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

被引:59
作者
Montemartini, M
Nogoceke, E
Singh, M
Steinert, P
Flohé, L
Kalisz, HM
机构
[1] Gesell Biotechnol Forsch GmbH, D-38124 Braunschweig, Germany
[2] Tech Univ Braunschweig, Dept Physiol Chem, D-38124 Braunschweig, Germany
关键词
D O I
10.1074/jbc.273.9.4864
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Tryparedoxin peroxidase from Crithidia fasciculata is an essential component of the trypanothione-dependent hydroperoxide metabolism in the trypanosomatids (Nogoceke, E., Gommel, D, U,, Kie beta, 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-l, The enzyme unequivocally belongs to the peroxiredoxin family of proteins, whose members have been found in all phyla, A phylo genetic tree comprising 47 protein and DNA sequences showed tryparedoxin peroxidase and a homologous Trypanosoma blucei sequence to form a distinct molecular clade, The consensus sequence: x(n)Ax(5-6)Fx(9)Gx(3)Vx(2)Fx(1)Px(2)Fx(1)FVCPTEx(21)Sx(1)Dx(7)Wx(16-19)Dx(15-16)Gx(3)Rx(2)Fx(2)Dx(27)Ax(1)Qx(4-11)Cx(1-3)Wx(n) was demonstrated by alignment of the sequences of tryparedoxin peroxidase and 8 other peroxiredoxins with established peroxidase function.
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页码:4864 / 4871
页数:8
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