His-tagged tryparedoxin peroxidase of Trypanosoma cruzi as a tool for drug screening.
Tryparedoxin peroxidase has recently been identified as a constituent of the complex peroxidase system in the trypanosomatid Crithidia fasciculata [Nogoceke E, Gommel DU, Kiess M, Kalisz HM, Flohe L (1997) Biol Chem 378: 827-836]. In trypanosomatids, hydroperoxides are reduced at the expense of NADPH by means of a cascade of three oxidoreductases: the flavoprotein trypanothione reductase, tryparedoxin and tryparedoxin peroxidase. Inhibitors of these enzymes are presumed to be trypanocidal drugs. Here, we present the heterologous expression of a putative tryparedoxin peroxidase gene of Trypanosoma cruzi (accession no AJ012101) as an N-terminally His-tagged protein (TcH6TXNPx). The product was purified with a high yield (8.75 mg from 11 fermentation broth of A(600)2.1) from the cytosolic fraction of sonified Escherichia coli BL21(DE3)[pET22b( + )/TcH6TXNPx] by metal-chelating chromatography. TcH6TXNPx proved to be fully active when tested with heterologous tryparedoxins of C. fasciculata (His-tagged TXN1H6 and TXN2H6). TcH6TXNPx displayed ping-pong kinetics with a k(cat) of 1.7 s(-1) and limiting Km values of 51.8 microM and 1.7 microM for t-butyl hydroperoxide and CfTXN2H6, respectively.
Guerrero
S A
SA
Department of Biochemistry, Technical University of Braunschweig, Germany.
Lopez
J A
JA
Steinert
P
P
Montemartini
M
M
Kalisz
H M
HM
Colli
W
W
Singh
M
M
Alves
M J
MJ
Flohé
L
L
D000595:Amino Acid Sequence; D000818:Animals; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006639:Histidine / Q000737:chemistry; D008969:Molecular Sequence Data; D010544:Peroxidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015800:Protozoan Proteins; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014349:Trypanosoma cruzi / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D020122:tert-Butylhydroperoxide / Q000378:metabolism*
D004791:Enzyme Inhibitors; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D006639:Histidine; D020122:tert-Butylhydroperoxide; D010544:Peroxidases; C110959:tryparedoxin peroxidase