RESUMO
Transketolase (TKT) is part of the non-oxidative branch of the pentose phosphate pathway (PPP). Here we describe the impact of removing this enzyme from the pathogenic protozoan Leishmania mexicana. Whereas the deletion had no obvious effect on cultured promastigote forms of the parasite, the Δtkt cells were not virulent in mice. Δtkt promastigotes were more susceptible to oxidative stress and various leishmanicidal drugs than wild-type, and metabolomics analysis revealed profound changes to metabolism in these cells. In addition to changes consistent with those directly related to the role of TKT in the PPP, central carbon metabolism was substantially decreased, the cells consumed significantly less glucose, flux through glycolysis diminished, and production of the main end products of metabolism was decreased. Only minor changes in RNA abundance from genes encoding enzymes in central carbon metabolism, however, were detected although fructose-1,6-bisphosphate aldolase activity was decreased two-fold in the knock-out cell line. We also showed that the dual localisation of TKT between cytosol and glycosomes is determined by the C-terminus of the enzyme and by engineering different variants of the enzyme we could alter its sub-cellular localisation. However, no effect on the overall flux of glucose was noted irrespective of whether the enzyme was found uniquely in either compartment, or in both.
Assuntos
Leishmania mexicana/patogenicidade , Leishmaniose Cutânea/metabolismo , Leishmaniose Cutânea/parasitologia , Metaboloma , Transcetolase/metabolismo , Virulência , Animais , Glicólise , Estágios do Ciclo de Vida , Metabolômica , Camundongos , Camundongos Endogâmicos BALB C , Monócitos/metabolismo , Monócitos/parasitologia , Estresse Oxidativo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Deleção de Sequência , Transcetolase/genéticaRESUMO
L-3,4-dihydroxyphenylalanine (L-DOPA) is an aromatic compound employed for the treatment of Parkinson's disease. Metabolic engineering was applied to generate Escherichia coli strains for the production of L-DOPA from glucose by modifying the phosphoenolpyruvate:sugar phosphotransferase system (PTS) and aromatic biosynthetic pathways. Carbon flow was directed to the biosynthesis of L-tyrosine (L-Tyr), an L-DOPA precursor, by transforming strains with compatible plasmids carrying genes encoding a feedback-inhibition resistant version of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, transketolase, the chorismate mutase domain from chorismate mutase-prephenate dehydratase from E. coli and cyclohexadienyl dehydrogenase from Zymomonas mobilis. The effects on L-Tyr production of PTS inactivation (PTS(-) gluc(+) phenotype), as well as inactivation of the regulatory protein TyrR, were evaluated. PTS inactivation caused a threefold increase in the specific rate of L-Tyr production (q( L-Tyr)), whereas inactivation of TyrR caused 1.7- and 1.9-fold increases in q( L-Tyr) in the PTS(+) and the PTS(-) gluc(+) strains, respectively. An 8.6-fold increase in L-Tyr yield from glucose was observed in the PTS(-) gluc(+) tyrR (-) strain. Expression of hpaBC genes encoding the enzyme 4-hydroxyphenylacetate 3-hydroxylase from E. coli W in the strains modified for L-Tyr production caused the synthesis of L-DOPA. One of such strains, having the PTS(-) gluc(+) tyrR (-) phenotype, displayed the best production parameters in minimal medium, with a specific rate of L-DOPA production of 13.6 mg/g/h, L-DOPA yield from glucose of 51.7 mg/g and a final L-DOPA titer of 320 mg/l. In a batch fermentor culture in rich medium this strain produced 1.51 g/l of L-DOPA in 50 h.
Assuntos
Escherichia coli/metabolismo , Glucose/metabolismo , Levodopa/biossíntese , 3-Desoxi-7-Fosfo-Heptulonato Sintase/genética , 3-Desoxi-7-Fosfo-Heptulonato Sintase/metabolismo , Corismato Mutase/genética , Corismato Mutase/metabolismo , Escherichia coli/genética , Engenharia Metabólica , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Plasmídeos , Prefenato Desidratase/genética , Prefenato Desidratase/metabolismo , Prefenato Desidrogenase/genética , Prefenato Desidrogenase/metabolismo , Transcetolase/genética , Transcetolase/metabolismo , Tirosina/biossíntese , Zymomonas/enzimologiaRESUMO
Transketolase has been characterized in Leishmania mexicana. A gene encoding this enzyme was identified and cloned. The gene was expressed in Escherichia coli and the protein was purified and characterized. An apparent K(m) of 2.75 mM for ribose 5-phosphate was determined. X-ray crystallography was used to determine the three-dimensional structure of the enzyme to a resolution of 2.2 A (1 A identical with 0.1 nm). The C-terminus of the protein contains a type-1 peroxisome-targeting signal, suggestive of a possible glycosomal subcellular localization. Subcellular localization experiments performed with promastigote forms of the parasite revealed that the protein was predominantly cytosolic, although a significant component of the total activity was associated with the glycosomes. Transketolase is thus the first enzyme of the nonoxidative branch of the pentose phosphate pathway whose presence has been demonstrated in a peroxisome-like organelle.
Assuntos
Leishmania mexicana/química , Leishmania mexicana/enzimologia , Transcetolase/metabolismo , Sequência de Aminoácidos/genética , Animais , Clonagem Molecular , Cristalografia por Raios X/métodos , DNA de Protozoário/genética , Leishmania mexicana/crescimento & desenvolvimento , Microcorpos/química , Microcorpos/enzimologia , Dados de Sequência Molecular , Peroxissomos/química , Peroxissomos/enzimologia , Sinais Direcionadores de Proteínas/fisiologia , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Transcetolase/biossíntese , Transcetolase/química , Transcetolase/genéticaRESUMO
O termo alcoolismo refere-se aos sintomas que se desenvolvem a partir do consumo inadequado de álcool. Por ser o álcool a principal droga psicoativa utilizada no mundo, é de se esperar que o desenvolvimento da doença dependa da interaçäo entre fatores neurobiológicos e psicossociais. Diversos estudos têm mostrado que o alcoolismo é mais frequente nas famílias de alcoolitas do que na populaçäo em geral. Entretanto, além das influências genéticas, os fatores ambientais também säo responsáveis pela agregaçäo familiar observada, caracterizando, assim, uma herança multifatorial. A subdivisäo do alcoolismo nos tipos 1 e 2 tem permitido uma melhor compreensäo da doença do ponto de vista etiopatogênico e uma melhor abordagem da mesma. Apesar das diferenças metodológicas, a grande maioria dos estudos familiares sugere que os fatores säo muito importantes na determinaçäo do alcoolismo. Já foram identificados os seguintes fatores predisponentes ao alcoolismo: o alelo A1 do gene do receptor de dopammina DRD2, a reduçäo da atividade da MAO-B plaquetária, e a reduçäo da amplitude da onda P300 nos ERPs. Também evidenciou-se que uma anomalia da enzima transcetolase predispöe à Síndrome de Wernicke-Korsakoff. Além disto, foram identificados fatores protetores à doença, como a presença dos alelos ADH2*2 e ALDH2*2 dos genes das enzimas álcool desidrogenase, rspectivamente. Apesar da identificaçäo desses fatores associados ao alcoolismo, pouco ainda se sabe sobre o modo pelo qual eles interagem entre si e, portanto, muitos estudos ainda seräo necessários para um melhor esclarecimento do assunto