Trypanothione-dependent glyoxalase I in Trypanosoma cruzi
Résumé
The glyoxalase system, comprising glyoxalase I and glyoxalase II, is a ubiquitous pathway that detoxifies highly reactive aldehydes such as methylglyoxal, using glutathione as a co-factor. Recent studies of Leishmania major glyoxalase I and Trypanosoma brucei glyoxalase II have revealed a unique dependence upon the trypanosomatid thiol trypanothione as a co-factor. This difference suggests that the trypanothione-dependent glyoxalase system may be an attractive target for rational drug design against the trypanosomatid parasites. Here we describe the cloning, expression and kinetic characterisation of glyoxalase I from Trypanosoma cruzi. Like L. major glyoxalase I, recombinant T. cruzi glyoxalase I showed a preference for nickel as its metal co-factor. In contrast to the Leishmania enzyme, T. cruzi glyoxalase I was far less fastidious in its choice of metal co-factor efficiently utilising cobalt, manganese and zinc. T. cruzi glyoxalase I isomerised hemithioacetal adducts of trypanothione more than 2400-times more efficiently than glutathione adducts with the methylglyoxal adducts 2-3 fold better substrates than the equivalent phenylglyoxal adducts. However, glutathionylspermidine hemithioacetal adducts were most efficiently isomerised and the glutathionylspermidine-based inhibitor S-4-bromobenzylglutathionylspermidine was found to be a potent linear competitive inhibitor of the T. cruzi enzyme with a K i} of 5.4 ± 0.6 {mu}M. Prediction algorithms, combined with subcellular fractionation, suggest that T. cruzi glyoxalase I localises to the cytosol but also the mitochondria of T. cruzi epimastigotes. The contrasting substrate specificities of human and trypanosomatid glyoxalase enzymes, confirmed in this study, suggest that the glyoxalase system may be an attractive target for anti-trypanosomal chemotherapy.
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