Sequence analysis of the tryparedoxin peroxidase gene from Crithidia fasciculata and its functional expression in Escherichia coli.
Tryparedoxin peroxidase from Crithidia fasciculata is an essential component of the trypanothione-dependent hydroperoxide metabolism in the trypanosomatids (Nogoceke, E., Gommel, D. U., Kiebeta, M., Kalisz, H. M., and Flohé, 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: xnAx5-6Fx9Gx3Vx2Fx1Px2Fx1FVCPTEx21Sx1Dx7Wx16-19Dx15- 16Gx3Rx2Fx2Dx27Ax 1Qx4-11Cx1-3Wxn was demonstrated by alignment of the sequences of tryparedoxin peroxidase and 8 other peroxiredoxins with established peroxidase function.[1]References
- Sequence analysis of the tryparedoxin peroxidase gene from Crithidia fasciculata and its functional expression in Escherichia coli. Montemartini, M., Nogoceke, E., Singh, M., Steinert, P., Flohé, L., Kalisz, H.M. J. Biol. Chem. (1998) [Pubmed]
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