Sequence, heterologous expression and functional characterization of tryparedoxin1 from Crithidia fasciculata.
Tryparedoxin (TXN) has recently been discovered as a constituent of the complex peroxidase system in the trypanosomatid Crithidia fasciculata [Nogoceke et 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 proteins 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 CXXC motif and three more remote residues presumed to contribute to the folding pattern of the thioredoxin-type proteins. The TXNs thus form a distinct molecular clade within the thioredoxin superfamily. TXN1 was expressed in Escherichia coli BL21 (DE3)pLysS as a C-terminally extended and His-tagged protein, isolated by chelate chromatography and characterized functionally. The recombinant product exhibited a kinetic pattern identical with, and kinetic 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 potential trypanocidal agents.
Guerrero
S A
SA
Department of Biochemistry, Technical University of Braunschweig, Germany.
Flohé
L
L
Kalisz
H M
HM
Montemartini
M
M
Nogoceke
E
E
Hecht
H J
HJ
Steinert
P
P
Singh
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D016831:Crithidia fasciculata / Q000737:chemistry*; D004220:Disulfides / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013879:Thioredoxins / Q000737:chemistry* / Q000235:genetics
D004220:Disulfides; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C114110:tryparedoxin; D013879:Thioredoxins