RESUMEN
Acetate:succinate CoA-transferases (ASCT) are acetate-producing enzymes in hydrogenosomes, anaerobically functioning mitochondria and in the aerobically functioning mitochondria of trypanosomatids. Although acetate is produced in the hydrogenosomes of a number of anaerobic microbial eukaryotes such as Trichomonas vaginalis, no acetate producing enzyme has ever been identified in these organelles. Acetate production is the last unidentified enzymatic reaction of hydrogenosomal carbohydrate metabolism. We identified a gene encoding an enzyme for acetate production in the genome of the hydrogenosome-containing protozoan parasite T. vaginalis. This gene shows high similarity to Saccharomyces cerevisiae acetyl-CoA hydrolase and Clostridium kluyveri succinyl-CoA:CoA-transferase. Here we demonstrate that this protein is expressed and is present in the hydrogenosomes where it functions as the T. vaginalis acetate:succinate CoA-transferase (TvASCT). Heterologous expression of TvASCT in CHO cells resulted in the expression of an active ASCT. Furthermore, homologous overexpression of the TvASCT gene in T. vaginalis resulted in an equivalent increase in ASCT activity. It was shown that the CoA transferase activity is succinate-dependent. These results demonstrate that this acetyl-CoA hydrolase/transferase homolog functions as the hydrogenosomal ASCT of T. vaginalis. This is the first hydrogenosomal acetate-producing enzyme to be identified. Interestingly, TvASCT does not share any similarity with the mitochondrial ASCT from Trypanosoma brucei, the only other eukaryotic succinate-dependent acetyl-CoA-transferase identified so far. The trichomonad enzyme clearly belongs to a distinct class of acetate:succinate CoA-transferases. Apparently, two completely different enzymes for succinate-dependent acetate production have evolved independently in ATP-generating organelles.
Asunto(s)
Coenzima A Transferasas/metabolismo , Orgánulos/enzimología , Trichomonas vaginalis/enzimología , Acetatos/metabolismo , Acetilcoenzima A/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Antiprotozoarios/inmunología , Especificidad de Anticuerpos , Células CHO , Cromatografía por Intercambio Iónico , Coenzima A Transferasas/química , Coenzima A Transferasas/aislamiento & purificación , Cricetinae , Cricetulus , Genes Protozoarios , Cinética , Datos de Secuencia Molecular , Péptidos/química , Transporte de Proteínas , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Fracciones Subcelulares/enzimología , Ácido Succínico/metabolismo , Trichomonas vaginalis/genéticaRESUMEN
We investigated whether substrate availability influences the type of energy metabolism in procyclic Trypanosoma brucei. We show that absence of glycolytic substrates (glucose and glycerol) does not induce a shift from a fermentative metabolism to complete oxidation of substrates. We also show that glucose (and even glycolysis) is not essential for normal functioning and proliferation of pleomorphic procyclic T. brucei cells. Furthermore, absence of glucose did not result in increased degradation of amino acids. Variations in availability of glucose and glycerol did result, however, in adaptations in metabolism in such a way that the glycosome was always in redox balance. We argue that it is likely that, in procyclic cells, phosphoglycerate kinase is located not only in the cytosol, but also inside glycosomes, as otherwise an ATP deficit would occur in this organelle. We demonstrate that procyclic T. brucei uses parts of the Krebs cycle for purposes other than complete degradation of mitochondrial substrates. We suggest that citrate synthase plus pyruvate dehydrogenase and malate dehydrogenase are used to transport acetyl-CoA units from the mitochondrion to the cytosol for the biosynthesis of fatty acids, a process we show to occur in proliferating procyclic cells. The part of the Krebs cycle consisting of alpha-ketoglutarate dehydrogenase and succinyl-CoA synthetase was used for the degradation of proline and glutamate to succinate. We also demonstrate that the subsequent enzymes of the Krebs cycle, succinate dehydrogenase and fumarase, are most likely used for conversion of succinate into malate, which can then be used in gluconeogenesis.
Asunto(s)
Bioquímica/métodos , Ciclo del Ácido Cítrico , Oxígeno/metabolismo , Trypanosoma brucei brucei/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Animales , Línea Celular , Cromatografía Líquida de Alta Presión , Citosol/metabolismo , Metabolismo Energético , Ácidos Grasos/química , Ácidos Grasos/metabolismo , Fermentación , Glucosa/química , Glucosa/metabolismo , Ácido Glutámico/química , Glicerol/química , Glicerol/metabolismo , Glucólisis , Modelos Biológicos , Modelos Químicos , Oxidación-Reducción , Prolina/química , Especificidad por Sustrato , Ácido Succínico/química , Treonina/química , Factores de Tiempo , Trypanosoma brucei brucei/químicaRESUMEN
Acetyl:succinate CoA-transferase (ASCT) is an acetate-producing enzyme shared by hydrogenosomes, mitochondria of trypanosomatids, and anaerobically functioning mitochondria. The gene encoding ASCT in the protozoan parasite Trypanosoma brucei was identified as a new member of the CoA transferase family. Its assignment to ASCT activity was confirmed by 1) a quantitative correlation of protein expression and activity upon RNA interference-mediated repression, 2) the absence of activity in homozygous Deltaasct/Deltaasct knock out cells, 3) mitochondrial colocalization of protein and activity, 4) increased activity and acetate excretion upon transgenic overexpression, and 5) depletion of ASCT activity from lysates upon immunoprecipitation. Genetic ablation of ASCT produced a severe growth phenotype, increased glucose consumption, and excretion of beta-hydroxybutyrate and pyruvate, indicating accumulation of acetyl-CoA. Analysis of the excreted end products of (13)C-enriched and (14)C-labeled glucose metabolism showed that acetate excretion was only slightly reduced. Adaptation to ASCT deficiency, however, was an infrequent event at the population level, indicating the importance of this enzyme. These studies show that ASCT is indeed involved in acetate production, but is not essential, as apparently it is not the only enzyme that produces acetate in T. brucei.
Asunto(s)
Coenzima A Transferasas/genética , Glucosa/metabolismo , Trypanosoma brucei brucei/enzimología , Animales , Coenzima A Transferasas/química , Coenzima A Transferasas/fisiología , Mitocondrias/enzimología , Resonancia Magnética Nuclear Biomolecular , Interferencia de ARNRESUMEN
Anaerobic chytridiomycete fungi possess hydrogenosomes, which generate hydrogen and ATP, but also acetate and formate as end-products of a prokaryotic-type mixed-acid fermentation. Notably, the anaerobic chytrids Piromyces and Neocallimastix use pyruvate:formate lyase (PFL) for the catabolism of pyruvate, which is in marked contrast to the hydrogenosomal metabolism of the anaerobic parabasalian flagellates Trichomonas vaginalis and Tritrichomonas foetus, because these organisms decarboxylate pyruvate with the aid of pyruvate:ferredoxin oxidoreductase (PFO). Here, we show that the chytrids Piromyces sp. E2 and Neocallimastix sp. L2 also possess an alcohol dehydrogenase E (ADHE) that makes them unique among hydrogenosome-bearing anaerobes. We demonstrate that Piromyces sp. E2 routes the final steps of its carbohydrate catabolism via PFL and ADHE: in axenic culture under standard conditions and in the presence of 0.3% fructose, 35% of the carbohydrates were degraded in the cytosol to the end-products ethanol, formate, lactate and succinate, whereas 65% were degraded via the hydrogenosomes to acetate and formate. These observations require a refinement of the previously published metabolic schemes. In particular, the importance of the hydrogenase in this type of hydrogenosome has to be revisited.
Asunto(s)
Acetiltransferasas/metabolismo , Alcohol Deshidrogenasa/metabolismo , Etanol/metabolismo , Proteínas Fúngicas/metabolismo , Piromyces/enzimología , Alcohol Deshidrogenasa/genética , Secuencia de Aminoácidos , Clonación Molecular , Metabolismo Energético , Fermentación , Proteínas Fúngicas/genética , Datos de Secuencia Molecular , Filogenia , Alineación de SecuenciaRESUMEN
The importance of a functional Krebs cycle for energy generation in the procyclic stage of Trypanosoma brucei was investigated under physiological conditions during logarithmic phase growth of a pleomorphic parasite strain. Wild type procyclic cells and mutants with targeted deletion of the gene coding for aconitase were derived by synchronous in vitro differentiation from wild type and mutant (Delta aco::NEO/Delta aco::HYG) bloodstream stage parasites, respectively, where aconitase is not expressed and is dispensable. No differences in intracellular levels of glycolytic and Krebs cycle intermediates were found in procyclic wild type and mutant cells, except for citrate that accumulated up to 90-fold in the mutants, confirming the absence of aconitase activity. Surprisingly, deletion of aconitase did not change differentiation nor the growth rate or the intracellular ATP/ADP ratio in those cells. Metabolic studies using radioactively labeled substrates and NMR analysis demonstrated that glucose and proline were not degraded via the Krebs cycle to CO(2). Instead, glucose was degraded to acetate, succinate, and alanine, whereas proline was degraded to succinate. Importantly, there was absolutely no difference in the metabolic products released by wild type and aconitase knockout parasites, and both were for survival strictly dependent on respiration via the mitochondrial electron transport chain. Hence, although the Krebs cycle enzymes are present, procyclic T. brucei do not use Krebs cycle activity for energy generation, but the mitochondrial respiratory chain is essential for survival and growth. We therefore propose a revised model of the energy metabolism of procyclic T. brucei.
Asunto(s)
Aconitato Hidratasa/metabolismo , Metabolismo Energético/fisiología , Trypanosoma brucei brucei/fisiología , Aconitato Hidratasa/deficiencia , Aconitato Hidratasa/genética , Animales , Ciclo del Ácido Cítrico , Eliminación de Gen , Glucosa/metabolismo , Glutamato Deshidrogenasa/deficiencia , Glutamato Deshidrogenasa/genética , Glutamato Deshidrogenasa/metabolismo , Isocitrato Deshidrogenasa/deficiencia , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Complejo Cetoglutarato Deshidrogenasa/deficiencia , Complejo Cetoglutarato Deshidrogenasa/genética , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Espectroscopía de Resonancia Magnética , Cianuro de Potasio/farmacología , Transfección , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crecimiento & desarrolloRESUMEN
Mitochondria are usually considered to be the powerhouses of the cell and to be responsible for the aerobic production of ATP. However, many eukaryotic organisms are known to possess anaerobically functioning mitochondria, which differ significantly from classical aerobically functioning mitochondria. Recently, functional and phylogenetic studies on some enzymes involved clearly indicated an unexpected evolutionary relationship between these anaerobically functioning mitochondria and the classical aerobic type. Mitochondria evolved by an endosymbiotic event between an anaerobically functioning archaebacterial host and an aerobic alpha-proteobacterium. However, true anaerobically functioning mitochondria, such as found in parasitic helminths and some lower marine organisms, most likely did not originate directly from the pluripotent ancestral mitochondrion, but arose later in evolution from the aerobic type of mitochondria after these were already adapted to an aerobic way of life by losing their anaerobic capacities. This review will focus on some biochemical and evolutionary aspects of these fermentative mitochondria, with special attention to fumarate reductase, the synthesis of the rhodoquinone involved, and the enzymes involved in acetate production (acetate : succinate CoA-transferase and succinyl CoA-synthetase).