Characterization of a novel non-canonical thioredoxin from Entamoeba histolytica specific to cystine reduction

Entamoeba histolytica, a unicellular parasite, has negligible glutathione levels and instead relies on cysteine as its primary intracellular thiol. It has a functional thioredoxin (TRX) system, which consists of four canonical TRXs and thioredoxin reductase (TRXR). We identified a coding sequence for a putative non-canonical TRX with a WCKDC redox-active motif (EhTRX212, Uniprot M2QBU7) within the E. histolytica HM-1:IMSS genome. We produced the recombinant protein and conducted its biochemical characterization. Steady-state kinetic assays revealed that EhTRX212 is not reduced by EhTRXR nor directly reduces protein disulfides. Instead, EhTRX212 catalyzes the reduction of cystine, S-nitrosocysteine, and cysteine-derived heterodisulfides through a coupled reaction with EhTRX8 (a canonical TRX) and EhTRXR. Complementary pre-steady-state kinetics, using stopped-flow methodology, showed that the reduction of cystine by EhTRX212 follows a biphasic temporal progression, consistent with a thiol-disulfide exchange mechanism. The first phase (nucleophilic cysteine attack on the cystine disulfide) followed second-order kinetics (k = 2.4 × 10⁶ M^-1 s^-1), while the second phase (mixed disulfide resolution) followed first-order kinetics (k = 11 s^-1). Chemical modifications, on the amino group of the cysteine in disulfide substrates, impaired reduction by EhTRX212. Finally, confocal microscopy and digitonin subcellular fractionation experiments localized EhTRX212 to the trophozoite cytoplasm. This study strongly supports the existence of a novel class of TRXs with high specificity for cystine reduction, expanding our understanding of TRX function in E. histolytica and suggesting important roles for these proteins in the redox metabolism of this parasitic pathogen.