Insight to the catalytic mechanism and identification of structural/functional elements in highly conserved regions of trypanothione synthetases

COMINI, MA; SALAME, M; BLANCATO, VS; GUERRERO, SA; FLOHÉ, L

Rosario, Argentina

Congreso; XX Reunión de la Sociedad Argentina de Protozoología; 2004

Sociedad Argentina de Protozoología

Parasites of the family Trypanosomatidae are responsible for some of the most devastating and prevalent diseases of humans and domestic animals. There is an urgent need for novel trypanocidal drugs considering that there is no prospect for a vaccine in the short-term, and the current chemotherapy is inefficient, toxic and costly. In this regard, metabolic pathways that are of vital importance for pathogens but absent in their hosts, like biosynthesis and use of trypanothione deserve particular interest. Trypanothione synthetase (TryS) is a multifunctional enzyme that has been reported to catalyse the entire synthesis of trypanothione in Crithidia fasciculata, Trypanosoma cruzi and T. brucei, and has also been suggested in Leishmania spp. Moreover, TryS has recently been validated as a most attractive drug target for African trypanosomes. In order to progress further in the characterization of such relevant trypanocidal candidates, we conducted steady-state kinetic studies, limited proteolysis, and site directed mutagenesis analysis on the available TrySs. Our studies suggest that: i) TrySs operate via a concerted-substitution mechanism for the ligation of glutathione to glutathionylspermidine, ii) TrySs posses in the C-terminal domain a module essential for synthetase activity, iii) TrySs contain non-regular secondary structures (resembling of W- and P-loop motifs) that interact with the substrates, iv) two arginine residues located in these motifs are essential in binding substrates and, hence, in enzyme function. Thus, these results constitute the first insight into the mechanism and amino acidic residues involved in catalysis in this family of enzymes.