RESUMEN
Lysine acetylation is an evolutionarily conserved protein modification that changes protein functions and plays an essential role in many cellular processes, such as central metabolism, transcriptional regulation, chemotaxis, and pathogen virulence. It can alter DNA binding, enzymatic activity, protein-protein interactions, protein stability, or protein localization. In prokaryotes, lysine acetylation occurs non-enzymatically and by the action of lysine acetyltransferases (KAT). In enzymatic acetylation, KAT transfers the acetyl group from acetyl-CoA (AcCoA) to the lysine side chain. In contrast, acetyl phosphate (AcP) is the acetyl donor of chemical acetylation. Regardless of the acetylation type, the removal of acetyl groups from acetyl lysines occurs only enzymatically by lysine deacetylases (KDAC). KATs are grouped into three main superfamilies based on their catalytic domain sequences and biochemical characteristics of catalysis. Specifically, members of the GNAT are found in eukaryotes and prokaryotes and have a core structural domain architecture. These enzymes can acetylate small molecules, metabolites, peptides, and proteins. This review presents current knowledge of acetylation mechanisms and functional implications in bacterial metabolism, pathogenicity, stress response, translation, and the emerging topic of protein acetylation in the gut microbiome. Additionally, the methods used to elucidate the biological significance of acetylation in bacteria, such as relative quantification and stoichiometry quantification, and the genetic code expansion tool (CGE), are reviewed.
Asunto(s)
Bacterias , Proteínas Bacterianas , Procesamiento Proteico-Postraduccional , Acetilación , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Bacterias/metabolismo , Bacterias/genética , Lisina/metabolismo , Lisina Acetiltransferasas/metabolismo , Lisina Acetiltransferasas/genética , Acetilcoenzima A/metabolismoRESUMEN
In brief: Pyruvate metabolism is one of the main metabolic pathways during oocyte maturation. This study demonstrates that pyruvate metabolism also regulates the epigenetic and molecular maturation in bovine oocytes. Abstract: Pyruvate, the final product of glycolysis, undergoes conversion into acetyl-CoA within the mitochondria of oocytes, serving as a primary fuel source for the tricarboxylic acid (TCA) cycle. The citrate generated in the TCA cycle can be transported to the cytoplasm and converted back into acetyl-CoA. This acetyl-CoA can either fuel lipid synthesis or act as a substrate for histone acetylation. This study aimed to investigate how pyruvate metabolism influences lysine 9 histone 3 acetylation (H3K9ac) dynamics and RNA transcription in bovine oocytes during in vitro maturation (IVM). Bovine cumulus-oocyte complexes were cultured in vitro for 24 h, considering three experimental groups: Control (IVM medium only), DCA (IVM supplemented with sodium dichloroacetate, a stimulant of pyruvate oxidation into acetyl-CoA), or IA (IVM supplemented with sodium iodoacetate, a glycolysis inhibitor). The results revealed significant alterations in oocyte metabolism in both treatments, promoting the utilization of lipids as an energy source. These changes during IVM affected the dynamics of H3K9ac, subsequently influencing the oocyte's transcriptional activity. In the DCA and IA groups, a total of 148 and 356 differentially expressed genes were identified, respectively, compared to the control group. These findings suggest that modifications in pyruvate metabolism trigger the activation of metabolic pathways, particularly lipid metabolism, changing acetyl-CoA availability and H3K9ac levels, ultimately impacting the mRNA content of in vitro matured bovine oocytes.
Asunto(s)
Histonas , Técnicas de Maduración In Vitro de los Oocitos , Animales , Bovinos , Femenino , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Técnicas de Maduración In Vitro de los Oocitos/métodos , Acetilcoenzima A/metabolismo , Histonas/metabolismo , Oocitos/metabolismo , Ácido Pirúvico/farmacología , Ácido Pirúvico/metabolismo , Epigénesis Genética , Células del CúmuloRESUMEN
The epigenetic reprogramming that occurs during the earliest stages of embryonic development has been described as crucial for the initial events of cell specification and differentiation. Recently, the metabolic status of the embryo has gained attention as one of the main factors coordinating epigenetic events. In this work, we investigate the link between pyruvate metabolism and epigenetic regulation by culturing bovine embryos from day 5 in the presence of dichloroacetate (DCA), a pyruvate analog that increases the pyruvate to acetyl-CoA conversion, and iodoacetate (IA), which inhibits the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), leading to glycolysis inhibition. After 8 h of incubation, both DCA and IA-derived embryos presented higher mitochondrial membrane potential. Nevertheless, in both cases, lower levels of acetyl-CoA, ATP-citrate lyase and mitochondrial membrane potential were found in blastocysts, suggesting an adaptative metabolic response, especially in the DCA group. The metabolic alteration found in blastocysts led to changes in the global pattern of H3K9 and H3K27 acetylation and H3K27 trimethylation. Transcriptome analysis revealed that such alterations resulted in molecular differences mainly associated to metabolic processes, establishment of epigenetic marks, control of gene expression and cell cycle. The latter was further confirmed by the alteration of total cell number and cell differentiation in both groups when compared to the control. These results corroborate previous evidence of the relationship between the energy metabolism and the epigenetic reprogramming in preimplantation bovine embryos, reinforcing that the culture system is decisive for precise epigenetic reprogramming, with consequences for the molecular control and differentiation of cells.
Asunto(s)
Epigénesis Genética , Transcriptoma , Femenino , Embarazo , Animales , Bovinos , Acetilcoenzima A/metabolismo , Desarrollo Embrionario/genética , Blastocisto/metabolismo , Perfilación de la Expresión Génica , Piruvatos/metabolismoRESUMEN
The malaria parasite Plasmodium falciparum possesses a unique Acetyl-CoA Synthetase (PfACS), which provides acetyl moieties for different metabolic and regulatory cellular pathways. We characterized PfACS and studied its role focusing on epigenetic modifications using the var gene family as reporter genes. For this, mutant lines to modulate plasmodial ACS expression by degron-mediated protein degradation and ribozyme-induced transcript decay were created. Additionally, an inhibitor of the human Acetyl-CoA Synthetase 2 was tested for its effectiveness in interfering with PfACS. The knockdown of PfACS or its inhibition resulted in impaired parasite growth. Decreased levels of PfACS also led to differential histone acetylation patterns, altered variant gene expression, and concomitantly decreased cytoadherence of infected red blood cells containing knocked-down parasites. Further, ChIP analysis revealed the presence of PfACS in many loci in ring stage parasites, underscoring its involvement in the regulation of chromatin. Due to its central function in the plasmodial metabolism and significant differences to human ACS, PfACS is an interesting target for drug development.
Asunto(s)
Parásitos , Plasmodium falciparum , Acetilcoenzima A , Animales , Cromatina , Humanos , Ligasas , Plasmodium falciparum/genéticaRESUMEN
BACKGROUND: Freezing human biopsies is common in clinical practice for storage. However, this technique disrupts mitochondrial membranes, hampering further analyses of respiratory function. To contribute to laboratorial diagnosis of mitochondrial diseases, this study sought to develop a respirometry approach using O2k (Oroboros Ins.) to measure the whole electron transport chain (ETC) activity in homogenates of frozen skeletal muscle biopsies. PATIENTS AND METHODS: We enrolled 16 patients submitted to muscle biopsy in the process of routine diagnostic investigation: four with mitochondrial disease and severe mitochondrial dysfunction; seven with exercise intolerance and multiple deletions of mitochondrial DNA, presenting mild to moderate mitochondrial dysfunction; five without mitochondrial disease, as controls. Whole homogenates of muscle fragments were prepared using grinder-type equipment. O2 consumption rates were normalized using citrate synthase activity. RESULTS: Transmission electron microscopy confirmed mitochondrial membrane discontinuation, indicating increased permeability of mitochondrial membranes in homogenates from frozen biopsies. O2 consumption rates in the presence of acetyl-CoA lead to maximum respiratory rates sensitive to rotenone, malonate and antimycin. This protocol of acetyl-CoA-driven respiration (ACoAR), applied in whole homogenates of frozen muscle, was sensitive enough to identify ETC abnormality, even in patients with mild to moderate mitochondrial dysfunction. We demonstrated adequate repeatability of ACoAR and found significant correlation between O2 consumption rates and enzyme activity assays of individual ETC complexes. CONCLUSIONS: We present preliminary data on a simple, low cost and reliable procedure to measure respiratory function in whole homogenates of frozen skeletal muscle biopsies, contributing to diagnosis of mitochondrial diseases in humans.
Asunto(s)
Acetilcoenzima A/metabolismo , Mitocondrias Musculares/metabolismo , Enfermedades Mitocondriales/diagnóstico , Músculo Esquelético/metabolismo , Consumo de Oxígeno , Adolescente , Adulto , Biopsia , Respiración de la Célula , Niño , Técnicas de Laboratorio Clínico/métodos , Criopreservación , Transporte de Electrón , Femenino , Humanos , Síndrome MELAS/diagnóstico , Síndrome MELAS/metabolismo , Masculino , Potencial de la Membrana Mitocondrial , Enfermedades Mitocondriales/metabolismo , Membranas Mitocondriales/metabolismo , Músculo Esquelético/patología , Oftalmoplejía Externa Progresiva Crónica/diagnóstico , Oftalmoplejía Externa Progresiva Crónica/metabolismo , Fosforilación Oxidativa , Permeabilidad , Manejo de Especímenes , Adulto JovenRESUMEN
Iterative type I polyketide synthases (PKS) are megaenzymes essential to the biosynthesis of an enormously diverse array of bioactive natural products. Each PKS contains minimally three functional domains, ß-ketosynthase (KS), acyltransferase (AT), and acyl carrier protein (ACP), and a subset of reducing domains such as ketoreductase (KR), dehydratase (DH), and enoylreductase (ER). The substrate selection, condensation reactions, and ß-keto processing of the polyketide growing chain are highly controlled in a programmed manner. However, the structural features and mechanistic rules that orchestrate the iterative cycles, processing domains functionality, and chain termination in this kind of megaenzymes are often poorly understood. Here, we present a biochemical and functional characterization of the KS and the AT domains of a PKS from the mallard duck Anas platyrhynchos (ApPKS). ApPKS belongs to an animal PKS family phylogenetically more related to bacterial PKS than to metazoan fatty acid synthases. Through the dissection of the ApPKS enzyme into mono- to didomain fragments and its reconstitution in vitro, we determined its substrate specificity toward different starters and extender units. ApPKS AT domain can effectively transfer acetyl-CoA and malonyl-CoA to the ApPKS ACP stand-alone domain. Furthermore, the KS and KR domains, in the presence of Escherichia coli ACP, acetyl-CoA, and malonyl-CoA, showed the ability to catalyze the chain elongation and the ß-keto reduction steps necessary to yield a 3-hydroxybutyryl-ACP derivate. These results provide new insights into the catalytic efficiency and specificity of this uncharacterized family of PKSs.
Asunto(s)
Acetilcoenzima A/metabolismo , Malonil Coenzima A/metabolismo , Sintasas Poliquetidas/metabolismo , Acilación , Animales , Dominio Catalítico , Patos , Cinética , Filogenia , Sintasas Poliquetidas/química , Sintasas Poliquetidas/genética , Dominios Proteicos , Especificidad por SustratoRESUMEN
Mycobacterium tuberculosis produces a large number of structurally diverse lipids that have been implicated in the pathogenicity, persistence and antibiotic resistance of this organism. Most building blocks involved in the biosynthesis of all these lipids are generated by acyl-CoA carboxylases whose subunit composition and physiological roles have not yet been clearly established. Inconclusive data in the literature refer to the exact protein composition and substrate specificity of the enzyme complex that produces the long-chain α-carboxy-acyl-CoAs, which are substrates involved in the last step of condensation mediated by the polyketide synthase 13 to synthesize mature mycolic acids. Here we have successfully reconstituted the long-chain acyl-CoA carboxylase (LCC) complex from its purified components, the α subunit (AccA3), the ε subunit (AccE5) and the two ß subunits (AccD4 and AccD5), and demonstrated that the four subunits are essential for its activity. Furthermore, we also showed by substrate competition experiments and the use of a specific inhibitor that the AccD5 subunit's role in the carboxylation of the long acyl-CoAs, as part of the LCC complex, was structural rather than catalytic. Moreover, AccD5 was also able to carboxylate its natural substrates, acetyl-CoA and propionyl-CoA, in the context of the LCC enzyme complex. Thus, the supercomplex formed by these four subunits has the potential to generate the main substrates, malonyl-CoA, methylmalonyl-CoA and α-carboxy-C24-26 -CoA, used as condensing units for the biosynthesis of all the lipids present in this pathogen.
Asunto(s)
Proteínas Bacterianas/metabolismo , Ligasas de Carbono-Carbono/metabolismo , Mycobacterium tuberculosis/metabolismo , Sintasas Poliquetidas/metabolismo , Subunidades de Proteína/metabolismo , Acetilcoenzima A/metabolismo , Acilcoenzima A/metabolismo , Proteínas Bacterianas/genética , Ligasas de Carbono-Carbono/genética , Clonación Molecular , Pruebas de Enzimas , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Cinética , Malonil Coenzima A/metabolismo , Mycobacterium tuberculosis/genética , Ácidos Micólicos/metabolismo , Sintasas Poliquetidas/genética , Ingeniería de Proteínas , Subunidades de Proteína/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
In this study, the respiratory activity and carbon usage of the mutant strain of A. vinelandii AT6, impaired in poly-ß-hydroxybutyrate (PHB) production, and their relationship with the synthesis of alginate were evaluated. The alginate yield and the specific oxygen uptake rate were higher (2.5-fold and 62 %, respectively) for the AT6 strain, compared to the control strain (ATCC 9046), both in shake flasks cultures and in bioreactor, under fixed dissolved oxygen tension (1 %). In contrast, the degree of acetylation was similar in both strains. These results, together with the analysis of carbon usage (% C-mol), suggest that in the case of the AT6 strain, the flux of acetyl-CoA (precursor molecule for PHB biosynthesis and alginate acetylation) was diverted to the respiratory chain passing through the tricarboxylic acids cycle, and an important % C-mol was directed through alginate biosynthesis, up to 25.9 % and to a lesser extent, to biomass production (19.7 %).
Asunto(s)
Azotobacter vinelandii/metabolismo , Carbono/metabolismo , Acetilcoenzima A/metabolismo , Alginatos , Azotobacter vinelandii/genética , Ácido Glucurónico/biosíntesis , Ácidos Hexurónicos , Hidroxibutiratos/metabolismo , Mutación , Oxígeno/metabolismo , Poliésteres/metabolismoRESUMEN
BACKGROUND: Astaxanthin is a potent antioxidant with increasing biotechnological interest. In Xanthophyllomyces dendrorhous, a natural source of this pigment, carotenogenesis is a complex process regulated through several mechanisms, including the carbon source. X. dendrorhous produces more astaxanthin when grown on a non-fermentable carbon source, while decreased astaxanthin production is observed in the presence of high glucose concentrations. In the present study, we used a comparative proteomic and metabolomic analysis to characterize the yeast response when cultured in minimal medium supplemented with glucose (fermentable) or succinate (non-fermentable). RESULTS: A total of 329 proteins were identified from the proteomic profiles, and most of these proteins were associated with carotenogenesis, lipid and carbohydrate metabolism, and redox and stress responses. The metabolite profiles revealed 92 metabolites primarily associated with glycolysis, the tricarboxylic acid cycle, amino acids, organic acids, sugars and phosphates. We determined the abundance of proteins and metabolites of the central pathways of yeast metabolism and examined the influence of these molecules on carotenogenesis. Similar to previous proteomic-stress response studies, we observed modulation of abundance from several redox, stress response, carbohydrate and lipid enzymes. Additionally, the accumulation of trehalose, absence of key ROS response enzymes, an increased abundance of the metabolites of the pentose phosphate pathway and tricarboxylic acid cycle suggested an association between the accumulation of astaxanthin and oxidative stress in the yeast. Moreover, we observed the increased abundance of late carotenogenesis enzymes during astaxanthin accumulation under succinate growth conditions. CONCLUSIONS: The use of succinate as a carbon source in X. dendrorhous cultures increases the availability of acetyl-CoA for the astaxanthin production compared with glucose, likely reflecting the positive regulation of metabolic enzymes of the tricarboxylic acid and glyoxylate cycles. The high metabolite level generated in this pathway could increase the cellular respiration rate, producing reactive oxygen species, which induces carotenogenesis.
Asunto(s)
Basidiomycota/metabolismo , Carbono/metabolismo , Metaboloma/fisiología , Proteoma/análisis , Acetilcoenzima A/metabolismo , Basidiomycota/crecimiento & desarrollo , Carotenoides/metabolismo , Electroforesis en Gel Bidimensional , Proteínas Fúngicas/metabolismo , Glucosa/metabolismo , Metabolómica , Estrés Oxidativo , Proteómica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Ácido Succínico/metabolismo , Xantófilas/biosíntesisRESUMEN
Investiga o estatuto e as condições de emergência da categoria nosológica de síndrome do coração irritável presente nos discursos médicos anglo-americanos na segunda metade do século XIX. No contexto da Guerra Civil Americana, examina elementos sócio-históricos que configuraram a atenção médica sobre os sintomas de ordem cardíaca de soldados. Destacam-se os valores morais de médicos-militares frente aos sintomas de medo em combatentes, assim como as hipóteses etiológicas britânicas e norte-americanas que consolidaram o estatuto nosológico do sofrimento dos soldados com palpitações. Propõe análise da especificidade da síndrome do coração irritável frente às categorias nosológicas do medo descritas pela nosologia psiquiátrica atual.
This paper examines the characteristics and the conditions for the emergence of the nosological category known as irritable heart syndrome to be found in Anglo-American medical literature in the second half of the nineteenth century. In the context of the American Civil War, it looks at some of the socio-historical elements, which comprised the medical care given to certain cardiac symptoms shown by soldiers. It emphasizes the moral values influencing the medical attitudes of military physicians towards symptoms of fear experienced by combatants, as well as the British and American etiological theories, which contributed to the nosological characterization of the suffering of soldiers afflicted with palpitations. Finally, it offers a brief analysis of the specific nature of the medical category known as irritable heart syndrome in the light of the categories of fear described by current psychiatric nosology.
Asunto(s)
Acetilcoenzima A/fisiología , Aspergillus nidulans/metabolismo , Proteínas Fúngicas/fisiología , Fenilacetatos/metabolismo , Proteínas Represoras/fisiología , Ácido Acético/farmacología , Transporte BiológicoRESUMEN
Phytoplasmas ('Candidatus Phytoplasma') are insect-vectored plant pathogens. The genomes of these bacteria are small with limited metabolic capacities making them dependent on their plant and insect hosts for survival. In contrast to mycoplasmas and other relatives in the class Mollicutes, phytoplasmas encode genes for malate transporters and malic enzyme (ME) for conversion of malate into pyruvate. It was hypothesized that malate is probably a major energy source for phytoplasmas as these bacteria are limited in the uptake and processing of carbohydrates. In this study, we investigated the metabolic capabilities of 'Candidatus (Ca.) phytoplasma' aster yellows witches'-broom (AYWB) malic enzyme (ME). We found that AYWB-ME has malate oxidative decarboxylation activity, being able to convert malate to pyruvate and CO2 with the reduction of either NAD or NADP, and displays distinctive kinetic mechanisms depending on the relative concentration of the substrates. AYWB-ME activity was strictly modulated by the ATP/ADP ratio, a feature which has not been found in other ME isoforms characterized to date. In addition, we found that the 'Ca. Phytoplasma' AYWB PduL-like enzyme (AYWB-PduL) harbours phosphotransacetylase activity, being able to convert acetyl-CoA to acetyl phosphate downstream of pyruvate. ATP also inhibited AYWB-PduL activity, as with AYWB-ME, and the product of the reaction catalysed by AYWB-PduL, acetyl phosphate, stimulated AYWB-ME activity. Overall, our data indicate that AYWB-ME and AYWB-PduL activities are finely coordinated by common metabolic signals, like ATP/ADP ratios and acetyl phosphate, which support their participation in energy (ATP) and reducing power [NAD(P)H] generation from malate in phytoplasmas.
Asunto(s)
Metabolismo Energético , Regulación Enzimológica de la Expresión Génica , Malato Deshidrogenasa/metabolismo , Malatos/metabolismo , Fosfato Acetiltransferasa/metabolismo , Phytoplasma/enzimología , Phytoplasma/metabolismo , Acetilcoenzima A/metabolismo , Dióxido de Carbono/metabolismo , Regulación Bacteriana de la Expresión Génica , NAD/metabolismo , NADP/metabolismo , Phytoplasma/genética , Ácido Pirúvico/metabolismoRESUMEN
Mycobacteria contain a large variety of fatty acids which are used for the biosynthesis of several complex cell wall lipids that have been implicated in the ability of the organism to resist host defenses. The building blocks for the biosynthesis of all these lipids are provided by a fairly complex set of acyl-CoA carboxylases (ACCases) whose subunit composition and roles within these organisms have not yet been clearly established. Previous biochemical and structural studies provided strong evidences that ACCase 5 from Mycobacterium tuberculosis is formed by the AccA3, AccD5 and AccE5 subunits and that this enzyme complex carboxylates acetyl-CoA and propionyl-CoA with a clear substrate preference for the latest. In this work we used a genetic approach to unambiguously demonstrate that the products of both accD5 and accE5 genes are essential for the viability of Mycobacterium smegmatis. By obtaining a conditional mutant on the accD5-accE5 operon, we also demonstrated that the main physiological role of this enzyme complex was to provide the substrates for fatty acid and mycolic acid biosynthesis. Furthermore, enzymatic and biochemical analysis of the conditional mutant provided strong evidences supporting the notion that AccD5 and/or AccE5 have an additional role in the carboxylation of long chain acyl-CoA prior to mycolic acid condensation. These studies represent a significant step towards a better understanding of the roles of ACCases in mycobacteria and confirm ACCase 5 as an interesting target for the development of new antimycobacterial drugs.
Asunto(s)
Ligasas de Carbono-Carbono/genética , Pared Celular/genética , Lípidos/biosíntesis , Mycobacterium smegmatis/genética , Acetilcoenzima A , Acilcoenzima A , Secuencia de Aminoácidos , Pared Celular/metabolismo , Ácidos Grasos/genética , Ácidos Grasos/metabolismo , Lipogénesis , Mycobacterium smegmatis/metabolismo , Ácidos Micólicos/metabolismoRESUMEN
The biosynthesis of poly-3-hydroxybutyrate (P3HB), a biodegradable bio-plastic, requires acetyl-CoA as precursor and NADPH as cofactor. Escherichia coli has been used as a heterologous production model for P3HB, but metabolic pathway analysis shows a deficiency in maintaining high levels of NADPH and that the acetyl-CoA is mainly converted to acetic acid by native pathways. In this work the pool of NADPH was increased 1.7-fold in E. coli MG1655 through plasmid overexpression of the NADP(+)-dependent glyceraldehyde 3-phosphate dehydrogenase gene (gapN) from Streptococcus mutans (pTrcgapN). Additionally, by deleting the main acetate production pathway (ackA-pta), the acetic acid production was abolished, thus increasing the acetyl-CoA pool. The P3HB biosynthetic pathway was heterologously expressed in strain MG1655 Δack-pta/pTrcgapN, using an IPTG inducible vector with the P3HB operon from Azotobacter vinelandii (pPHB Av ). Cultures were performed in controlled fermentors using mineral medium with glucose as the carbon source. Accordingly, the mass yield of P3HB on glucose increased to 73 % of the maximum theoretical and was 30 % higher when compared to the progenitor strain (MG1655/pPHB Av ). In comparison with the wild type strain expressing pPHB Av , the specific accumulation of PHB (gPHB/gDCW) in MG1655 Δack-pta/pTrcgapN/pPHB Av increased twofold, indicating that as the availability of NADPH is raised and the production of acetate abolished, a P3HB intracellular accumulation of up to 84 % of the E. coli dry weight is attainable.
Asunto(s)
Acetilcoenzima A/metabolismo , Vías Biosintéticas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Hidroxibutiratos/metabolismo , Ingeniería Metabólica , NADP/metabolismo , Poliésteres/metabolismo , Azotobacter vinelandii/enzimología , Azotobacter vinelandii/genética , Reactores Biológicos/microbiología , Medios de Cultivo/química , Escherichia coli/enzimología , Eliminación de Gen , Gliceraldehído 3-Fosfato Deshidrogenasa (NADP+)/genética , Gliceraldehído 3-Fosfato Deshidrogenasa (NADP+)/metabolismo , Plásmidos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptococcus mutans/enzimología , Streptococcus mutans/genéticaRESUMEN
In recent decades, a number of hypotheses on the autotrophic origin of life have been presented. These proposals invoke the emergence of reaction networks leading from CO or CO2 to the organic molecules required for life. It has also been suggested that the last (universal) common ancestor (LCA or LUCA) of all extant cell lineages was a chemolitho-autotrophic thermophilic anaerobe. The antiquity of some carbon fixation pathways, the phylogenetic basal distribution of some autotrophic organisms, and the catalytic properties of iron-sulfur minerals have been advanced in support of these ideas. Here we critically examine the phylogenetic distribution and evolution of enzymes that are essential for two of the most ancient autotrophic means of metabolism: the reductive tricarboxylic acid (rTCA) cycle and the reductive acetyl-CoA pathway. Phylogenetic analysis of citryl-CoA synthetase and of citryl-CoA lyase, key enzymatic components of the rTCA cycle, and of CO dehydrogenase/acetyl-CoA synthase, a key enzyme in the reductive acetyl-CoA pathway, revealed that all three enzymes have undergone major lateral transfer events and therefore cannot be used as proof of the LCA's metabolic abilities nor as evidence of an autotrophic origin of life.
Asunto(s)
Acetilcoenzima A/genética , Bacterias/clasificación , Bacterias/enzimología , Proteínas Bacterianas/genética , Evolución Biológica , Filogenia , Acetilcoenzima A/metabolismo , Procesos Autotróficos , Bacterias/genética , Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Ciclo del Ácido Cítrico , Datos de Secuencia Molecular , Ácidos Tricarboxílicos/metabolismoRESUMEN
Toxicity of vanadium on cells is one of the less studied effects. This prompted us to study the structural effects induced on neuroblastoma and erythrocytes by vanadium (V) sodium metavanadate. This salt was incubated with mice cholinergic neuroblastoma cells and intact human erythrocytes. To learn whether metavanadate interacts with membrane lipid bilayers it was incubated with bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE). These are phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. Exposure of neuroblastoma cells to metavanadate showed significant decreases in cell viability as well as in cell number correlating with inhibition of aconitase activity. In scanning electron microscopy (SEM) and defocusing microscopy (DM) it was observed that induced on erythrocytes the formation of echinocytes. However, no effects were obtained when metavanadate was made to interact with DMPC and DMPE multibilayers and liposomes, assays performed by X-ray diffraction and differential scanning calorimetry (DSC), respectively. These results imply that the effects of metavanadate on erythrocytes are through interactions with proteins located in the membrane outer moiety, and could still involve other minor lipid components as well. Also, partly unsaturated lipids could interact differently the fully saturated chains in the model systems.
Asunto(s)
Eritrocitos/efectos de los fármacos , Neuronas/efectos de los fármacos , Vanadatos/farmacología , Acetilcoenzima A/metabolismo , Animales , Rastreo Diferencial de Calorimetría , Diferenciación Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Dimiristoilfosfatidilcolina/química , Eritrocitos/citología , Humanos , Liposomas , Ratones , Microscopía Electrónica de Rastreo , Neuroblastoma , Neuronas/citología , Neuronas/metabolismo , Fosfatidiletanolaminas/química , Vanadatos/química , Vanadatos/toxicidad , Difracción de Rayos XRESUMEN
Research on biological influence of vanadium has gained major importance because it exerts potent toxic, mutagenic, and genotoxic effects on a wide variety of biological systems. However, hematological toxicity is one of the less studied effects. The lack of information on this issue prompted us to study the structural effects induced on the human erythrocyte membrane by vanadium (V). Sodium orthovanadate was incubated with intact erythrocytes, isolated unsealed human erythrocyte membranes (IUM) and molecular models of the erythrocyte membrane. The latter consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. This report presents evidence in order that orthovanadate interacted with red cell membranes as follows: a) in scanning electron microscopy (SEM) studies it was observed that morphological changes on human erythrocytes were induced; b) fluorescence spectroscopy experiments in isolated unsealed human erythrocyte membranes (IUM) showed that an increase in the molecular dynamics and/or water content at the shallow depth of the lipids glycerol backbone at concentrations as low as 50µM was produced; c) X-ray diffraction studies showed that orthovanadate 0.25-1mM range induced increasing structural perturbation to DMPE; d) somewhat similar effects were observed by differential scanning calorimetry (DSC) with the exception of the fact that DMPC pretransition was shown to be affected; and e) fluorescence spectroscopy experiments performed in DMPC large unilamellar vesicles (LUV) showed that at very low concentrations induced changes in DPH fluorescence anisotropy at 18°C. Additional experiments were performed in mice cholinergic neuroblastoma SN56 cells; a statistically significant decrease of cell viability was observed on orthovanadate in low or moderate concentrations.
Asunto(s)
Eritrocitos/metabolismo , Neuroblastoma/metabolismo , Sodio/farmacología , Vanadatos/farmacología , Acetilcoenzima A/química , Animales , Anisotropía , Rastreo Diferencial de Calorimetría/métodos , Línea Celular Tumoral , Supervivencia Celular , Dimiristoilfosfatidilcolina/química , Eritrocitos/efectos de los fármacos , Humanos , Técnicas In Vitro , Lípidos/química , Ratones , Microscopía Electrónica de Rastreo/métodos , Fosfatidiletanolaminas/química , Espectrometría de Fluorescencia/métodos , Temperatura , Liposomas Unilamelares/química , Vanadio/farmacologíaRESUMEN
Introduccion: la aciduria glutarica tipo II, o deficiencia multiple de acil-CoA deshidrogenasas,es un trastorno causado por deficiencia de la flavoproteina de transferencia de electrones,de su oxidorreductasa o de ambas; se trata de una enfermedad metabolica autosomica recesiva, caracterizada por acidosis, hipoglicemia, aciduria organica, olor a pies sudados y malformaciones en cerebro y riñones. Objetivo: analizar las tasas de oxidacion de sustratos tritiados por fibroblastos de pacientescon aciduria glutarica tipo II. Materiales y metodos: se incubaron fibroblastos de dos pacientes con aciduria glutarica tipoII y de 20 controles en presencia de acidos palmitico y miristico tritiados. Resultados: se encontro muy deprimida (16%-18%) la oxidacion de los sustratos tritiados porlos fibroblastos procedentes de pacientes con aciduria glutarica tipo II en comparacion con los controles. Conclusion: la prueba estudiada permite la confirmacion in vitro del diagnostico de aciduriaglutarica tipo II.
Introduction: Glutaric aciduria type II (GA II), or multiple acyl-CoA dehydrogenase deficiency, is a disorder caused by deficiency of either electron transport flavoprotein or electron transport flavoprotein oxyreductase. It is an autsomal recessive metabolic disease, characterized by acidosis, hypoglycemia, organic aciduria, sweat-sock odour, and malformations in brain and kidneys. Objective: To analyse the oxidation rate of tritiated substrates by fibroblasts of patients with GA II. Materials and methods: Fibroblasts of two patients with GA II were incubated with tritiated palmitic and myristic acids. Results: Oxidation of tritiated substrates by fibroblasts of patients with GA II was very depressed (16%-18%) in comparison with controls. Conclusion: Diagnosis of GA II may be confirmed in vitro by the studied test.
Asunto(s)
Humanos , Acetilcoenzima A , Ácidos Grasos , Oxidorreductasas/deficienciaRESUMEN
The first committed step of fatty acid and polyketides biosynthesis, the biotin-dependent carboxylation of an acyl-CoA, is catalyzed by acyl-CoA carboxylases (ACCases) such as acetyl-CoA carboxylase (ACC) and propionyl-CoA carboxylase (PCC). ACC and PCC in Streptomyces coelicolor are homologue multisubunit complexes that can carboxylate different short chain acyl-CoAs. While ACC is able to carboxylate acetyl-, propionyl-, or butyryl-CoA with approximately the same specificity, PCC only recognizes propionyl- and butyryl-CoA as substrates. How ACC and PCC have such different specificities toward these substrates is only partially understood. To further understand the molecular basis of how the active site residues can modulate the substrate recognition, we mutated D422, N80, R456, and R457 of PccB, the catalytic beta subunit of PCC. The crystal structures of six PccB mutants and the wild type crystal structure were compared systematically to establish the sequence-structure-function relationship that correlates the observed substrate specificity toward acetyl-, propionyl-, and butyryl-CoA with active site geometry. The experimental data confirmed that D422 is a key determinant of substrate specificity, influencing not only the active site properties but further altering protein stability and causing long-range conformational changes. Mutations of N80, R456, and R457 lead to variations in the quaternary structure of the beta subunit and to a concomitant loss of enzyme activity, indicating the importance of these residues in maintaining the active protein conformation as well as a critical role in substrate binding.
Asunto(s)
Ligasas de Carbono-Carbono , Acetilcoenzima A/genética , Acetilcoenzima A/metabolismo , Acetil-CoA Carboxilasa/genética , Acetil-CoA Carboxilasa/metabolismo , Acilcoenzima A/química , Acilcoenzima A/genética , Acilcoenzima A/metabolismo , Secuencia de Bases , Biotina/genética , Biotina/metabolismo , Ligasas de Carbono-Carbono/química , Ligasas de Carbono-Carbono/genética , Ligasas de Carbono-Carbono/metabolismo , Catálisis , Genotipo , Metilmalonil-CoA Descarboxilasa/química , Metilmalonil-CoA Descarboxilasa/genética , Metilmalonil-CoA Descarboxilasa/metabolismo , Conformación Proteica , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Especificidad por Sustrato/genéticaRESUMEN
Escherichia coli phosphotransacetylase (Pta) catalyzes the reversible interconversion of acetyl-CoA and acetyl phosphate. Both compounds are critical in E. coli metabolism, and acetyl phosphate is also involved in the regulation of certain signal transduction pathways. Along with acetate kinase, Pta plays an important role in acetate production when E. coli grows on rich medium; alternatively, it is involved in acetate utilization at high acetate concentrations. E. coli Pta is composed of three different domains, but only the C-terminal one, called PTA_PTB, is specific for all Ptas. In the present work, the characterization of E. coli Pta and deletions from the N-terminal region were performed. E. coli Pta acetyl phosphate-forming and acetyl phosphate-consuming reactions display different maximum activities, and are differentially regulated by pyruvate and phosphoenolpyruvate. These compounds activate acetyl phosphate production, but inhibit acetyl-CoA production, thus playing a critical role in defining the rates of the two Pta reactions. The characterization of three truncated Ptas, which all display Pta activity, indicates that the substrate-binding site is located at the C-terminal PTA_PTB domain. However, the N-terminal P-loop NTPase domain is involved in expression of the maximal catalytic activity, stabilization of the hexameric native state, and Pta activity regulation by NADH, ATP, phosphoenolpyruvate, and pyruvate. The truncated protein Pta-F3 was able to complement the growth on acetate of an E. coli mutant defective in acetyl-CoA synthetase and Pta, indicating that, although not regulated by metabolites, the Pta C-terminal domain is active in vivo.
Asunto(s)
Acetato Quinasa/metabolismo , Acetilcoenzima A/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiología , Organofosfatos/metabolismo , Fosfato Acetiltransferasa/metabolismo , Catálisis , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Cinética , Modelos Biológicos , Fosfato Acetiltransferasa/genética , Estructura Terciaria de Proteína/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal/genéticaRESUMEN
AIMS: Analysis of the physiology and metabolism of Escherichia coli arcA and creC mutants expressing a bifunctional alcohol-acetaldehyde dehydrogenase from Leuconostoc mesenteroides growing on glycerol under oxygen-restricted conditions. The effect of an ldhA mutation and different growth medium modifications was also assessed. METHODS AND RESULTS: Expression of adhE in E. coli CT1061 [arcA creC(Con)] resulted in a 1.4-fold enhancement in ethanol synthesis. Significant amounts of lactate were produced during micro-oxic cultures and strain CT1061LE, in which fermentative lactate dehydrogenase was deleted, produced up to 6.5 +/- 0.3 g l(-1) ethanol in 48 h. Escherichia coli CT1061LE derivatives resistant to >25 g l(-1) ethanol were obtained by metabolic evolution. Pyruvate and acetaldehyde addition significantly increased both biomass and ethanol concentrations, probably by overcoming acetyl-coenzyme A (CoA) shortage. Yeast extract also promoted growth and ethanol synthesis, and this positive effect was mainly attributable to its vitamin content. Two-stage bioreactor cultures were conducted in a minimal medium containing 100 microg l(-1) calcium d-pantothenate to evaluate oxic acetyl-CoA synthesis followed by a switch into fermentative conditions. Ethanol reached 15.4 +/- 0.9 g l(-1) with a volumetric productivity of 0.34 +/- 0.02 g l(-1) h(-1). CONCLUSIONS: Escherichia coli responded to adhE over-expression by funnelling carbon and reducing equivalents into a highly reduced metabolite, ethanol. Acetyl-CoA played a key role in micro-oxic ethanol synthesis and growth. SIGNIFICANCE AND IMPACT OF THE STUDY: Insight into the micro-oxic metabolism of E. coli growing on glycerol is essential for the development of efficient industrial processes for reduced biochemicals production from this substrate, with special relevance to biofuels synthesis.