SEQUENCE, HETEROLOGOUS EXPRESSION AND FUNCTIONAL-CHARACTERIZATION OF A NOVEL TRYPAREDOXIN FROM CRITHIDIA-FASCICULATA

Tryparedoxin has recently been discovered as a constituent of the tryp anosomal peroxidase system catalysing the reduction of a peroxiredoxin -type peroxidase by trypanothione [Nogoceke et al, (1997) Biol, Chem. 378, 827-836] and has attracted interest as a potential molecular targ et for the development of trypanocidal agents. Here we describe the fi rst isolation of a novel gene from Crithidia fasciculata encoding a di fferent tryparedoxin designated tryparedoxin II, The deduced amino aci d sequence of tryparedoxin II (accession number AF055986) differs subs tantially from the partial sequence reported for the tryparedoxin desc ribed previously and now renamed tryparedoxin I. It shares the sequenc e motif Vx(3)FSAxWCPPCR shown to represent the catalytic site in trypa redoxin I [Gommel et al, (1997) Eur. J. Biochem, 248, 913-918] with mo use nucleoredoxin (accession number X92750), and a thioredoxin-like ge ne product of Caenorhabditis elegans (accession number U23511), Depend ing on which ATG is considered functional as translation start codon, tryparedoxin II, with 150 or 165 amino acid residues, is 50% larger th an the typical thioredoxins. The tryparedoxins appear phylogenetically related to the thioredoxins, but sequence similarities are restricted to the active site motifs and their intimate neighbourhood. His-tagge d tryparedoxin II expressed in E. coli exhibited ping-pong kinetics in the trypanothione:peroxiredoxin assay with kinetic parameters (K-M pe roxiredoxin = 4.2 mu M, K-M trypanothione = 33 mu M, V-max/[E] = 952 m in(-1)) similar to those reported for tryparedoxin I [Gommel et al, (1 997) Eur. J. Biochem, 248, 913-918], The co-existence of two distinct tryparedoxins in C. fasciculata suggests diversified biological roles of this novel type of protein, which in trypanosomatids may substitute for the pleiotropic redox catalyst thioredoxin.