Sequence, heterologous expression and functional characterization of tryparedoxin1 from Crithidia fasciculata

Tryparedoxin (TXN) has recently been discovered as a constituent of the com plex peroxidase system in the trypanosomatid Crithidia fasciculata [Nogocek e ed al. (1997) Biol. Chem. 378, 827-836] where it catalyzes the reduction of a peroxiredoxin-type peroxidase by trypanothione. Here we report on the full-length DNA sequence of the TXN previously isolated from C. fasciculata (TXN1). The deduced amino acid sequence comprises 147 residues and matches with all the peptide sequences of fragments obtained from TXN1. It shares a characteristic sequence motif YFSAxWCPPCR with some thioredoxin-related p roteins of unknown function. This motif is homologous with the CXXC motif, which characterizes the thioredoxin superfamily of proteins and is known to catalyze disulfide reductions. Sequence conservations between TXNs and the typical thioredoxins are restricted to the intimate environment of the CXX C motif and three more remote residues presumed to contribute to the foldin g pattern of the thioredoxin-type proteins. The TXNs thus form a distinct m olecular clade within the thioredoxin superfamily. TXN1 was expressed in Es cherichia coli BL21(DE3)pLysS as a C-terminally extended and His-tagged pro tein, isolated by chelate chromatography and characterized functionally. Th e recombinant product exhibited a kinetic pattern identical with, and kinet ic parameters similar to those of the authentic enzyme in the trypanothione /peroxiredoxin oxidoreductase assay. The recombinant TXN1 can therefore be considered a valuable tool for the screening of specific inhibitors as pote ntial trypanocidal agents.