RESUMO
BACKGROUND: Entamoeba histolytica, an intestinal parasitic protozoan that usually lives and multiplies within the human gut, is the causative agent of amoebiasis. To date, de novo glutathione biosynthesis and its associated enzymes have not been identified in the parasite. Cysteine has been proposed to be the main intracellular thiol. METHODS: Using bioinformatics tools to search for glutaredoxin homologs in the E. histolytica genome database, we identified a coding sequence for a putative Grx-like small protein (EhGLSP) in the E. histolytica HM-1:IMSS genome. We produced the recombinant protein and performed its biochemical characterization. RESULTS: Through in vitro experiments, we observed that recombinant EhGLSP could bind GSH and L-Cys as ligands. However, the protein exhibited very low GSH-dependent disulfide reductase activity. Interestingly, via UV-Vis spectroscopy and chemical analysis, we detected that recombinant EhGLSP (freshly purified from Escherichia coli cells by IMAC) was isolated together with a redox-labile [FeS] bio-inorganic complex, suggesting that this protein could have some function linked to the metabolism of this cofactor. Western blotting showed that EhGLSP protein levels were modulated in E. histolytica cells exposed to exogenous oxidative species and metronidazole, suggesting that this protein cooperates with the antioxidant mechanisms of this parasite. CONCLUSIONS AND GENERAL SIGNIFICANCE: Our findings support the existence of a new metabolic actor in this pathogen. To the best of our knowledge, this is the first report on this protein class in E. histolytica.
Assuntos
Entamoeba histolytica , Parasitos , Animais , Humanos , Entamoeba histolytica/genética , Entamoeba histolytica/metabolismo , Parasitos/metabolismo , Anaerobiose , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Proteínas de Protozoários/metabolismoRESUMO
Peroxiredoxins (Prxs) have been shown to be important enzymes for trypanosomatids, counteracting oxidative stress and promoting cell infection and intracellular survival. In this work, we investigate the in vitro sensitivity to overoxidation and the overoxidation dynamics of Trypanosoma cruzi Prxs in parasites in culture and in the infection context. We showed that recombinant m-TXNPx, in contrast to what was observed for c-TXNPx, exists as low molecular mass forms in the overoxidized state. We observed that T. cruzi Prxs were overoxidized in epimastigotes treated with oxidants, and a significant proportion of the overoxidized forms were still present at least 24 h after treatment suggesting that these forms are not actively reversed. In in vitro infection experiments, we observed that Prxs are overoxidized in amastigotes residing in infected macrophages, demonstrating that inactivation of at least part of the Prxs by overoxidation occurs in a physiological context. We have shown that m-TXNPx has a redox-state-dependent chaperone activity. This function may be related to the increased thermotolerance observed in m-TXNPx-overexpressing parasites. This study suggests that despite the similarity between protozoan and mammalian Prxs, T. cruzi Prxs have different oligomerization dynamics and sensitivities to overoxidation, which may have implications for their function in the parasite life cycle and infection process.
RESUMO
Trypanosoma cruzi is the causal agent of Chagas Disease and is a unicellular parasite that infects a wide variety of mammalian hosts. The parasite exhibits auxotrophy by L-Met; consequently, it must be acquired from the extracellular environment of the host, either mammalian or invertebrate. Methionine (Met) oxidation produces a racemic mixture (R and S forms) of methionine sulfoxide (MetSO). Reduction of L-MetSO (free or protein-bound) to L-Met is catalyzed by methionine sulfoxide reductases (MSRs). Bioinformatics analyses identified the coding sequence for a free-R-MSR (fRMSR) enzyme in the genome of T. cruzi Dm28c. Structurally, this enzyme is a modular protein with a putative N-terminal GAF domain linked to a C-terminal TIP41 motif. We performed detailed biochemical and kinetic characterization of the GAF domain of fRMSR in combination with mutant versions of specific cysteine residues, namely, Cys12, Cys98, Cys108, and Cys132. The isolated recombinant GAF domain and full-length fRMSR exhibited specific catalytic activity for the reduction of free L-Met(R)SO (non-protein bound), using tryparedoxins as reducing partners. We demonstrated that this process involves two Cys residues, Cys98 and Cys132. Cys132 is the essential catalytic residue on which a sulfenic acid intermediate is formed. Cys98 is the resolutive Cys, which forms a disulfide bond with Cys132 as a catalytic step. Overall, our results provide new insights into redox metabolism in T. cruzi, contributing to previous knowledge of L-Met metabolism in this parasite.
Assuntos
Metionina Sulfóxido Redutases , Trypanosoma cruzi , Metionina Sulfóxido Redutases/genética , Metionina Sulfóxido Redutases/química , Metionina Sulfóxido Redutases/metabolismo , Trypanosoma cruzi/genética , Oxirredução , Cisteína/química , Metionina/metabolismoRESUMO
Endo-ß-1,3-glucanases from several organisms have attracted much attention in recent years because of their capability for in vitro degrading ß-1,3-glucan as a critical step for both biofuels production and short-chain oligosaccharides synthesis. In this study, we biochemically characterized a putative endo-ß-1,3-glucanase (EgrGH64) belonging to the family GH64 from the single-cell protist Euglena gracilis. The gene coding for the enzyme was heterologously expressed in a prokaryotic expression system supplemented with 3% (v/v) ethanol to optimize the recombinant protein right folding. Thus, the produced enzyme was highly purified by immobilized-metal affinity and gel filtration chromatography. The enzymatic study demonstrated that EgrGH64 could hydrolyze laminarin (KM 23.5 mg ml-1,kcat 1.20 s-1) and also, but with less enzymatic efficiency, paramylon (KM 20.2 mg ml-1,kcat 0.23 ml mg-1 s-1). The major product of the hydrolysis of both substrates was laminaripentaose. The enzyme could also use ramified ß-glucan from the baker's yeast cell wall as a substrate (KM 2.10 mg ml-1, kcat 0.88 ml mg-1 s-1). This latter result, combined with interfacial kinetic analysis evidenced a protein's greater efficiency for the yeast polysaccharide, and a higher number of hydrolysis sites in the ß-1,3/ß-1,6-glucan. Concurrently, the enzyme efficiently inhibited the fungal growth when used at 1.0 mg/mL (15.4 µM). This study contributes to assigning a correct function and determining the enzymatic specificity of EgrGH64, which emerges as a relevant biotechnological tool for processing ß-glucans.
Assuntos
Euglena gracilis , Cinética , Polissacarídeos/metabolismo , Hidrólise , Saccharomyces cerevisiae/metabolismo , Especificidade por SubstratoRESUMO
Euglena gracilis is a eukaryotic single-celled and photosynthetic organism grouped under the kingdom Protista. This phytoflagellate can accumulate the carbon photoassimilate as a linear ß-1,3-glucan chain called paramylon. This storage polysaccharide can undergo degradation to provide glucose units to obtain ATP and reducing power both in aerobic and anaerobic growth conditions. Our group has recently characterized an essential enzyme for accumulating the polysaccharide, the UDP-glucose pyrophosphorylase (Biochimie vol 154, 2018, 176-186), which catalyzes the synthesis of UDP-glucose (the substrate for paramylon synthase). Additionally, the identification of nucleotide sequences coding for putative UDP-sugar pyrophosphorylases suggests the occurrence of an alternative source of UDP-glucose. In this study, we demonstrate the active involvement of both pyrophosphorylases in paramylon accumulation. Using techniques of single and combined knockdown of transcripts coding for these proteins, we evidenced a substantial decrease in the polysaccharide synthesis from 39 ± 7 µg/106 cells determined in the control at day 21st of growth. Thus, the paramylon accumulation in Euglena gracilis cells decreased by 60% and 30% after a single knockdown of the expression of genes coding for UDP-glucose pyrophosphorylase and UDP-sugar pyrophosphorylase, respectively. Besides, the combined knockdown of both genes resulted in a ca. 65% reduction in the level of the storage polysaccharide. Our findings indicate the existence of a physiological dependence between paramylon accumulation and the partitioning of sugar nucleotides into other metabolic routes, including the Leloir pathway's functionality in Euglena gracilis.
Assuntos
Metabolismo dos Carboidratos , Euglena gracilis , Genética Reversa , Euglena gracilis/genética , Euglena gracilis/metabolismo , Glucanos/biossíntese , Glucanos/genéticaRESUMO
BACKGROUND: Methionine (Met) oxidation leads to a racemic mixture of R and S forms of methionine sulfoxide (MetSO). Methionine sulfoxide reductases (Msr) are enzymes that can reduce specifically each isomer of MetSO, both free and protein-bound. The Met oxidation could change the structure and function of many proteins, not only of those redox-related but also of others involved in different metabolic pathways. Until now, there is no information about the presence or function of Msrs enzymes in Leptospira interrogans. METHODS: We identified genes coding for putative MsrAs (A1 and A2) and MsrB in L. interrogans serovar Copenhageni strain Fiocruz L1-130 genome project. From these, we obtained the recombinant proteins and performed their functional characterization. RESULTS: The recombinant L. interrogans MsrB catalyzed the reduction of Met(R)SO using glutaredoxin and thioredoxin as reducing substrates and behaves like a 1-Cys Msr (without resolutive Cys residue). It was able to partially revert the in vitro HClO-dependent inactivation of L. interrogans catalase. Both recombinant MsrAs reduced Met(S)SO, being the recycle mediated by the thioredoxin system. LinMsrAs were more efficient than LinMsrB for free and protein-bound MetSO reduction. Besides, LinMsrAs are enzymes involving a Cys triad in their catalytic mechanism. LinMsrs showed a dual localization, both in cytoplasm and periplasm. CONCLUSIONS AND GENERAL SIGNIFICANCE: This article brings new knowledge about redox metabolism in L. interrogans. Our results support the occurrence of a metabolic pathway involved in the critical function of repairing oxidized macromolecules in this pathogen.
Assuntos
Citoplasma/química , Leptospira interrogans/genética , Metionina Sulfóxido Redutases/genética , Metionina/metabolismo , Sequência de Aminoácidos/genética , Catálise , Citoplasma/enzimologia , Genoma Bacteriano/genética , Humanos , Leptospira interrogans/enzimologia , Metionina/química , Metionina/genética , Metionina Sulfóxido Redutases/química , Metionina Sulfóxido Redutases/ultraestrutura , Oxirredução , Homologia de Sequência de Aminoácidos , Estereoisomerismo , Especificidade por SubstratoRESUMO
Amoebiasis is caused by the protozoan Entamoeba histolytica that affects millions of people throughout the world. The standard treatment is metronidazole, however, this drug causes several side effects, and is also mutagenic and carcinogenic. Therefore, the search for therapeutic alternatives is necessary. Quinoxaline 1,4-di-N-oxides (QdNOs) derivatives have been shown to exhibit activity against different protozoan. In the present study, the effects of esters of quinoxaline-7-carboxylate 1,4-di-N-oxide (7-carboxylate QdNOs) derivatives on E. histolytica proliferation, morphology, ultrastructure, and oxidative stress were evaluated, also their potential as E. histolytica thioredoxin reductase (EhTrxR) inhibitors was analyzed. In vitro tests showed that 12 compounds from n-propyl and isopropyl series, were more active (IC50 = 0.331 to 3.56 µM) than metronidazole (IC50 = 4.5 µM). The compounds with better biological activity have a bulky, trifluoromethyl and isopropyl group at R1-, R2-, and R3-position, respectively. The main alterations found in trophozoites treated with some of these compounds included changes in chromatin, cell granularity, redistribution of vacuoles with cellular debris, and an increase in reactive oxygen species. Interestingly, docking studies suggested that 7-carboxylate QdNOs derivatives could interact with amino acid residues of the NADPH-binding domain and/or the redox-active site of EhTrxR. Enzymatic assays demonstrated that selected 7-carboxylate QdNOs inhibits EhTrxR disulfide reductase activity, and diaphorase activity shows that these compounds could act as electron acceptor substrates for the enzyme. Taken together, these data indicate that among the mechanisms involved in the antiamoebic effect of the 7-carboxylate QdNOs derivatives studied, is the induction of oxidative stress and the inhibition of EhTrxR activity.
Assuntos
Entamoeba histolytica/efeitos dos fármacos , Quinoxalinas/farmacologia , Tiorredoxina Dissulfeto Redutase/antagonistas & inibidores , Óxidos N-Cíclicos , Entamoeba histolytica/enzimologia , Ésteres , Humanos , Metronidazol/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Quinolinas , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND: Glutathione (GSH) plays a role as a main antioxidant metabolite in all eukaryotes and many prokaryotes. Most of the organisms synthesize GSH by a pathway involving two enzymatic reactions, each one consuming one molecule of ATP. In a first step mediated by glutamate-cysteine ligase (GCL), the carboxylate of l-glutamic acid reacts with l-cysteine to form the dipeptide γ-glutamylcysteine (γ-GC). The second step involves the addition of glycine to the C-terminal of γ-GC catalyzed by glutathione synthetase (GS). In many bacteria, such as in the pathogen Leptospira interrogans, the main intracellular thiol has not yet been identified and the presence of GSH is not clear. METHODS: We performed the molecular cloning of the genes gshA and gshB from L. interrogans; which respectively code for GCL and GS. After heterologous expression of the cloned genes we recombinantly produced the respective proteins with high degree of purity. These enzymes were exhaustively characterized in their biochemical properties. In addition, we determined the contents of GSH and the activity of related enzymes (and proteins) in cell extracts of the bacterium. RESULTS: We functionally characterized GCL and GS, the two enzymes putatively involved in GSH synthesis in L. interrogans serovar Copenhageni. LinGCL showed higher substrate promiscuity (was active in presence of l-glutamic acid, l-cysteine and ATP, and also with GTP, l-aspartic acid and l-serine in lower proportion) unlike LinGS (which was only active with γ-GC, l-glycine and ATP). LinGCL is significantly inhibited by γ-GC and GSH, the respective intermediate and final product of the synthetic pathway. GSH showed inhibitory effect over LinGS but with a lower potency than LinGCL. Going further, we detected the presence of GSH in L. interrogans cells grown under basal conditions and also determined enzymatic activity of several GSH-dependent/related proteins in cell extracts. CONCLUSIONS: and General Significance. Our results contribute with novel insights concerning redox metabolism in L. interrogans, mainly supporting that GSH is part of the antioxidant defense in the bacterium.
Assuntos
Proteínas de Bactérias/metabolismo , Glutamato-Cisteína Ligase/metabolismo , Glutationa Sintase/metabolismo , Glutationa/metabolismo , Leptospira interrogans/metabolismo , Proteínas de Bactérias/genética , Clonagem Molecular , Glutamato-Cisteína Ligase/genética , Glutationa Sintase/genética , Leptospira interrogans/genética , Leptospira interrogans/crescimento & desenvolvimento , OxirreduçãoRESUMO
BACKGROUND: Chagas cardiomyopathy, caused by Trypanosoma cruzi infection, continues to be a neglected illness, and has a major impact on global health. The parasite undergoes several stages of morphological and biochemical changes during its life cycle, and utilizes an elaborated antioxidant network to overcome the oxidants barrier and establish infection in vector and mammalian hosts. Trypanothione synthetase (TryS) catalyzes the biosynthesis of glutathione-spermidine adduct trypanothione (T(SH)2) that is the principal intracellular thiol-redox metabolite in trypanosomatids. METHODS AND RESULTS: We utilized genetic overexpression (TryShi) and pharmacological inhibition approaches to examine the role of TryS in T. cruzi proliferation, tolerance to oxidative stress and resistance to anti-protozoal drugs. Our data showed the expression and activity of TryS was increased in all morphological stages of TryShi (vs. control) parasites. In comparison to controls, the TryShi epimastigotes (insect stage) recorded shorter doubling time, and both epimastigotes and infective trypomastigotes of TryShi exhibited 36-71% higher resistance to H2O2 (50-1000⯵M) and heavy metal (1-500⯵M) toxicity. Treatment with TryS inhibitors (5-30⯵M) abolished the proliferation and survival advantages against H2O2 pressure in a dose-dependent manner in both TryShi and control parasites. Further, epimastigote and trypomastigote forms of TryShi (vs. control) T. cruzi tolerated higher doses of benznidazole and nifurtimox, the drugs currently administered for acute Chagas disease treatment. CONCLUSIONS: TryS is essential for proliferation and survival of T. cruzi under normal and oxidant stress conditions, and provides an advantage to the parasite to develop resistance against currently used anti-trypanosomal drugs. TryS indispensability has been chemically validated with inhibitors that may be useful for drug combination therapy against Chagas disease.
Assuntos
Amida Sintases/metabolismo , Antioxidantes/metabolismo , Cardiomiopatia Chagásica/parasitologia , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/fisiologia , Amida Sintases/genética , Animais , Antiprotozoários/uso terapêutico , Proliferação de Células , Células Cultivadas , Cardiomiopatia Chagásica/tratamento farmacológico , Resistência a Medicamentos , Humanos , Oxirredução , Estresse Oxidativo , Proteínas de Protozoários/genética , Transgenes/genéticaRESUMO
Many oligo and polysaccharides (including paramylon) are critical in the Euglena gracilis life-cycle and they are synthesized by glycosyl transferases using UDP-glucose as a substrate. Herein, we report the molecular cloning of a gene putatively coding for a UDP-glucose pyrophosphorylase (EgrUDP-GlcPPase) in E. gracilis. After heterologous expression of the gene in Escherichia coli, the recombinant enzyme was characterized structural and functionally. Highly purified EgrUDP-GlcPPase exhibited a monomeric structure, able to catalyze synthesis of UDP-glucose with a Vmax of 3350 U.mg-1. Glucose-1P and UTP were the preferred substrates, although the enzyme also used (with lower catalytic efficiency) TTP, galactose-1P and mannose-1P. Oxidation by hydrogen peroxide inactivated the enzyme, an effect reversed by reduction with dithiothreitol or thioredoxin. The redox process would involve sulfenic acid formation, since no pair of the 7 cysteine residues is close enough in the 3D structure of the protein to form a disulfide bridge. Electrophoresis studies suggest that, after oxidation, the enzyme arranges in many enzymatically inactive structural conformations; which were also detected in vivo. Finally, confocal fluorescence microscopy provided evidence for a cytosolic (mainly in the flagellum) localization of the enzyme.
Assuntos
Metabolismo dos Carboidratos , Euglena gracilis/enzimologia , Glucanos/química , UTP-Glucose-1-Fosfato Uridililtransferase/química , Catálise , Glucanos/metabolismo , Cinética , Domínios Proteicos , UTP-Glucose-1-Fosfato Uridililtransferase/metabolismoRESUMO
Visceral leishmaniasis is a public health problem worldwide. The early diagnosis in dogs is crucial, since they are an epidemiologically relevant reservoir of the disease. The aim of a field study is to early identify the disease allowing rapid intervention to reduce its effects. We propose an immunoagglutination test as a visual in situ method for diagnosis of canine visceral leishmaniasis. Latex-protein complexes were sensitized by covalent coupling of a chimeric recombinant antigen of Leishmania spp. onto polystyrene latex with carboxyl functionality. The reaction time and the antigen concentration under which the immunoagglutination assay shows greater discrimination between the responses of a positive control serum and a negative control serum were determined. Then, the latex-protein complexes were evaluated as a visual diagnostic tool with a panel of 170 sera. The test may be read between 2 and 5 min and can be performed even using sera with elevated concentration of lipids, bilirubin or with variable percentage of hemolysis. The sensitivity, the specificity and the diagnostic accuracy were 78%; 100% and >80%, respectively. The visual immunoagglutination test is of potential application as a method for field studies because it shows results in less than 5 min, it is easy to implement and does not require sophisticated equipment.
Assuntos
Anticorpos Antiprotozoários/sangue , Doenças do Cão/diagnóstico , Testes de Fixação do Látex/veterinária , Leishmania infantum/imunologia , Leishmaniose Visceral/veterinária , Animais , Antígenos de Protozoários/imunologia , Western Blotting/veterinária , Reservatórios de Doenças , Doenças do Cão/parasitologia , Cães , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/parasitologia , Proteínas Recombinantes/imunologia , Sensibilidade e EspecificidadeRESUMO
To combat the deleterious effects that oxidation of the sulfur atom in methionine to sulfoxide may bring, aerobic cells express repair pathways involving methionine sulfoxide reductases (MSRs) to reverse the above reaction. Here, we show that Trypanosoma brucei, the causative agent of African trypanosomiasis, expresses two distinct trypanothione-dependent MSRs that can be distinguished from each other based on sequence, sub-cellular localisation and substrate preference. One enzyme found in the parasite's cytosol, shows homology to the MSRA family of repair proteins and preferentially metabolises the S epimer of methionine sulfoxide. The second, which contains sequence motifs present in MSRBs, is restricted to the mitochondrion and can only catalyse reduction of the R form of peptide-bound methionine sulfoxide. The importance of these proteins to the parasite was demonstrated using functional genomic-based approaches to produce cells with reduced or elevated expression levels of MSRA, which exhibited altered susceptibility to exogenous H2O2. These findings identify new reparative pathways that function to fix oxidatively damaged methionine within this medically important parasite.
Assuntos
Metionina Sulfóxido Redutases/genética , Metionina/análogos & derivados , Metionina/metabolismo , Proteínas de Protozoários/genética , Trypanosoma brucei brucei/genética , Sequência de Aminoácidos , Biocatálise , Citosol/efeitos dos fármacos , Citosol/enzimologia , Expressão Gênica , Teste de Complementação Genética , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Isoenzimas/genética , Isoenzimas/metabolismo , Metionina Sulfóxido Redutases/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Oxirredução , Proteínas de Protozoários/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Estereoisomerismo , Especificidade por Substrato , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/enzimologiaRESUMO
Glucitol, also known as sorbitol, is a major photosynthetic product in plants from the Rosaceae family. This sugar alcohol is synthesized from glucose-6-phosphate by the combined activities of aldose-6-phosphate reductase (Ald6PRase) and glucitol-6-phosphatase. In this work we show the purification and characterization of recombinant Ald6PRase from peach leaves. The recombinant enzyme was inhibited by glucose-1-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate and orthophosphate. Oxidizing agents irreversibly inhibited the enzyme and produced protein precipitation. Enzyme thiolation with oxidized glutathione protected the enzyme from insolubilization caused by diamide, while incubation with NADP+ (one of the substrates) completely prevented enzyme precipitation. Our results suggest that Ald6PRase is finely regulated to control carbon partitioning in peach leaves.
Assuntos
Aldeído Redutase/metabolismo , Folhas de Planta/enzimologia , Proteínas de Plantas/metabolismo , Prunus domestica/enzimologia , Aldeído Redutase/antagonistas & inibidores , Aldeído Redutase/genética , Frutosedifosfatos/metabolismo , Frutosedifosfatos/farmacologia , Frutosefosfatos/metabolismo , Frutosefosfatos/farmacologia , Glucofosfatos/metabolismo , Glucofosfatos/farmacologia , Dissulfeto de Glutationa/metabolismo , Hexosefosfatos/metabolismo , Hexosefosfatos/farmacologia , Immunoblotting , Cinética , Modelos Biológicos , NADP/metabolismo , Oxidantes/metabolismo , Oxidantes/farmacologia , Fosfatos/metabolismo , Fosfatos/farmacologia , Filogenia , Folhas de Planta/genética , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Prunus domestica/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Compostos de Sulfidrila/metabolismoRESUMO
BACKGROUND: Entamoeba histolytica, an intestinal parasite that is the causative agent of amoebiasis, is exposed to elevated amounts of highly toxic reactive oxygen and nitrogen species during tissue invasion. A flavodiiron protein and a rubrerythrin have been characterized in this human pathogen, although their physiological reductants have not been identified. METHODS: The present work deals with biochemical studies performed to reach a better understanding of the kinetic and structural properties of rubredoxin reductase and two ferredoxins from E. histolytica. RESULTS: We complemented the characterization of two different metabolic pathways for O2 and H2O2 detoxification in E. histolytica. We characterized a novel amoebic protein with rubredoxin reductase activity that is able to catalyze the NAD(P)H-dependent reduction of heterologous rubredoxins, amoebic rubrerythrin and flavodiiron protein but not ferredoxins. In addition, the protein exhibited an NAD(P)H oxidase activity, which generates hydrogen peroxide from molecular oxygen. We describe how different ferredoxins were also efficient reducing substrates for both flavodiiron protein and rubrerythrin. CONCLUSIONS: The enzymatic systems herein characterized could contribute to the in vivo detoxification of O2 and H2O2, playing a key role for the parasite defense against reactive oxidant species. GENERAL SIGNIFICANCE: To the best of our knowledge this is the first characterization of a eukaryotic rubredoxin reductase, including a novel kinetic study on ferredoxin-dependent reduction of flavodiiron and rubrerythrin proteins.
Assuntos
Entamoeba histolytica/enzimologia , NADH NADPH Oxirredutases/metabolismo , Proteínas de Protozoários/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Clonagem Molecular , Entamoeba histolytica/genética , Hemeritrina/metabolismo , Peróxido de Hidrogênio/metabolismo , Cinética , NADH NADPH Oxirredutases/genética , NADP/metabolismo , Oxirredução , Oxigênio/metabolismo , Proteínas de Protozoários/genética , Proteínas Recombinantes/metabolismo , Rubredoxinas/metabolismoRESUMO
BACKGROUND: Mycobacterium tuberculosis is a pathogenic prokaryote adapted to survive in hostile environments. In this organism and other Gram-positive actinobacteria, the metabolic pathways of glycogen and trehalose are interconnected. RESULTS: In this work we show the production, purification and characterization of recombinant enzymes involved in the partitioning of glucose-1-phosphate between glycogen and trehalose in M. tuberculosis H37Rv, namely: ADP-glucose pyrophosphorylase, glycogen synthase, UDP-glucose pyrophosphorylase and trehalose-6-phosphate synthase. The substrate specificity, kinetic parameters and allosteric regulation of each enzyme were determined. ADP-glucose pyrophosphorylase was highly specific for ADP-glucose while trehalose-6-phosphate synthase used not only ADP-glucose but also UDP-glucose, albeit to a lesser extent. ADP-glucose pyrophosphorylase was allosterically activated primarily by phosphoenolpyruvate and glucose-6-phosphate, while the activity of trehalose-6-phosphate synthase was increased up to 2-fold by fructose-6-phosphate. None of the other two enzymes tested exhibited allosteric regulation. CONCLUSIONS: Results give information about how the glucose-1-phosphate/ADP-glucose node is controlled after kinetic and regulatory properties of key enzymes for mycobacteria metabolism. GENERAL SIGNIFICANCE: This work increases our understanding of oligo and polysaccharides metabolism in M. tuberculosis and reinforces the importance of the interconnection between glycogen and trehalose biosynthesis in this human pathogen.
Assuntos
Glucofosfatos/metabolismo , Glicogênio/biossíntese , Redes e Vias Metabólicas , Mycobacterium tuberculosis/metabolismo , Trealose/biossíntese , Regulação Alostérica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Glucose-1-Fosfato Adenililtransferase/genética , Glucose-1-Fosfato Adenililtransferase/metabolismo , Glucose-6-Fosfato/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Glicogênio Sintase/genética , Glicogênio Sintase/metabolismo , Cinética , Modelos Biológicos , Mycobacterium tuberculosis/enzimologia , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , UTP-Glucose-1-Fosfato Uridililtransferase/genética , UTP-Glucose-1-Fosfato Uridililtransferase/metabolismoRESUMO
Little is known about the mechanisms by which Leptospira interrogans, the causative agent of leptospirosis, copes with oxidative stress at the time it establishes persistent infection within its human host. We report the molecular cloning of a gene encoding a 2-Cys peroxiredoxin (LinAhpC) from this bacterium. After bioinformatic analysis we found that LinAhpC contains the characteristic GGIG and YF motifs present in peroxiredoxins that are sensitive to overoxidation (mainly eukaryotic proteins). These motifs are absent in insensitive prokaryotic enzymes. Recombinant LinAhpC showed activity as a thioredoxin peroxidase with sensitivity to overoxidation by H2O2 (Chyp 1% ~30 µM at pH 7.0 and 30°C). So far, Anabaena 2-Cys peroxiredoxin, Helicobacter pylori AhpC, and LinAhpC are the only prokaryotic enzymes studied with these characteristics. The properties determined for LinAhpC suggest that the protein could be critical for the antioxidant defense capacity in L. interrogans.
Assuntos
Proteínas de Bactérias/química , Leptospira interrogans/enzimologia , Peroxirredoxinas/química , Sequência de Aminoácidos , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Escherichia coli , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Oxirredução , Peroxirredoxinas/biossíntese , Peroxirredoxinas/genética , FilogeniaRESUMO
In Trypanosoma cruzi, the modification of thiols by glutathionylation-deglutathionylation and its potential relation to protective, regulatory or signaling functions have been scarcely explored. Herein we characterize a dithiolic glutaredoxin (TcrGrx), a redox protein with deglutathionylating activity, having potential functionality to control intracellular homeostasis of protein and non-protein thiols. The catalytic mechanism followed by TcrGrx was found dependent on thiol concentration. Results suggest that TcrGrx operates as a dithiolic or a monothiolic Grx, depending on GSH concentration. TcrGrx functionality to mediate reduction of protein and non-protein disulfides was studied. TcrGrx showed a preference for glutathionylated substrates respect to protein disulfides. From in vivo assays involving TcrGrx overexpressing parasites, we observed the contribution of the protein to increase the general resistance against oxidative damage and intracellular replication of the amastigote stage. Also, studies performed with epimastigotes overexpressing TcrGrx strongly suggest the involvement of the protein in a cellular pathway connecting an apoptotic stimulus and apoptotic-like cell death. Novel information is presented about the participation of this glutaredoxin not only in redox metabolism but also in redox signaling pathways in T. cruzi. The influence of TcrGrx in several parasite physiological processes suggests novel insights about the protein involvement in redox signaling.
Assuntos
Glutarredoxinas/metabolismo , Redes e Vias Metabólicas , Proteínas de Protozoários/metabolismo , Tolueno/análogos & derivados , Trypanosoma cruzi/metabolismo , Apoptose , Biocatálise , Western Blotting , Citosol/enzimologia , Glutarredoxinas/genética , Glutationa/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Oxirredução , Proteínas de Protozoários/genética , Especificidade por Substrato , Tolueno/metabolismo , Trypanosoma cruzi/citologia , Trypanosoma cruzi/genéticaRESUMO
This paper addresses the identification, cloning, expression, purification and functional characterization of thioredoxin reductase from Babesia bovis, the etiological agent of babesiosis. The work deals with in vitro steady state kinetic studies and other complementary analyses of the thioredoxin reductase found in the pathogenic protist. Thioredoxin reductase from B. bovis was characterized as a homodimeric flavoprotein that catalyzes the NADPH-dependent reduction of Trx with a high catalytic efficiency. Moreover, the enzyme exhibited a disulfide reductase activity using DTNB as substrate, being this activity highly sensitive to inhibition by Eosin B. The thioredoxin reductase/thioredoxin system can reduce oxidized glutathione and S-nitrosoglutathione. Our in vitro data suggest that antioxidant defense in B. bovis could be supported by this enzyme. We have performed an enzymatic characterization, searching for targets for rational design of inhibitors. This work contributes to the better understanding of the redox biochemistry occurring in the parasite.
Assuntos
Babesia bovis/enzimologia , Proteínas de Protozoários/química , Tiorredoxina Dissulfeto Redutase/química , Sequência de Aminoácidos , Antibacterianos/farmacologia , Antiprotozoários/química , Clonagem Molecular , Sequência Conservada , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Fluoresceínas/química , Dissulfeto de Glutationa/química , Canamicina/farmacologia , Cinética , Dados de Sequência Molecular , NADP/química , Oxirredução , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/biossíntese , Tiorredoxina Dissulfeto Redutase/antagonistas & inibidores , Tiorredoxina Dissulfeto Redutase/biossínteseRESUMO
Tryparedoxins (TXNs) are multipurpose oxidoreductases from trypanosomatids that transfer reducing equivalents from trypanothione to various thiol proteins. In Trypanosoma cruzi, two genes coding for TXN-like proteins have been identified: TXNI, previously characterized as a cytoplasmic protein, and TXNII, a putative tail-anchored membrane protein. In this work, we performed a comparative functional characterization of T. cruzi TXNs. Particularly, we cloned the gene region coding for the soluble version of TXNII for its heterologous expression. The truncated recombinant protein (without its 22 C-terminal transmembrane amino acids) showed TXN activity. It was also able to transfer reducing equivalents from trypanothione, glutathione, or dihydrolipoamide to various acceptors, including methionine sulfoxide reductases and peroxiredoxins. The results support the occurrence and functionality of a second tryparedoxin, which appears as a new component in the redox scenario for T. cruzi.
Assuntos
Glutationa/metabolismo , Tiorredoxinas/genética , Trypanosoma cruzi/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Expressão Gênica , Glutationa/análogos & derivados , Oxirredução , Proteína Dissulfeto Redutase (Glutationa) , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Homologia de Sequência de Aminoácidos , Espermidina/análogos & derivados , Espermidina/metabolismo , Tiorredoxinas/metabolismoRESUMO
BACKGROUND: Entamoeba histolytica, an intestinal protozoan that is the causative agent of amoebiasis, is exposed to elevated amounts of highly toxic reactive oxygen and nitrogen species during tissue invasion. Thioredoxin reductase catalyzes the reversible transfer of reducing equivalents between NADPH and thioredoxin, a small protein that plays key metabolic functions in maintaining the intracellular redox balance. METHODS: The present work deals with in vitro steady state kinetic studies aimed to reach a better understanding of the kinetic and structural properties of thioredoxin reductase from E. histolytica (EhTRXR). RESULTS: Our results support that native EhTRXR is a homodimeric covalent protein that is able to catalyze the NAD(P)H-dependent reduction of amoebic thioredoxins and S-nitrosothiols. In addition, the enzyme exhibited NAD(P)H dependent oxidase activity, which generates hydrogen peroxide from molecular oxygen. The enzyme can reduce compounds like methylene blue, quinones, ferricyanide or nitro-derivatives; all alternative substrates displaying a relative high capacity to inhibit disulfide reductase activity of EhTRXR. CONCLUSIONS AND GENERAL SIGNIFICANCE: Interestingly, EhTRXR exhibited kinetic and structural properties that differ from other low molecular weight TRXR. The TRX system could play an important role in the parasite defense against reactive species. The latter should be critical during the extra intestinal phase of the amoebic infection. So far we know, this is the first in depth characterization of EhTRXR activity and functionality.