RESUMO
In this paper, we report the subcellular distribution of phosphoglycerate kinase (PGK) in epimastigotes of Trypanosoma cruzi. Approximately 80% of the PGK activity was found in the cytosol, 20% in the glycosomes. Western blot analysis suggested that two isoenzymes of 56 and 48 kDa, respectively, are responsible for the glycosomal PGK activity, whereas the cytosolic activity should be attributed to a single PGK of 48 kDa. In analogy to the situation previously reported for PGK in Trypanosoma brucei, these isoenzymes were called PGKA, C and B, respectively. However, in T. cruzi, PGKA seems not to be a minor enzyme like its counterpart in T. brucei. Whereas PGKC behaved as a soluble glycosomal matrix protein, PGKA appeared to be present at the inner surface of the organelle's membrane. After alkaline carbonate treatment, the enzyme remained associated with the particulate fraction of the organelles. Upon solubilization of glycosomes with Triton X-114, PGKA was recovered from the detergent phase, indicating its (partial) hydrophobic character and therefore, a possible hydrophobic interaction with the membrane. The PGKA gene was cloned and sequenced, but the predicted amino-acid sequence did not reveal an obvious clue as to the mechanism by which the enzyme is attached to the glycosomal membrane.
Assuntos
Fosfoglicerato Quinase/metabolismo , Frações Subcelulares/enzimologia , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Citosol/enzimologia , Isoenzimas/metabolismo , Microcorpos/enzimologia , Dados de Sequência Molecular , Fosfoglicerato Quinase/genética , Análise de Sequência de DNA , Trypanosoma cruzi/genética , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
Phytomonas sp. contains two malate dehydrogenase isoforms, a mitochondrial isoenzyme with a high specificity for oxaloacetate and a glycosomal isozyme that acts on a broad range of substrates (Uttaro, A. D., and Opperdoes, F.R. (1997) Mol. Biochem. Parasitol. 89, 51-59). Here, we show that the low specificity of the latter isoenzyme is the result of a number of recent gene duplications that gave rise to a family of glycosomal 2-hydroxyacid dehydrogenase genes. Two of these genes were cloned, sequenced, and overexpressed in Escherichia coli. Although both gene products have 322 amino acids, share 90.4% identical residues, and have a similar hydrophobicity profile and net charge, their kinetic properties were strikingly different. One isoform behaved as a real malate dehydrogenase with a high specificity for oxaloacetate, whereas the other showed no activity with oxaloacetate but was able to reduce other oxoacids, such as phenyl pyruvate, 2-oxoisocaproate, 2-oxovalerate, 2-oxobutyrate, 2-oxo-4-methiolbutyrate, and pyruvate.
Assuntos
Oxirredutases do Álcool/metabolismo , Sequência Conservada/genética , Microcorpos/enzimologia , Família Multigênica/genética , Trypanosomatina/enzimologia , Oxirredutases do Álcool/química , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/isolamento & purificação , Sequência de Aminoácidos , Animais , Southern Blotting , Clonagem Molecular , Dosagem de Genes , Genes Duplicados/genética , Genes de Protozoários/genética , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Cinética , Malato Desidrogenase/química , Malato Desidrogenase/genética , Malato Desidrogenase/isolamento & purificação , Malato Desidrogenase/metabolismo , Dados de Sequência Molecular , Oxaloacetatos/metabolismo , Filogenia , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Especificidade por Substrato , Trypanosomatina/genéticaRESUMO
BACKGROUND: NAD-dependent glycerol-3-phosphate dehydrogenase (GPDH) catalyzes the interconversion of dihydroxyacetone phosphate and L-glycerol-3-phosphate. Although the enzyme has been characterized and cloned from a number of sources, until now no three-dimensional structure has been determined for this enzyme. Although the utility of this enzyme as a drug target against Leishmania mexicana is yet to be established, the critical role played by GPDH in the long slender bloodstream form of the related kinetoplastid Trypanosoma brucei makes it a viable drug target against sleeping sickness. RESULTS: The 1.75 A crystal structure of apo GPDH from L. mexicana was determined by multiwavelength anomalous diffraction (MAD) techniques, and used to solve the 2.8 A holo structure in complex with NADH. Each 39 kDa subunit of the dimeric enzyme contains a 189-residue N-terminal NAD-binding domain and a 156-residue C-terminal substrate-binding domain. Significant parts of both domains share structural similarity with plant acetohydroxyacid isomeroreductase. The discovery of extra, fatty-acid like, density buried inside the C-terminal domain indicates a possible post-translational modification with an associated biological function. CONCLUSIONS: The crystal structure of GPDH from L. mexicana is the first structure of this enzyme from any source and, in view of the sequence identity of 63%, serves as a valid model for the T. brucei enzyme. The differences between the human and trypanosomal enzymes are extensive, with only 29% sequence identity between the parasite and host enzyme, and support the feasibility of exploiting the NADH-binding site to develop selective inhibitors against trypanosomal GPDH. The structure also offers a plausible explanation for the observed inhibition of the T. brucei enzyme by melarsen oxide, the active form of the trypanocidal drugs melarsoprol and cymelarsan.
Assuntos
Glicerolfosfato Desidrogenase/química , Leishmania mexicana/enzimologia , Modelos Moleculares , Proteínas de Protozoários/química , Animais , Sítios de Ligação , Cristalografia por Raios X , Dimerização , Desenho de Fármacos , Evolução Molecular , Glicerol-3-Fosfato Desidrogenase (NAD+) , Glicerolfosfato Desidrogenase/genética , Glicerolfosfato Desidrogenase/metabolismo , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Homologia de Sequência de Aminoácidos , Tripanossomicidas/química , Tripanossomicidas/metabolismoRESUMO
The NAD-dependent glycerol-3-phosphate dehydrogenases (G3PDH, EC 1.1.1.8) of Trypanosoma brucei and Leishmania mexicana are thought to have different roles in carbohydrate metabolism. Here the physicochemical and kinetic properties of natural G3PDH from T. brucei with the recombinant homologue of L. mexicana which share 63% positional identity are compared. Despite their supposed different functions in energy metabolism of the parasites the two G3PDHs have remarkably similar properties, including pH optima and K(m) value for dihydroxyacetone phosphate (DHAP) and NADH in the formation of glycerol 3-phosphate (G3P) and for NAD+ and G3P in the reverse reaction. Both enzymes are subject inhibition by dihydroxyacetone phosphate at concentrations above 0.2 mM and are inhibited by the trypanocidal drugs suramin and melarsen oxide at sub-micromolar concentrations.
Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Leishmania mexicana/enzimologia , Trypanosoma brucei brucei/enzimologia , Animais , Fosfato de Di-Hidroxiacetona/metabolismo , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática , Escherichia coli/enzimologia , Gliceraldeído-3-Fosfato Desidrogenases/química , Gliceraldeído-3-Fosfato Desidrogenases/genética , Glicerofosfatos/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Leishmania mexicana/genética , NAD/metabolismo , NADP/metabolismo , Concentração Osmolar , Proteínas Recombinantes/metabolismo , Cloreto de Sódio/farmacologiaAssuntos
Frutose-Bifosfato Aldolase/genética , Leishmania mexicana/genética , Proteínas Recombinantes/isolamento & purificação , Sequência de Aminoácidos , Animais , Clonagem Molecular , Escherichia coli/genética , Frutose-Bifosfato Aldolase/antagonistas & inibidores , Frutose-Bifosfato Aldolase/isolamento & purificação , Genes de Protozoários , Leishmania mexicana/enzimologia , Dados de Sequência Molecular , Peroxissomos/enzimologia , Homologia de Sequência de Aminoácidos , Suramina/farmacologia , Tripanossomicidas/farmacologiaRESUMO
Glycolysis occupies a central role in cellular metabolism, and is of particular importance for the catabolic production of ATP in protozoan parasites such as Leishmania and Trypanosoma. In these organisms pyruvate kinase plays a key regulatory role, and is unique in responding to fructose 2,6-bisphosphate as allosteric activator. The determination of the first eukaryotic pyruvate kinase crystal structure in the T-state is reported. A comparison of the leishmania and yeast R-state enzymes reveals fewer differences than the previous comparison of Escherichia coli T-state and rabbit muscle non-allosteric enzymes. Structural changes related to the allosteric transition can therefore be distinguished from those that are a consequence of the inherent wide structural divergence between bacterial and mammalian proteins. The allosteric transition involves significant changes in a tightly packed array of eight alpha helices at the interface near the catalytic site. At the other interface the allosteric transition appears to be accompanied by the bending of a ten-stranded intersubunit beta sheet adjacent to the effector site. Helix Calpha1 makes contacts to the N-terminal helical domain and bridges both interfaces. A comparison of the effector sites of the leishmania and yeast enzymes reveals the structural basis for the different effector specificity. Two loops comprising residues 443-453 and 480-489 adopt very different conformations in the two enzymes, and Lys453 and His480 that are a feature of trypanosomatid enzymes provide probable ligands for the 2-phospho group of the effector molecule. These differences offer an opportunity for the design of drugs that would bind to the trypanosomatid enzymes but not to those of the mammalian host.
Assuntos
Leishmania mexicana/enzimologia , Piruvato Quinase/química , Regulação Alostérica , Sequência de Aminoácidos , Animais , Domínio Catalítico/genética , Cristalografia por Raios X , Leishmania mexicana/genética , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , Coelhos , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/genéticaRESUMO
The structure of the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from glycosomes of the parasite Trypanosoma cruzi, causative agent of Chagas' disease, is reported. The final model at 2.8 A includes the bound cofactor NAD+ and 90 water molecules per monomer and resulted in an Rfactor of 20.1%, Rfree = 22.3%, with good geometry indicators. The structure has no ions bound at the active site resulting in a large change in the side chain conformation of Arg249 which as a consequence forms a salt bridge to Asp210 in the present structure. We propose that this conformational change could be important for the reaction mechanism and possibly a common feature of many GAPDH structures. Comparison with the human enzyme indicates that interfering with this salt bridge could be a new approach to specific inhibitor design, as the equivalent to Asp210 is a leucine in the mammalian enzymes.
Assuntos
Inibidores Enzimáticos/química , Gliceraldeído-3-Fosfato Desidrogenases/química , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Trypanosoma cruzi/enzimologia , Animais , Desenho de Fármacos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Gliceraldeído-3-Fosfato Desidrogenases/antagonistas & inibidores , Humanos , Modelos Moleculares , NAD/metabolismo , Fosfatos/química , Conformação Proteica , Sulfatos/químicaRESUMO
In Leishmania mexicana two genes were detected coding for different isoforms of the glycolytic enzyme phosphoglycerate kinase. This situation contrasts with that observed in other Trypanosomatidae (Trypanosoma brucei, Trypanosoma congolense, Crithidia fasciculata) analyzed previously, which all contain three different genes coding for isoenzymes A, B and C, respectively. All attempts to detect in L. mexicana a type A PGK, or a gene encoding it, proved unsuccesful. We have cloned and characterized the genes PGKB and PGKC. They code for polypeptides of 416 and 478 amino acids with a molecular mass of 45146 and 51318 Da, respectively. The two polypeptides are 99% identical. PGKC is characterized by a 62 residue C-terminal extension with alternating stretches of hydrophobic and charged, mainly positive amino acids. As in other Trypanosomatidae, PGKB is located in the cytosol, PGKC in the glycosomes. However, Leishmania mexicana distinguishes itself from other trypanosomatids by the simultaneous expression of these isoenzymes: approximately 80% of PGK activity is found in the cytosol and 20% in the glycosomes, both in promastigotes and in the amastigote-like form of the parasite.
Assuntos
Genes de Protozoários , Leishmania mexicana/genética , Fosfoglicerato Quinase/genética , Sequência de Aminoácidos , Animais , Southern Blotting , Western Blotting , Clonagem Molecular , Citosol/enzimologia , Expressão Gênica , Glicólise , Ponto Isoelétrico , Isoenzimas/química , Isoenzimas/genética , Leishmania mexicana/enzimologia , Leishmania mexicana/crescimento & desenvolvimento , Leishmania mexicana/ultraestrutura , Dados de Sequência Molecular , Organelas/enzimologia , Fosfoglicerato Quinase/análise , Fosfoglicerato Quinase/biossíntese , Fosfoglicerato Quinase/química , Reação em Cadeia da Polimerase , Análise de Sequência , Trypanosomatina/enzimologia , Trypanosomatina/genéticaRESUMO
A polyclonal antiserum raised against the purified glycosomal glycerol-3-phosphate dehydrogenase of Trypanosoma brucei brucei has been used to identify the corresponding cDNA clone in a T.b. brucei expression library. This cDNA was subsequently used to obtain genomic clones containing glycerol-3-phosphate dehydrogenase genes. Two tandemly arranged genes were detected in these clones. Characterization of one of the genes showed that it codes for a polypeptide of 353 amino acids, with a molecular mass of 37,651 Da and a calculated net charge of +8. Using the T.b. brucei gene as a probe, a corresponding glycerol-3-phosphate dehydrogenase gene was also identified in a genomic library of Leishmania mexicana mexicana. The L.m. mexicana gene codes for a polypeptide of 365 amino acids, with a molecular mass of 39,140 Da and a calculated net charge of +8. The amino-acid sequences of both polypeptides are 63% identical and carry a type-1 peroxisomal targeting signal (PTS1) SKM and -SKL at their respective C-termini. Moreover, the L.m. mexicana polypeptide also carries a short N-terminal extension reminiscent of a mitochondrial transit sequence. Subcellular localisation analysis showed that in L.m. mexicana the glycerol-3-phosphate dehydrogenase activity co-fractionated both with mitochondria and with glycosomes. This is not the case in T. brucei, where the enzyme is predominantly glycosomal. The two trypanosomatid sequences resemble their prokaryotic homologues (32-36%) more than their eukaryotic counterparts (25-31%) and carry typical prokaryotic signatures. The possible reason for this prokaryotic nature of a trypanosomatid glycerol-3-phosphate dehydrogenase is discussed.
Assuntos
Glicerolfosfato Desidrogenase/química , Leishmania mexicana/enzimologia , Trypanosoma brucei brucei/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Expressão Gênica , Glicerol-3-Fosfato Desidrogenase (NAD+) , Glicerolfosfato Desidrogenase/genética , Leishmania mexicana/genética , Dados de Sequência Molecular , NAD/metabolismo , Filogenia , Homologia de Sequência , Trypanosoma brucei brucei/genéticaRESUMO
The gene coding for the glycosomal glyceraldehyde-3-phosphate dehydrogenase from Leishmania mexicana has been cloned into vector pET3A and expressed as a soluble and active protein in Escherichia coli BL21(DE3) in which the endogenous gene has been inactivated by mutation. The recombinant enzyme was purified to near homogeneity by ammonium sulphate precipitation, followed by hydrophobic and cation-exchange chromatography. From a 1-L culture of E. coli cells, 25 mg purified protein was obtained with a specific activity of 125 units/mg. The recombinant protein restores the natural E. coli phenotype when expressed at low level. The enzyme has also been partially purified from glycosomes of cultured L. mexicana promastigotes. The recombinant and the native proteins show identical mobilities on SDS/PAGE, and have the same isoelectric point and similar pH-activity profiles. The kinetics of both enzymes are very similar, the most important aspect being their lower apparent affinity for the cofactor NAD when compared to all other homologous enzymes studied, with the exception of glycosomal glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma brucei.