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J. Biol. Chem., Vol. 280, Issue 1, 564-570, January 7, 2005

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Peroxiredoxin-linked Detoxification of Hydroperoxides in Toxoplasma gondii^*

Susan E. Akerman¶ and Sylke Müller¶||

From the Division of Biological Chemistry and Molecular Microbiology, School of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom and Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom

The apicomplexan parasite Toxoplasma gondii is highly susceptible to oxidative stress caused by tert-butyl-hydroperoxide, juglone, and phenazine methylsulfate with IC₅₀ 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., Schlüter, D., Clayton, C., and Soldati, D. (2004) Mol. Microbiol. 51, 47-61). TgTrx-Px1 showed K_m values for H₂O₂ 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 10⁶-10⁷ 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 µM depending on the concentration of the non-inhibitory substrate thioredoxin. TgTrx-Px2 protected glutamine synthetase from inactivation by Fe³⁺/DTT, showing that it is an active peroxiredoxin.

Received for publication, June 8, 2004 , and in revised form, September 30, 2004.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Funded by a Wellcome Trust Prize Studentship.

^|| A Wellcome Trust Senior Fellow in Basic Biomedical Science. To whom correspondence should be addressed: Infection and Immunity, Institute of Biomedical and Life Sciences, Joseph Black Building, University of Glasgow, Glasgow G12 8QQ, UK. Tel.: 44-141-330-2383; Fax: 44-141-330-4600; E-mail: s.muller{at}bio.gla.ac.uk.

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