Sequence analysis of the tryparedoxin peroxidase gene from Crithidia fasciculata and its functional expression in Escherichia coli

Abstract

Tryparedoxin peroxidase from Crithidia fasciculata is an essential component of the trypanothione-dependent hydroperoxide metabolism in the trypanosomatids (Nogoceke, E., Gommel, D. U., Kiess, M., Kalisz, H. M., and Flohe, L. (1997) Biol. Chem. 378, 827-836). The tryparedoxin peroxidase gene and its flanking regions have been isolated and sequenced from a C. fasciculata genomic DNA library. It consists of an open reading frame of 564 base pairs encoding a protein of 188 amino acid residues. The gene, modified to encode 6 additional histidine residues, was expressed in Escherichia coli and the recombinant protein was purified to homogeneity by metal chelating chromatography. Recombinant tryparedoxin peroxidase has a subunit molecular mass of 21884 ± 22 and contains two isoforms of pI 6.2 and 6.3. It exhibits a kinetic pattern identical to that of the authentic tryparedoxin peroxidase and has a similar specific activity of 2.51 units mg-1. The enzyme unequivocally belongs to the peroxiredoxin family of proteins, whose members have been found in all phyla. A phylogenetic tree comprising 47 protein and DNA sequences showed tryparedoxin peroxidase and a homologous Trypanosoma brucei sequence to form a distinct molecular clade. The consensus sequence: xn(n)Ax(5-6)Fx9Gx3Vx2Fx1Px2Fx1FVCPTEx21Sx1Dx7Wx16-19Dx15-166x3Rx2Fx2-Dx27Ax1Qx4-11Cx1-3Wx(n) was demonstrated by alignment of the sequences of tryparedoxin peroxidase and 8 other peroxiredoxins with established peroxidase function.