Peroxiredoxin-linked detoxification of hydroperoxides in Toxoplasma gondii.
The apicomplexan parasite Toxoplasma gondii is highly susceptible to oxidative stress caused by tert-butyl-hydroperoxide, juglone, and phenazine methylsulfate with IC(50) in the nanomolar range. Using dichlorofluorescein diacetate, a detector of endogenous oxidative stress, it was shown that juglone and phenazine methylsulfate are potentially toxic to the parasites without affecting the host cells. These results demonstrate that T. gondii is vulnerable to oxidative challenge that results from disruption of its redox balance and so this could be an effective approach to therapeutic intervention. This study has characterized redox active and antioxidant peroxidases belonging to the class of 1-Cys and 2-Cys peroxiredoxins that play crucial roles in maintaining redox balance. The tachyzoite stages of T. gondii express thioredoxin (TgTrx), 1-Cys peroxiredoxin (TgTrx-Px2), and a 2-Cys peroxiredoxin (TgTrx-Px1) and immunofluorescent studies revealed that all three proteins are located in the cytosol of the parasite confirming previous studies on TgTrx-Px1 (Kwok, L.Y., Schluter, D., Clayton, C., and Soldati, D. (2004) Mol. Microbiol. 51, 47-61). TgTrx-Px1 showed K(m) values for H(2)O(2) and tert-butyl hydroperoxide in the nanomolar range, emphasizing the great affinity of the protein for theses substrates. Moreover, the catalytic efficiency of TgTrx-Px1 for these substrates at 10(6)-10(7) M(-1) s(-1) is unusually high, which qualifies the enzyme as an extremely potent antioxidant. Kinetic analyses revealed that TgTrx-Px1 is inhibited by tert-butyl hydroperoxide, and apparent inhibition constants were determined to be between 33 and 35.6 microm depending on the concentration of the non-inhibitory substrate thioredoxin. TgTrx-Px2 protected glutamine synthetase from inactivation by Fe(3+)/DTT, showing that it is an active peroxiredoxin.
Akerman
Susan E
SE
Division of Biological Chemistry and Molecular Microbiology, School of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.
Müller
Sylke
S
D000818:Animals; D006861:Hydrogen Peroxide / Q000378:metabolism*; D008658:Inactivation, Metabolic; D007700:Kinetics; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010544:Peroxidases / Q000378:metabolism*; D054464:Peroxiredoxins; D013879:Thioredoxins / Q000378:metabolism; D014122:Toxoplasma / Q000378:metabolism*
D013879:Thioredoxins; D006861:Hydrogen Peroxide; D010544:Peroxidases; D054464:Peroxiredoxins