RESUMO
Trypanosoma cruzi, the causative agent of Chagas' disease, belongs to the Trypanosomatidae family. The parasite undergoes multiple morphological and metabolic changes during its life cycle, in which it can use both glucose and amino acids as carbon and energy sources. The glycolytic pathway is peculiar in that its first six or seven steps are compartmentalized in glycosomes, and has a two-branched auxiliary glycosomal system functioning beyond the intermediate phosphoenolpyruvate (PEP) that is also used in the cytosol as substrate by pyruvate kinase. The pyruvate phosphate dikinase (PPDK) is the first enzyme of one branch, converting PEP, PPi, and AMP into pyruvate, Pi, and ATP. Here we present a kinetic study of PPDK from T. cruzi that reveals its hysteretic behavior. The length of the lag phase, and therefore the time for reaching higher specific activity values is affected by the concentration of the enzyme, the presence of hydrogen ions and the concentrations of the enzyme's substrates. Additionally, the formation of a more active PPDK with more complex structure is promoted by it substrates and the cation ammonium, indicating that this enzyme equilibrates between the monomeric (less active) and a more complex (more active) form depending on the medium. These results confirm the hysteretic behavior of PPDK and are suggestive for its functioning as a regulatory mechanism of this auxiliary pathway. Such a regulation could serve to distribute the glycolytic flux over the two auxiliary branches as a response to the different environments that the parasite encounters during its life cycle.
Assuntos
Doença de Chagas/parasitologia , Piruvato Ortofosfato Diquinase/metabolismo , Trypanosoma cruzi/enzimologia , Monofosfato de Adenosina/metabolismo , Difosfatos/metabolismo , Glucose/metabolismo , Glicólise , Concentração de Íons de Hidrogênio , Cinética , Microcorpos/enzimologia , Fosfoenolpiruvato/metabolismo , Piruvato Ortofosfato Diquinase/química , Piruvatos/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
Activation of phosphoenolpyruvate carboxylase (PEPC) enzymes by glucose 6-phosphate (G6P) and other phospho-sugars is of major physiological relevance. Previous kinetic, site-directed mutagenesis and crystallographic results are consistent with allosteric activation, but the existence of a G6P-allosteric site was questioned and competitive activation-in which G6P would bind to the active site eliciting the same positive homotropic effect as the substrate phosphoenolpyruvate (PEP)-was proposed. Here, we report the crystal structure of the PEPC-C4 isozyme from Zea mays with G6P well bound into the previously proposed allosteric site, unambiguously confirming its existence. To test its functionality, Asp239-which participates in a web of interactions of the protein with G6P-was changed to alanine. The D239A variant was not activated by G6P but, on the contrary, inhibited. Inhibition was also observed in the wild-type enzyme at concentrations of G6P higher than those producing activation, and probably arises from G6P binding to the active site in competition with PEP. The lower activity and cooperativity for the substrate PEP, lower activation by glycine and diminished response to malate of the D239A variant suggest that the heterotropic allosteric activation effects of free-PEP are also abolished in this variant. Together, our findings are consistent with both the existence of the G6P-allosteric site and its essentiality for the activation of PEPC enzymes by phosphorylated compounds. Furthermore, our findings suggest a central role of the G6P-allosteric site in the overall kinetics of these enzymes even in the absence of G6P or other phospho-sugars, because of its involvement in activation by free-PEP.
Assuntos
Glucose-6-Fosfato/química , Fosfoenolpiruvato Carboxilase/química , Fosfoenolpiruvato/química , Proteínas de Plantas/química , Zea mays/enzimologia , Regulação Alostérica , Domínio Catalítico , Glucose-6-Fosfato/metabolismo , Cinética , Fosfoenolpiruvato/metabolismo , Fosfoenolpiruvato Carboxilase/genética , Fosfoenolpiruvato Carboxilase/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Zea mays/genéticaRESUMO
Laboratory and industrial cultures of Escherichia coli employ media containing glucose which is mainly transported and phosphorylated by the phosphotransferase system (PTS). In these strains, 50% of the phosphoenolpyruvate (PEP), which results from the catabolism of transported glucose, is used as a phosphate donor for its phosphorylation and translocation by the PTS. This characteristic of the PTS limits the production of industrial biocommodities that have PEP as a precursor. Furthermore, when E. coli is exposed to carbohydrate mixtures, the PTS prevents expression of catabolic and non-PTS transport genes by carbon catabolite repression and inducer exclusion. In this contribution, we discuss the main strategies developed to overcome these potentially limiting effects in production strains. These strategies include adaptive laboratory evolution selection of PTS(-) Glc(+) mutants, followed by the generation of strains that recover their ability to grow with glucose as a carbon source while allowing the simultaneous consumption of more than one carbon source. We discuss the benefits of using alternative glucose transport systems and describe the application of these strategies to E. coli strains with specific genetic modifications in target pathways. These efforts have resulted in significant improvements in the production of diverse biocommodities, including aromatic metabolites, biofuels and organic acids.
Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Metabólica , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Biocombustíveis , Transporte Biológico , Repressão Catabólica , Evolução Molecular Direcionada , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/metabolismo , Glucose/metabolismo , Fenótipo , Fosfoenolpiruvato/metabolismo , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , FosforilaçãoRESUMO
In a previous phylogenetic study of the family of pyruvate kinase, we found one cluster with Glu117 and another with Lys117. Those sequences with Glu117 have Thr113 and are K+-dependent, whereas those with Lys117 have Leu113 and are K+-independent. The carbonyl oxygen of Thr113 is one of the residues that coordinate K+ in the active site. Even though the side chain of Thr113 does not participate in binding K+, the strict co-evolution between position 117 and 113 suggests that T113 may be the result of the evolutionary pressure to maintain the selectivity of pyruvate kinase activity for K+. Thus, we explored if the replacement of Thr113 by Leu alters the characteristics of the K+ binding site. We found that the polarity of the residue 113 is central in the partition of K+ into its site and that the substitution of Thr for Leu changes the ion selectivity for the monovalent cation with minor changes in the binding of the substrates. Therefore, Thr113 is instrumental in the selectivity of pyruvate kinase for K+.
Assuntos
Potássio/metabolismo , Piruvato Quinase/química , Piruvato Quinase/metabolismo , Difosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , Domínio Catalítico , Dimetil Sulfóxido/metabolismo , Ativação Enzimática , Íons , Magnésio/metabolismo , Modelos Moleculares , Músculos/enzimologia , Proteínas Mutantes/metabolismo , Fosfoenolpiruvato/metabolismo , Coelhos , Especificidade por Substrato , Termodinâmica , Água/metabolismoRESUMO
The probiotic Lactobacillus casei catabolizes galacto-N-biose (GNB) and lacto-N-biose (LNB) by using a transport system and metabolic routes different from those of Bifidobacterium. L. casei contains a gene cluster, gnbREFGBCDA, involved in the metabolism of GNB, LNB and also N-acetylgalactosamine. Inactivation of gnbC (EIIC) or ptsI (Enzyme I) of the phosphoenolpyruvate : sugar phosphotransferase system (PTS) prevented the growth on those three carbohydrates, indicating that they are transported and phosphorylated by the same PTS(Gnb) . Enzyme activities and growth analysis with knockout mutants showed that GnbG (phospho-ß-galactosidase) hydrolyses both disaccharides. However, GnbF (N-acetylgalactosamine-6P deacetylase) and GnbE (galactosamine-6P isomerase/deaminase) are involved in GNB but not in LNB fermentation. The utilization of LNB depends on nagA (N-acetylglucosamine-6P deacetylase), showing that the N-acetylhexosamine moieties of GNB and LNB follow different catabolic routes. A lacAB mutant (galactose-6P isomerase) was impaired in GNB and LNB utilization, indicating that their galactose moiety is channelled through the tagatose-6P pathway. Transcriptional analysis showed that the gnb operon is regulated by substrate-specific induction mediated by the transcriptional repressor GnbR, which binds to a 26 bp DNA region containing inverted repeats exhibiting a 2T/2A conserved core. The data represent the first characterization of novel metabolic pathways for human milk oligosaccharides and glycoconjugate structures in Firmicutes.
Assuntos
Acetilglucosamina/análogos & derivados , Dissacarídeos/metabolismo , Lacticaseibacillus casei/genética , Lacticaseibacillus casei/metabolismo , Leite Humano/química , Mucosa/química , Família Multigênica , Fosfoenolpiruvato/metabolismo , Acetilglucosamina/metabolismo , Proteínas de Bactérias/metabolismo , Galactose/metabolismo , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Genes Bacterianos , Humanos , Mutação , Óperon , Polissacarídeos , Reação em Cadeia da Polimerase em Tempo Real , beta-Galactosidase/genéticaRESUMO
Two phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.49) isoforms of 74 and 65 kDa were found to coexist in vivo in pineapple leaves, a constitutive Crassulacean Acid Metabolism plant. The 65 kDa form was not the result of proteolytic cleavage of the larger form since extraction methods reported to prevent PEPCK proteolysis in other plant tissues failed to yield a single immunoreactive PEPCK polypeptide in leaf extracts. In this work, the smaller form of 65 kDa was purified to homogeneity and physically and kinetically characterized and showed parameters compatible with a fully active enzyme. The specific activity was nearly twice higher for decarboxylation of oxaloacetate when compared to carboxylation of phosphoenolpyruvate. Kinetic parameters fell within the range of those estimated for other plant PEPCKs. Its activity was affected by several metabolites, as shown by inhibition by 3-phosphoglycerate, citrate, malate, fructose-1,6-bisphosphate, l-asparagine and activation of the decarboxylating activity by succinate. A break in the Arrhenius plot at about 30°C indicates that PEPCK structure is responsive to changes in temperature. The results indicate that pineapple leaves contain two PEPCK forms. The biochemical characterization of the smaller isoform performed in this work suggests that it could participate in both carbon and nitrogen metabolism in vivo by acting as a decarboxylase.
Assuntos
Ananas/enzimologia , Fosfoenolpiruvato Carboxilase/isolamento & purificação , Proteínas de Plantas/isolamento & purificação , Descarboxilação , Ácido Oxaloacético/metabolismo , Fosfoenolpiruvato/metabolismo , Fosfoenolpiruvato Carboxilase/química , Fotossíntese/fisiologia , Folhas de Planta/química , Proteínas de Plantas/química , Isoformas de Proteínas , TemperaturaRESUMO
Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase is a key enzyme of the gluconeogenic pathway and catalyzes the decarboxylation of oxaloacetate and transfer of the gamma-phosphoryl group of ATP to yield PEP, ADP, and CO2 in the presence of a divalent metal ion. Previous experiments indicate that mutation of amino acid residues at metal site 1 decrease the enzyme catalytic efficiency and the affinity of the protein for PEP, evidencing the relevance of hydrogen-bond interactions between PEP and water molecules of the first coordination sphere of the metal ion for catalysis [Biochemistry 41 (2002) 12763]. To further understand the function of amino acid residues located in the PEP binding site, we have now addressed the catalytic importance of Arg70, whose guanidinium group is close to the PEP carboxyl group. Arg70 mutants of PEP carboxykinase were prepared, and almost unaltered kinetic parameters were found for the Arg70Lys PEP carboxykinase, while a decrease in 4-5 orders of magnitude for the catalytic efficiency was detected for the Arg70Gln and Arg70Met altered enzymes. To evaluate the enzyme interaction with PEP, the phosphopyridoxyl-derivatives of wild type, Arg70Lys, Arg70Gln, and Arg70Met S. cerevisiae PEP carboxykinase were prepared, and the change in the fluorescence emission of the probe upon PEP binding was used to obtain the dissociation equilibrium constant of the corresponding derivatized enzyme-PEP-Mn2+ complex. The titration experiments showed that a loss in 2.1 kcal/mol in PEP binding affinity is produced in the Arg70Met and Arg70Gln mutant enzymes. It is proposed that the electrostatic interaction between the guanidinium group of Arg70 and the carboxyl group of PEP is important for PEP binding and for further steps in catalysis.
Assuntos
Arginina/metabolismo , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Difosfato de Adenosina/metabolismo , Substituição de Aminoácidos , Catálise , Domínio Catalítico , Cinética , Mutagênese Sítio-Dirigida , Fosfoenolpiruvato/metabolismo , Fosfoenolpiruvato Carboxiquinase (ATP)/química , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , TermodinâmicaRESUMO
Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase is a key enzyme of the gluconeogenic pathway and catalyzes the decarboxylation of oxaloacetate and transfer of the gamma-phosphoryl group of ATP to yield PEP, ADP, and CO(2) in the presence of a divalent metal ion. Previous experiments have shown that mutation of amino acid residues at metal site 1 decrease the steady-state affinity of the enzyme for PEP, suggesting interaction of PEP with the metal ion [Biochemistry 41 (2002) 12763]. To more completely understand this enzyme interactions with substrate ligands, we have prepared the phosphopyridoxyl (P-pyridoxyl)-derivatives of wild type, Lys213Arg, and His233Gln S. cerevisiae PEP carboxykinase and used the changes in the fluorescence probe to determine the dissociation equilibrium constants of PEP, ATPMn(2-), and ADPMn(1-) from the corresponding derivatized enzyme-Mn(2+) complexes. Homology modeling of P-pyridoxyl-PEP carboxykinase and P-pyridoxyl-PEP carboxykinase-substrate complexes agree with experimental evidence indicating that the P-pyridoxyl group does not interfere with substrate binding. ATPMn(2-) binding is 0.8kcalmol(-1) more favorable than ADPMn(1-) binding to wild type P-pyridoxyl-enzyme. The thermodynamic data obtained in this work indicate that PEP binding is 2.3kcalmol(-1) and 3.2kcalmol(-1) less favorable for the Lys213Arg and His233Gln mutant P-pyridoxyl-PEP carboxykinases than for the wild type P-pyridoxyl-enzyme, respectively. The possible relevance of N and O ligands for Mn(2+) in relation to PEP binding and catalysis is discussed.
Assuntos
Fosfoenolpiruvato Carboxiquinase (ATP)/química , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Fosfoenolpiruvato/metabolismo , Saccharomyces cerevisiae/enzimologia , Substituição de Aminoácidos , Mutação , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Ligação Proteica , Espectrometria de Fluorescência , Coloração e Rotulagem , Homologia Estrutural de Proteína , Especificidade por SubstratoRESUMO
We report the kinetic characterization of a previously unidentified pyruvate kinase (PK) activity in extracts from Entamoeba histolytica trophozoites. This activity was about 74% of the activity of pyruvate phosphate dikinase. EhPK differed from most PKs in that its pH optimum was 5.5-6.5 and was inhibited by high PEP concentrations (1-5mM); these are concentrations at which PK is usually assayed. The optimal temperature was above 40 degrees C with negligible activity below 20 degrees C. EhPK exhibited hyperbolic kinetics with respect to both PEP (K(m) = 0.018 mM) and ADP (K(m) = 1.05 mM). However, it exhibited a sigmoidal behavior with respect to PEP at sub-saturating ADP concentrations. EhPK did not require monovalent cations for activity. Fructose-1,6 bisphosphate was a potent non-essential activator; it increased the affinity for ADP without modification of the V(max) or the affinity for PEP. Phosphate, citrate, malate, and alpha-ketoglutarate significantly inhibited EhPK activity. A putative EhPK gene fragment found in EhDNA was analyzed. The data indicate that E. histolytica trophozoites contain an active PK, which might contribute to the generation of glycolytic ATP for parasite survival.
Assuntos
Entamoeba histolytica/enzimologia , Piruvato Quinase/metabolismo , Difosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , Cricetinae , Eletroforese em Gel de Poliacrilamida , Entamoeba histolytica/genética , Concentração de Íons de Hidrogênio , Cinética , Abscesso Hepático Amebiano/parasitologia , Dados de Sequência Molecular , Fosfoenolpiruvato/metabolismo , Piruvato Quinase/química , Piruvato Quinase/genética , Piruvato Ortofosfato Diquinase/metabolismo , Coelhos , Alinhamento de Sequência , TemperaturaRESUMO
Bacillus sphaericus, a bacterium of biotechnological interest due to its ability to produce mosquitocidal toxins, is unable to use sugars as carbon source. However, ptsHI genes encoding HPr and EI proteins belonging to a PTS were cloned, sequenced and characterized. Both HPr and EI proteins were fully functional for phosphoenolpyruvate-dependent transphosphorylation in complementation assays using extracts from Staphylococcus aureus mutants for one of these proteins. HPr(His(6)) was purified from wild-type and a Ser46/Gln mutant of B. sphaericus, and used for in vitro phosphorylation experiments using extracts from either B. sphaericus or Bacillus subtilis as kinase source. The results showed that both phosphorylated forms, P-Ser46-HPr and P-His15-HPr, could be obtained. The findings also proved indirectly the existence of an HPr kinase activity in B. sphaericus. The genetic structure of these ptsHI genes has some unusual features, as they are co-transcribed with genes encoding metabolic enzymes related to N-acetylglucosamine (GlcNAc) catabolism (nagA, nagB and an undetermined orf2). In fact, this bacterium was able to utilize this amino sugar as carbon and energy source, but a ptsH null mutant had lost this characteristic. Investigation of GlcNAc uptake and streptozotocin inhibition in both a wild-type and a ptsH null mutant strain led to the proposal that GlcNAc is transported and phosphorylated by an EII(Nag) element of the PTS, as yet uncharacterized. In addition, GlcNAc-6-phosphate deacetylase and GlcN-6-phosphate deaminase activities were determined; both were induced in the presence of GlcNAc. These results, together with the authors' recent findings of the presence of a phosphofructokinase activity, are strongly indicative of a glycolytic pathway in B. sphaericus. They also open new possibilities for genetic improvements in industrial applications.