CONICET | Buscador de Institutos y Recursos Humanos

Entamoeba histolytica, a unicellular parasite, usually lives andmultiplies within the human gut, under reduced oxygen pressure. During tissueinvasion, the parasite is exposed to increased amounts of highly toxic reactiveoxygen species. E. histolytica lacksor has insignificant amounts of glutathione, with cysteine being the majorintracellular thiol. Previously, we characterized in E. histolytica its functionalthioredoxin system, composed of thioredoxins (TRXs) and thioredoxin reductase(TRXR). In this work, we present the kinetics characterization of non-canonicalthioredoxin (EhTRX212) with highspecificity for cystine reduction from E.histolytica. By steady-state kinetics assays, we observedthat EhTRX212 was unable toaccept reduction equivalents from EhTRXRdirectly and reduce protein disulfides. However, the EhTRX212 was able to catalyze the reductionof cystine, CySNO, and cysteine-derivate low molecular mass disulfidesvia EhTRX8 (a canonical TRX)and EhTRXR. Besides, EhTRX212 exhibited GSH-dependent cysteinereductase activity via a coupled enzymatic assay. Kinetic data indicated thatregeneration of EhTRX212 toits reduced form is more efficient using reduced EhTRX8 than GSH as a reducing substrate. Theobtained results are novelty because the EhTRX212is one of the first cases where it is observed a specific and efficientreduction equivalent transfer from a canonical TRX to another TRX. In acomplementary way, by pre-steady-state kinetics (using stopped-flowmethodology), we observed that the cystine-reduction by EhTRX212 follows a biphasic temporalprogression, consistent with a thiol-disulfide exchange mechanism. The firstphase (the nucleophilic cysteine attack on cystine disulfide) followedsecond-order kinetics (k =2.4 ·106 M-1s-1) and the second phase (themixed disulfide resolution) followed first-order kinetics (k = 11 s-1). The chemicalsubstitutions (for example, the N-acetylation) in cysteine moiety impair thereduction by EhTRX212 ofderivative low molecular mass disulfides, indicating a substrate specificity bycysteine-moiety in disulfide substrates. Finally, EhTRX212 has been localized in the cytosol oftrophozoites by confocal microscopy experiments. This work strongly supportsthe occurrence of a new class of TRX. Our results extend the knowledgeregarding TRX function in E. histolytica andsuggest that these proteins have critical roles in the redox metabolism of thispathogen parasite.