Publicação
Methylglyoxal metabolism in Leishmania infantum
| Resumo: | Leishmaniasis, caused by a Leishmania parasite belonging to the Trypanosomatidae family, are diseases affecting humans and other mammals. The most severe form of the disease is lethal if untreated, currently existing no vaccines or efficient therapies. The identification of new therapeutic targets is presently based on exploiting the biochemical differences between the parasite and the host. One of the main biochemical characteristics distinguishing trypanosomatids from other eukaryotic cells is the functional replacement of glutathione by trypanothione. In trypanosomatids, the glyoxalase system, comprising the enzymes glyoxalase I and glyoxalase II, depends on trypanothione to eliminate methylglyoxal, a toxic compound formed non-enzymatically during glycolysis. Hence, this is an excellent model system to understand trypanothione-dependent enzymes specificity at a kinetic and molecular level. The methylglyoxal metabolism in L. infantum study would not be complete without an account of aldose reductase, a NADPH-dependent enzyme also catabolising this toxic compound. This project includes an eclectic structural and biochemical study of the main enzymes involved in methylglyoxal catabolism, contributing for the knowledge of these complex parasites. Additionally, these enzymes’ activities complement each other in such a way that they can be synergistically exploited in the quest for new anti-leishmanial drug targets. The glyoxalase I gene from Leishmania infantum (LiGLO1) was isolated and cloned into an expression vector for bacteria. The recombinant protein was over-expressed in E. coli, purified and kinetically characterised. LiGLO1 showed to preferentially use the hemithioacetal derived from trypanothione, although it can also catalyse the same reaction with the glutathione-derived hemithioacetal. The recombinant protein was crystallised and its structure solved by molecular replacement, using the glyoxalase structure from L. major as a search model. Although the LiGLO1 structure is very similar to the L. major GLO1, as expected by its high homology, the metal observed at the active site is different. While LmGLO1 requires nickel for its activity, like glyoxalase I from prokaryotes, it was shown both by ICP and anomalous diffraction that LiGLO1 contains zinc in the active site, as its eukaryotic homologues. On the other hand, LiGLO1 has significant structural differences relatively to the human glyoxalase I enzyme. The glyoxalase II gene from L. infantum (LiGLO2) was also isolated and cloned in a bacterial expression vector. The recombinant protein was over-expressed in E. coli, purified and kinetically characterised, confirming its specificity towards trypanothione-derived thiolesters. LiGLO2 was crystallised and its structure solved by molecular replacement, using the glutathione-dependent human glyoxalase II structure as a search model, for its high sequence homology with the structurally unknown L. infantum protein. The determined structural model for LiGLO2 is very similar to its human counterpart. Highly conserved residues were identified in the active site, as well as specific residues of the L. infantum enzyme, being noteworthy the presence of the spermidine-binding Cys294 and Ile171, both absent from the human enzyme. The presence of a spermidine molecule on the LiGLO2 substrate binding site, together with sequence analysis, clarified the enzyme’s substrate specificity at a molecular level. Both ICP metal-analysis and the B factor values for the metal atoms revealed the presence of zinc and/or iron in the enzyme active site. A structure with D-lactate in the active site was obtained by crystal soaking with substrate. Superimposing both LiGLO2 structures, the localization of the trypanothione-derived thiolester in the substrate-binding site could be clearly inferred. Two of the residues forming the substrate- binding pocket, Tyr291 and Cys294, were subsequently replaced by the S-D-lactoylglutathione- binding residues found on the human enzyme, Arg249 and Lys252, respectively. Recombinant mutated LiGLO2 was over-expressed in E. coli. Kinetic analysis revealed that the enzyme’s substrate specificity was changed, catalysing the reaction with S-D-lactoylglutathione, and loosing affinity towards S-D-lactoyltrypanothione. These results show that the mutated residues are critical for the enzyme specificity. The aldose reductase gene from L. infantum (LiAKR) was identified for the first time in a trypanosomatid. It was isolated and cloned into an expression vector. Over-expression of the soluble recombinant protein in E. coli was only achieved by co-expression with chaperone systems. The LiAKR enzyme was kinetically characterised as a NADPH-dependent aldose reductase involved in the catabolism of methylglyoxal. This protein was recently crystallised, although the observed diffraction requires crystal optimization. |
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| Autores principais: | Barata, Lídia Isabel Sebastião, 1983- |
| Assunto: | Leishmania Infantum Metilglioxal Tripanotiono Aldose redutase Regulação bioquímica Teses de doutoramento - 2010 |
| Ano: | 2010 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | português |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | Leishmaniasis, caused by a Leishmania parasite belonging to the Trypanosomatidae family, are diseases affecting humans and other mammals. The most severe form of the disease is lethal if untreated, currently existing no vaccines or efficient therapies. The identification of new therapeutic targets is presently based on exploiting the biochemical differences between the parasite and the host. One of the main biochemical characteristics distinguishing trypanosomatids from other eukaryotic cells is the functional replacement of glutathione by trypanothione. In trypanosomatids, the glyoxalase system, comprising the enzymes glyoxalase I and glyoxalase II, depends on trypanothione to eliminate methylglyoxal, a toxic compound formed non-enzymatically during glycolysis. Hence, this is an excellent model system to understand trypanothione-dependent enzymes specificity at a kinetic and molecular level. The methylglyoxal metabolism in L. infantum study would not be complete without an account of aldose reductase, a NADPH-dependent enzyme also catabolising this toxic compound. This project includes an eclectic structural and biochemical study of the main enzymes involved in methylglyoxal catabolism, contributing for the knowledge of these complex parasites. Additionally, these enzymes’ activities complement each other in such a way that they can be synergistically exploited in the quest for new anti-leishmanial drug targets. The glyoxalase I gene from Leishmania infantum (LiGLO1) was isolated and cloned into an expression vector for bacteria. The recombinant protein was over-expressed in E. coli, purified and kinetically characterised. LiGLO1 showed to preferentially use the hemithioacetal derived from trypanothione, although it can also catalyse the same reaction with the glutathione-derived hemithioacetal. The recombinant protein was crystallised and its structure solved by molecular replacement, using the glyoxalase structure from L. major as a search model. Although the LiGLO1 structure is very similar to the L. major GLO1, as expected by its high homology, the metal observed at the active site is different. While LmGLO1 requires nickel for its activity, like glyoxalase I from prokaryotes, it was shown both by ICP and anomalous diffraction that LiGLO1 contains zinc in the active site, as its eukaryotic homologues. On the other hand, LiGLO1 has significant structural differences relatively to the human glyoxalase I enzyme. The glyoxalase II gene from L. infantum (LiGLO2) was also isolated and cloned in a bacterial expression vector. The recombinant protein was over-expressed in E. coli, purified and kinetically characterised, confirming its specificity towards trypanothione-derived thiolesters. LiGLO2 was crystallised and its structure solved by molecular replacement, using the glutathione-dependent human glyoxalase II structure as a search model, for its high sequence homology with the structurally unknown L. infantum protein. The determined structural model for LiGLO2 is very similar to its human counterpart. Highly conserved residues were identified in the active site, as well as specific residues of the L. infantum enzyme, being noteworthy the presence of the spermidine-binding Cys294 and Ile171, both absent from the human enzyme. The presence of a spermidine molecule on the LiGLO2 substrate binding site, together with sequence analysis, clarified the enzyme’s substrate specificity at a molecular level. Both ICP metal-analysis and the B factor values for the metal atoms revealed the presence of zinc and/or iron in the enzyme active site. A structure with D-lactate in the active site was obtained by crystal soaking with substrate. Superimposing both LiGLO2 structures, the localization of the trypanothione-derived thiolester in the substrate-binding site could be clearly inferred. Two of the residues forming the substrate- binding pocket, Tyr291 and Cys294, were subsequently replaced by the S-D-lactoylglutathione- binding residues found on the human enzyme, Arg249 and Lys252, respectively. Recombinant mutated LiGLO2 was over-expressed in E. coli. Kinetic analysis revealed that the enzyme’s substrate specificity was changed, catalysing the reaction with S-D-lactoylglutathione, and loosing affinity towards S-D-lactoyltrypanothione. These results show that the mutated residues are critical for the enzyme specificity. The aldose reductase gene from L. infantum (LiAKR) was identified for the first time in a trypanosomatid. It was isolated and cloned into an expression vector. Over-expression of the soluble recombinant protein in E. coli was only achieved by co-expression with chaperone systems. The LiAKR enzyme was kinetically characterised as a NADPH-dependent aldose reductase involved in the catabolism of methylglyoxal. This protein was recently crystallised, although the observed diffraction requires crystal optimization. |
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