Biochemical characterization of a trypanosome enzyme with glutathione-dependent peroxidase activity.
In most eukaryotes, glutathione-dependent peroxidases play a key role in the metabolism of peroxides. Numerous studies have reported that trypanosomatids lack this activity. Here we show that this is not the case, at least for the American trypanosome Trypanosoma cruzi. We have isolated a single-copy gene from T. cruzi with the potential to encode an 18 kDa enzyme, the sequence of which has highest similarity with glutathione peroxidases from plants. A recombinant form of the protein was purified following expression in Escherichia coli. The enzyme was shown to have peroxidase activity in the presence of glutathione/glutathione reductase but not in the presence of trypanothione/trypanothione reductase. It could metabolize a wide range of hydroperoxides (linoleic acid hydroperoxide and phosphatidylcholine hydroperoxide>cumene hydroperoxide>t-butyl hydroperoxide), but no activity towards hydrogen peroxide was detected. Enzyme activity could be saturated by glutathione when both fatty acid and short-chain organic hydroperoxides were used as substrate. For linoleic acid hydroperoxide, the rate-limiting step of this reaction is the reduction of the peroxidase by glutathione. With lower-affinity substrates such as t-butyl hydroperoxide, the rate-limiting step is the reduction of the oxidant. The data presented here identify a new arm of the T. cruzi oxidative defence system.
Wilkinson
S R
SR
Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London, WC1E 7HT, UK. shane.wilkinson@lshtm.ac.uk
Meyer
D J
DJ
Kelly
J M
JM
D000595:Amino Acid Sequence; D000818:Animals; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism; D005979:Glutathione Peroxidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D008041:Linoleic Acids / Q000378:metabolism; D008054:Lipid Peroxides / Q000378:metabolism*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013095:Spermidine / Q000031:analogs & derivatives* / Q000378:metabolism; D013379:Substrate Specificity; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000235:genetics; D020122:tert-Butylhydroperoxide / Q000378:metabolism
D008041:Linoleic Acids; D008054:Lipid Peroxides; D011993:Recombinant Fusion Proteins; C008285:linoleic acid hydroperoxide; D020122:tert-Butylhydroperoxide; C044809:trypanothione; D006861:Hydrogen Peroxide; D005979:Glutathione Peroxidase; D005978:Glutathione; D013095:Spermidine