RESUMO
Glyoxalase I and II form a ubiquitous glutathione-dependent pathway for the detoxification of reactive and mutagenic ketoaldehydes. Methylglyoxal produced as spontaneous by-product of glycolysis is probably the main physiological substrate. Consequently, African trypanosomes with their exorbitant glucose turnover were expected to have a most efficient detoxification system. Trypanosoma brucei possesses a trypanothione [bis(glutathionyl)spermidine]-dependent glyoxalase II but lacks a glyoxalase I gene. Methylglyoxal reductase as well as dehydrogenase activities are negligible. However, the concentrations of methylglyoxal and advanced glycation end products in the parasites are similar to those in different mammalian cells and the mechanism of methylglyoxal elimination remains elusive. Glyoxalase II is an abundant protein. Overexpression of the gene as well as RNA interference in bloodstream and procyclic cells did not result in a growth phenotype. Deletion of both alleles in procyclic parasites revealed that the enzyme is not essential at least under culture conditions. Recombinant glyoxalase II hydrolyzed the trypanothione-thioesters of methylglyoxal, glyoxal and 4,5-dioxovalerate, substrates of the classical glyoxalase system, with high efficiency. The absence of a glyoxalase I, however, renders these thioesters unlikely as physiological substrates. Here we show that trypanothione-thioesters can be generated from the respective coenzyme A derivative by transesterification. S-Acetyl- and S-propionyltrypanothione obtained by this spontaneous reaction proved to be excellent substrates of T. brucei glyoxalase II. This offers a function for the parasite glyoxalase II as general trypanothione thioesterase independent of ketoaldehyde detoxification.
Assuntos
Glutationa/análogos & derivados , Proteínas de Protozoários/metabolismo , Aldeído Pirúvico/metabolismo , Espermidina/análogos & derivados , Tioléster Hidrolases/metabolismo , Trypanosoma brucei brucei/enzimologia , Animais , Linhagem Celular , Glutationa/metabolismo , Humanos , Cinética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Espermidina/metabolismo , Especificidade por Substrato , Tioléster Hidrolases/química , Tioléster Hidrolases/genética , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Tripanossomíase Africana/parasitologiaRESUMO
The glyoxalase system is a ubiquitous pathway catalyzing the glutathione-dependent detoxication of ketoaldehydes such as methylglyoxal, which is mainly formed as a by-product of glycolysis. The gene encoding a glyoxalase II has been cloned from Trypanosoma brucei, the causative agent of African sleeping sickness. The deduced protein sequence contains the highly conserved metal binding motif THXHXDH but lacks three basic residues shown to fix the glutathione-thioester substrate in the crystal structure of human glyoxalase II. Recombinant T. brucei glyoxalase II hydrolyzes lactoylglutathione, but does not show saturation kinetics up to 5 mm with the classical substrate of glyoxalases II. Instead, the parasite enzyme strongly prefers thioesters of trypanothione (bis(glutathionyl)spermidine), which were prepared from methylglyoxal and trypanothione and analyzed by high performance liquid chromatography and mass spectrometry. Mono-(lactoyl)trypanothione and bis-(lactoyl)trypanothione are hydrolyzed by T. brucei glyoxalase II with k(cat)/K(m) values of 5 x 10(5) m(-1) s(-1) and 7 x 10(5) m(-1) s(-1), respectively, yielding d-lactate and regenerating trypanothione. Glyoxalase II occurs in the mammalian bloodstream and insect procyclic form of T. brucei and is the first glyoxalase II of the order of Kinetoplastida characterized so far. Our results show that the glyoxalase system is another pathway in which the nearly ubiquitous glutathione is replaced by the unique trypanothione in trypanosomatids.
