TCA cycle, protein catabolism and fatty acids oxidation were altered after in vitro NTZ exposure. In conclusion, NTZ induced a significant metabolic stress in the parasite indicating that it may be used as an alternative therapeutic choice for cysticercosis treatment. The use of metabolic approaches to establish comparisons between anti parasitic drugs mode of actions is proposed.

Asunto(s)

Antiparasitarios/farmacología , Taenia/efectos de los fármacos , Tiazoles/farmacología , Albendazol/análogos & derivados , Albendazol/farmacología , Análisis de Varianza , Animales , Antihelmínticos/farmacología , Citratos/metabolismo , Ciclo del Ácido Cítrico/efectos de los fármacos , Medios de Cultivo/química , Cysticercus/efectos de los fármacos , Cysticercus/metabolismo , Metabolismo Energético/efectos de los fármacos , Fumaratos/metabolismo , Ácidos Cetoglutáricos/metabolismo , Malatos/metabolismo , Neurocisticercosis/tratamiento farmacológico , Nitrocompuestos , Ácido Oxaloacético/metabolismo , Ácido Succínico/metabolismo , Taenia/metabolismo

RESUMEN

Herbaspirillum seropedicae is a plant growth promoting bacterium that is able to fix nitrogen and to colonize the surface and internal tissues of important crops. Nitrogen fixation in H. seropedicae is regulated at the transcriptional level by the prokaryotic enhancer binding protein NifA. The activity of NifA is negatively affected by oxygen and positively stimulated by interaction with GlnK, a PII signaling protein that monitors intracellular levels of the key metabolite 2-oxoglutarate (2-OG) and functions as an indirect sensor of the intracellular nitrogen status. GlnK is also subjected to a cycle of reversible uridylylation in response to intracellular levels of glutamine. Previous studies have established the role of the N-terminal GAF domain of NifA in intramolecular repression of NifA activity and the role of GlnK in relieving this inhibition under nitrogen-limiting conditions. However, the mechanism of this control of NifA activity is not fully understood. Here, we constructed a series of GlnK variants to elucidate the role of uridylylation and effector binding during the process of NifA activation. Our data support a model whereby GlnK uridylylation is not necessary to activate NifA. On the other hand, binding of 2-OG and MgATP to GlnK are very important for NifA activation and constitute the most important signal of cellular nitrogen status to NifA.

Asunto(s)

Proteínas Bacterianas/metabolismo , Herbaspirillum , Proteínas PII Reguladoras del Nitrógeno/metabolismo , Factores de Transcripción/metabolismo , Adenosina Trifosfato/metabolismo , Sitio Alostérico , Escherichia coli/metabolismo , Ácidos Cetoglutáricos/metabolismo , Mutagénesis , Proteínas PII Reguladoras del Nitrógeno/química , Proteínas PII Reguladoras del Nitrógeno/genética , Unión Proteica

RESUMEN

The nitrogen metabolism of Proteobacteria is controlled by the general Ntr system in response to nitrogen quality and availability. The PII proteins play an important role in this system by modulating the cellular metabolism through physical interaction with protein partners. Herbaspirillum seropedicae, a nitrogen-fixing bacterium, has two PII proteins paralogues, GlnB and GlnK. The interaction of H. seropedicae PII proteins with its targets is regulated by allosteric ligands and by reversible post-translational uridylylation. Both uridylylation and deuridylylation reactions are catalyzed by the same bifunctional enzyme, GlnD. The mechanism of regulation of GlnD activity is still not fully understood. Here, we characterized the regulation of deuridylylation activity of H. seropedicae GlnD in vitro. To this purpose, fully modified PII proteins were submitted to kinetics analysis of its deuridylylation catalyzed by purified GlnD. The deuridylylation activity was strongly stimulated by glutamine and repressed by 2-oxoglutarate and this repression was strong enough to overcome the glutamine stimulus of enzymatic activity. We also constructed and analyzed a truncated version of GlnD, lacking the C-terminal regulatory ACT domains. The GlnDΔACT protein catalyzed the futile cycle of uridylylation and deuridylylation of PII, regardless of glutamine and 2-oxoglutarate levels. The results presented here suggest that GlnD can sense the glutamine:2-oxoglutarate ratio and confirm that the ACT domains of GlnD are the protein sensors of environment clues of nitrogen availability.

Asunto(s)

Proteínas Bacterianas/metabolismo , Glutamina/metabolismo , Herbaspirillum/enzimología , Ácidos Cetoglutáricos/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Biocatálisis , Glutamina/química , Ácidos Cetoglutáricos/química , Cinética , Unión Proteica , Dominios Proteicos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación

RESUMEN

Taenia crassiceps cysticerci are used as experimental model to study the host-parasite relationship and treatment of cysticercosis. One of the described mode of actions of nitazoxanide (NTZ) is to block the pyruvate ferredoxine oxidoreductase (PFOR) enzyme which is an essential enzyme to the parasite metabolism. The aim of this study was to determine the in vivo influence of one dosage of NTZ on the energetic metabolism of T. crassiceps cysticerci. Thirty days after the intraperitoneal inoculation of T. crassiceps cysticerci, BALB/c mice were orally treated with 7.5 mg/kg of NTZ. The control group was treated with physiologic solution (NaCl 0.9%). After 24 h, the animals were euthanized and the cysticerci were removed, washed, and processed for biochemical analysis. The organic acids detection occurred through high-performance liquid chromatographic and spectrophotometric analysis. While there was no difference in the glucose dosages, it was possible to observe a significant increase in the lactate concentrations and a decrease in the pyruvate concentrations of the NTZ-treated groups when compared to the control group. Also, there was a decrease in the urea and alpha-ketoglutarate concentrations. This probably occurred due to the impairment of the parasite's PFOR and nitroreductases leading an impairment of the mitochondrial aerobic pathways. In conclusion, the in vivo NTZ treatment leads to an increase in the lactic fermentation and to a decrease in the protein catabolism in T. crassiceps cysticerci.

Asunto(s)

Anticestodos/uso terapéutico , Cisticercosis/tratamiento farmacológico , Taenia/efectos de los fármacos , Tiazoles/uso terapéutico , Anaerobiosis , Animales , Cromatografía Líquida de Alta Presión , Cisticercosis/parasitología , Metabolismo Energético/efectos de los fármacos , Femenino , Glucosa/metabolismo , Interacciones Huésped-Parásitos , Ácidos Cetoglutáricos/metabolismo , Ácido Láctico/metabolismo , Ratones , Ratones Endogámicos BALB C , Nitrocompuestos , Taenia/metabolismo

RESUMEN

Accumulation of 2-methylcitric acid (2MCA) is observed in methylmalonic and propionic acidemias, which are clinically characterized by severe neurological symptoms. The exact pathogenetic mechanisms of brain abnormalities in these diseases are poorly established and very little has been reported on the role of 2MCA. In the present work we found that 2MCA markedly inhibited ADP-stimulated and uncoupled respiration in mitochondria supported by glutamate, with a less significant inhibition in pyruvate plus malate respiring mitochondria. However, no alterations occurred when α-ketoglutarate or succinate was used as respiratory substrates, suggesting a defect on glutamate oxidative metabolism. It was also observed that 2MCA decreased ATP formation in glutamate plus malate or pyruvate plus malate-supported mitochondria. Furthermore, 2MCA inhibited glutamate dehydrogenase activity at concentrations as low as 0.5 mM. Kinetic studies revealed that this inhibitory effect was competitive in relation to glutamate. In contrast, assays of osmotic swelling in non-respiring mitochondria suggested that 2MCA did not significantly impair mitochondrial glutamate transport. Finally, 2MCA provoked a significant decrease in mitochondrial membrane potential and induced swelling in Ca(2+)-loaded mitochondria supported by different substrates. These effects were totally prevented by cyclosporine A plus ADP or ruthenium red, indicating induction of mitochondrial permeability transition. Taken together, our data strongly indicate that 2MCA behaves as a potent inhibitor of glutamate oxidation by inhibiting glutamate dehydrogenase activity and as a permeability transition inducer, disturbing mitochondrial energy homeostasis. We presume that 2MCA-induced mitochondrial deleterious effects may contribute to the pathogenesis of brain damage in patients affected by methylmalonic and propionic acidemias. We propose that brain glutamate oxidation is disturbed by 2-methylcitric acid (2MCA), which accumulates in tissues from patients with propionic and methylmalonic acidemias because of a competitive inhibition of glutamate dehydrogenase (GDH) activity. 2MCA also induced mitochondrial permeability transition (PT) and decreased ATP generation in brain mitochondria. We believe that these pathomechanisms may be involved in the neurological dysfunction of these diseases.

Asunto(s)

Citratos/farmacología , Ácido Glutámico/metabolismo , Mitocondrias/efectos de los fármacos , Adenosina Difosfato/farmacología , Adenosina Trifosfato/biosíntesis , Errores Innatos del Metabolismo de los Aminoácidos/metabolismo , Animales , Encéfalo/metabolismo , Calcio/farmacología , Ciclosporina/farmacología , Metabolismo Energético/efectos de los fármacos , Glutamato Deshidrogenasa/antagonistas & inhibidores , Humanos , Ácidos Cetoglutáricos/metabolismo , Malatos/metabolismo , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial , Poro de Transición de la Permeabilidad Mitocondrial , Fosforilación Oxidativa/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Acidemia Propiónica/metabolismo , Ácido Pirúvico/metabolismo , Ratas , Ratas Wistar

RESUMEN

The metabolite 2-oxoglutarate (also known as α-ketoglutarate, 2-ketoglutaric acid, or oxoglutaric acid) lies at the intersection between the carbon and nitrogen metabolic pathways. This compound is a key intermediate of one of the most fundamental biochemical pathways in carbon metabolism, the tricarboxylic acid (TCA) cycle. In addition, 2-oxoglutarate also acts as the major carbon skeleton for nitrogen-assimilatory reactions. Experimental data support the conclusion that intracellular levels of 2-oxoglutarate fluctuate according to nitrogen and carbon availability. This review summarizes how nature has capitalized on the ability of 2-oxoglutarate to reflect cellular nutritional status through evolution of a variety of 2-oxoglutarate-sensing regulatory proteins. The number of metabolic pathways known to be regulated by 2-oxoglutarate levels has increased significantly in recent years. The signaling properties of 2-oxoglutarate are highlighted by the fact that this metabolite regulates the synthesis of the well-established master signaling molecule, cyclic AMP (cAMP), in Escherichia coli.

Asunto(s)

Escherichia coli/metabolismo , Ácidos Cetoglutáricos/metabolismo , Redes y Vías Metabólicas , Archaea/genética , Archaea/metabolismo , Carbono/metabolismo , Ciclo del Ácido Cítrico , Cianobacterias/genética , Cianobacterias/metabolismo , Metabolismo Energético , Escherichia coli/genética , Redes y Vías Metabólicas/genética , Nitrógeno/metabolismo , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo

RESUMEN

Nitrogen metabolism in Proteobacteria is controlled by the Ntr system, in which PII proteins play a pivotal role, controlling the activity of target proteins in response to the metabolic state of the cell. Characterization of the binding of molecular effectors to these proteins can provide information about their regulation. Here, the binding of ATP, ADP and 2-oxoglutarate (2-OG) to the Herbaspirillum seropedicae PII proteins, GlnB and GlnK, was characterized using isothermal titration calorimetry. Results show that these proteins can bind three molecules of ATP, ADP and 2-OG with homotropic negative cooperativity, and 2-OG binding stabilizes the binding of ATP. Results also show that the affinity of uridylylated forms of GlnB and GlnK for nucleotides is significantly lower than that of the nonuridylylated proteins. Furthermore, fluctuations in the intracellular concentration of 2-OG in response to nitrogen availability are shown. Results suggest that under nitrogen-limiting conditions, PII proteins tend to bind ATP and 2-OG. By contrast, after an ammonium shock, a decrease in the 2-OG concentration is observed causing a decrease in the affinity of PII proteins for ATP. This phenomenon may facilitate the exchange of ATP for ADP on the ligand-binding pocket of PII proteins, thus it is likely that under low ammonium, low 2-OG levels would favor the ADP-bound state.

Asunto(s)

Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/metabolismo , Herbaspirillum/enzimología , Ácidos Cetoglutáricos/metabolismo , Nucleotidiltransferasas/metabolismo , Proteínas PII Reguladoras del Nitrógeno/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Calorimetría , Glutamato-Amoníaco Ligasa/química , Glutamato-Amoníaco Ligasa/genética , Glutamato-Amoníaco Ligasa/metabolismo , Herbaspirillum/fisiología , Cinética , Ligandos , Fijación del Nitrógeno , Nucleotidiltransferasas/química , Nucleotidiltransferasas/genética , Proteínas PII Reguladoras del Nitrógeno/química , Proteínas PII Reguladoras del Nitrógeno/genética , Proteínas Quinasas/química , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Estrés Fisiológico , Volumetría

RESUMEN

In this work, in silico flux balance analysis is used for predicting the metabolic behavior of Streptomyces clavuligerus during clavulanic acid production. To choose the best objective function for use in the analysis, three different optimization problems are evaluated inside the flux balance analysis formulation: (i) maximization of the specific growth rate, (ii) maximization of the ATP yield, and (iii) maximization of clavulanic acid production. Maximization of ATP yield showed the best predictions for the cellular behavior. Therefore, flux balance analysis using ATP as objective function was used for analyzing different scenarios of nutrient limitations toward establishing the effect of limiting the carbon, nitrogen, phosphorous, and oxygen sources on the growth and clavulanic acid production rates. Obtained results showed that ammonia and phosphate limitations are the ones most strongly affecting clavulanic acid biosynthesis. Furthermore, it was possible to identify the ornithine flux from the urea cycle and the α-ketoglutarate flux from the TCA cycle as the most determinant internal fluxes for promoting clavulanic acid production.

Asunto(s)

Ácido Clavulánico/biosíntesis , Análisis de Flujos Metabólicos/métodos , Streptomyces/metabolismo , Carbono/metabolismo , Ciclo del Ácido Cítrico , Ácidos Cetoglutáricos/metabolismo , Nitrógeno/metabolismo

RESUMEN

In this study, we used primary cultures of fish hepatic cells as a tool for evaluating the effects of environmental contamination. Primary hepatic cell cultures derived from the subtropical fish Metynnis roosevelti were exposed to different concentrations (0.275, 2.75 and 27.5 µg L(-1)) of the herbicides 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-chloro-2-methylphenoxyacetic acid (MCPA). Cellular respiratory activity was evaluated by polarography using three substrates: 0.5 M glucose, 0.5 M succinate and 0.5 M α-ketoglutarate. Significant changes were observed in cellular oxygen consumption with 0.5 M α-ketoglutarate. Even at low concentrations, 2,4-D and MCPA were potent uncouplers of oxidative phosphorylation. Primary cultures of M. roosevelti liver cells may provide a useful tool for the evaluation of environmental contaminant effects. A review of regulations regarding permitted concentrations of these herbicides is needed.

Asunto(s)

Ácido 2,4-Diclorofenoxiacético/toxicidad , Ácido 2-Metil-4-clorofenoxiacético/toxicidad , Herbicidas/toxicidad , Animales , Células Cultivadas , Characidae/metabolismo , Relación Dosis-Respuesta a Droga , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Ácidos Cetoglutáricos/metabolismo , Hígado/efectos de los fármacos , Polarografía/instrumentación , Polarografía/métodos , Pruebas de Toxicidad/métodos

RESUMEN

Glutamate (Glu) is a taste enhancer that contributes to the characteristic flavour of foods. In fruit of tomato (Solanum lycopersicum L.), the Glu content increases dramatically during the ripening process, becoming the most abundant free amino acid when the fruit become red. There is also a concomitant increase in NADH-dependent glutamate dehydrogenase (GDH) activity during the ripening transition. This enzyme is located in the mitochondria and catalyses the reversible amination of 2-oxoglutarate to Glu. To investigate the potential effect of GDH on Glu metabolism, the abundance of GDH was altered by artificial microRNA technology. Efficient silencing of all the endogenous SlGDH genes was achieved, leading to a dramatic decrease in total GDH activity. This decrease in GDH activity did not lead to any clear morphological or metabolic phenotype in leaves or green fruit. However, red fruit on the transgenic plants showed markedly reduced levels of Glu and a large increase in aspartate, glucose and fructose content in comparison to wild-type fruit. These results suggest that GDH is involved in the synthesis of Glu in tomato fruit during the ripening processes. This contrasts with the biological role ascribed to GDH in many other tissues and species. Overall, these findings suggest that GDH has a major effect on the control of metabolic composition during tomato fruit ripening, but not at other stages of development.

Asunto(s)

Glutamato Deshidrogenasa (NADP+)/metabolismo , Ácido Glutámico/metabolismo , NADP/metabolismo , Solanum lycopersicum/enzimología , Ácido Aspártico/metabolismo , Fructosa/metabolismo , Frutas/enzimología , Frutas/genética , Frutas/metabolismo , Regulación de la Expresión Génica de las Plantas , Silenciador del Gen , Glucosa/metabolismo , Glutamato Deshidrogenasa (NADP+)/genética , Ácidos Cetoglutáricos/metabolismo , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , MicroARNs/genética , Mitocondrias/enzimología , Especificidad de Órganos , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Especificidad de la Especie

RESUMEN

The aims of this study were to evaluate the effects of the addition of metabolic precursors and polydimethylsiloxane (PDMS) as an oxygen carrier to cultures of Bacillus subtilis BL53 during the production of γ-PGA. Kinetics analyses of cultivations of different media showed that B. subtilis BL53 is an exogenous glutamic acid-dependent strain. When the metabolic pathway precursors of γ-PGA synthesis, L-glutamine and a-ketoglutaric acid, were added to the culture medium, production of the biopolymer was increased by 20 % considering the medium without these precursors. The addition of 10 % of the oxygen carrier PDMS to cultures caused a two-fold increase in the volumetric oxygen mass transfer coefficient (kLa), improving γ-PGA production and productivity. Finally, bioreactor cultures of B. subtilis BL53 adopting the combination of optimized medium E, added of glutamine, α-ketoglutaric acid, and PDMS, showed a productivity of 1 g L(-1) h(-1) of g-PGA after only 24 h of cultivation. Results of this study suggest that the use of metabolic pathway precursors glutamine and a-ketolgutaric acid, combined with the addition of PDMS as an oxygen carrier in bioreactors, can improve γ-PGA production and productivity by Bacillus strains .

Asunto(s)

Bacillus subtilis/metabolismo , Dimetilpolisiloxanos/metabolismo , Redes y Vías Metabólicas , Ácido Poliglutámico/análogos & derivados , Medios de Cultivo/metabolismo , Ácido Glutámico/metabolismo , Glutamina/metabolismo , Ácidos Cetoglutáricos/metabolismo , Ácido Poliglutámico/biosíntesis

RESUMEN

The trimeric PII signal transduction proteins regulate the function of a variety of target proteins predominantly involved in nitrogen metabolism. ATP, ADP and 2-oxoglutarate (2-OG) are key effector molecules influencing PII binding to targets. Studies of PII proteins have established that the 20-residue T-loop plays a central role in effector sensing and target binding. However, the specific effects of effector binding on T-loop conformation have remained poorly documented. We present eight crystal structures of the Azospirillum brasilense PII protein GlnZ, six of which are cocrystallized and liganded with ADP or ATP. We find that interaction with the diphosphate moiety of bound ADP constrains the N-terminal part of the T-loop in a characteristic way that is maintained in ADP-promoted complexes with target proteins. In contrast, the interactions with the triphosphate moiety in ATP complexes are much more variable and no single predominant interaction mode is apparent except for the ternary MgATP/2-OG complex. These conclusions can be extended to most investigated PII proteins of the GlnB/GlnK subfamily. Unlike reported for other PII proteins, microcalorimetry reveals no cooperativity between the three binding sites of GlnZ trimers for any of the three effectors under carefully controlled experimental conditions.

Asunto(s)

Azospirillum brasilense/metabolismo , Proteínas Bacterianas/química , Nitrógeno/metabolismo , Termodinámica , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/metabolismo , Sitios de Unión , Calorimetría , Cristalografía por Rayos X , Ácidos Cetoglutáricos/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica

RESUMEN

The PII family comprises a group of widely distributed signal transduction proteins. The archetypal function of PII is to regulate nitrogen metabolism in bacteria. As PII can sense a range of metabolic signals, it has been suggested that the number of metabolic pathways regulated by PII may be much greater than described in the literature. In order to provide experimental evidence for this hypothesis a PII protein affinity column was used to identify PII targets in Azospirillum brasilense. One of the PII partners identified was the biotin carboxyl carrier protein (BCCP), a component of the acetyl-CoA carboxylase which catalyses the committed step in fatty acid biosynthesis. As BCCP had been previously identified as a PII target in Arabidopsis thaliana we hypothesized that the PII -BCCP interaction would be conserved throughout Bacteria. In vitro experiments using purified proteins confirmed that the PII -BCCP interaction is conserved in Escherichia coli. The BCCP-PII interaction required MgATP and was dissociated by increasing 2-oxoglutarate. The interaction was modestly affected by the post-translational uridylylation status of PII ; however, it was completely dependent on the post-translational biotinylation of BCCP.

Asunto(s)

Acetil-CoA Carboxilasa/metabolismo , Azospirillum brasilense/enzimología , Proteínas Bacterianas/metabolismo , Proteínas PII Reguladoras del Nitrógeno/metabolismo , Adenosina Trifosfato/metabolismo , Arabidopsis/enzimología , Escherichia coli/enzimología , Acido Graso Sintasa Tipo II/metabolismo , Ácidos Cetoglutáricos/metabolismo , Unión Proteica , Mapeo de Interacción de Proteínas

RESUMEN

Tellurite is toxic to most microorganisms because of its ability to generate oxidative stress. However, the way in which tellurite interferes with cellular processes is not fully understood to date. In this line, it was previously shown that tellurite-exposed cells displayed reduced activity of the α-ketoglutarate dehydrogenase complex (α-KGDH), which resulted in α-ketoglutarate (α-KG) accumulation. In this work, we assessed if α-KG accumulation in tellurite-exposed E. coli could also result from increased isocitrate dehydrogenase (ICDH) and glutamate dehydrogenase (GDH) activities, both enzymes involved in α-KG synthesis. Unexpectedly both activities were found to decrease in the presence of the toxicant, an observation that seems to result from the decreased transcription of icdA and gdhA genes (encoding ICDH and GDH, resp.). Accordingly, isocitrate levels were found to increase in tellurite-exposed E. coli. In the presence of the toxicant, cells lacking icdA or gdhA exhibited decreased reactive oxygen species (ROS) levels and higher tellurite sensitivity as compared to the wild type strain. Finally, a novel branch activity of ICDH as tellurite reductase is presented.

Asunto(s)

Escherichia coli/efectos de los fármacos , Isocitrato Deshidrogenasa/metabolismo , Ácidos Cetoglutáricos/metabolismo , Oxidorreductasas/metabolismo , Transcripción Genética/efectos de los fármacos , Escherichia coli/enzimología , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Telurio/toxicidad

RESUMEN

To determine the extent to which the supply of the precursor 2-oxoglutarate (2-OG) controls the synthesis of lysine in Saccharomyces cerevisiae growing exponentially in high glucose, top-down elasticity analysis was used. Three groups of reactions linked by 2-OG were defined. The 2-OG supply group comprised all metabolic steps leading to its formation, and the two 2-OG consumer groups comprised the enzymes and transporters involved in 2-OG transformation into lysine and glutamate and their further utilization for protein synthesis and storage. Various 2-OG steady-state concentrations that produced different fluxes to lysine and glutamate were attained using yeast mutants with increasing activities of Krebs cycle enzymes and decreased activities of Lys synthesis enzymes. The elasticity coefficients of the three enzyme groups were determined from the dependence of the amino acid fluxes on the 2-OG concentration. The respective degrees of control on the flux towards lysine (flux control coefficients) were determined from their elasticities, and were 1.1, 0.41 and -0.52 for the 2-OG producer group and the Lys and Glu branches, respectively. Thus, the predominant control exerted by the 2-OG supply on the rate of lysine synthesis suggests that over-expression of 2-OG producer enzymes may be a highly effective strategy to enhance Lys production.

Asunto(s)

Ácidos Cetoglutáricos/metabolismo , Lisina/biosíntesis , Saccharomyces cerevisiae/metabolismo , Ciclo del Ácido Cítrico/genética , Enzimas/genética , Enzimas/metabolismo , Cinética , Redes y Vías Metabólicas , Modelos Biológicos , Mutación , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo