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J. Biol. Chem., Vol. 278, Issue 30, 27612-27619, July 25, 2003

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Bis(glutathionyl)spermine and Other Novel Trypanothione Analogues in Trypanosoma cruzi^*

Mark R. Ariyanayagam, Sandra L. Oza, Angela Mehlert and Alan H. Fairlamb 

From the Division of Biological Chemistry and Molecular Microbiology, School of Life Sciences, Wellcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom

Trypanosomatids differ from other cells in their ability to conjugate glutathione with the polyamine spermidine to form the antioxidant metabolite trypanothione (N¹,N⁸-bis(glutathionyl)spermidine). In Trypanosoma cruzi, trypanothione is synthesized by an unusual trypanothione synthetase/amidase (TcTryS) that forms both glutathionylspermidine and trypanothione. Because T. cruzi is unable to synthesize putrescine and is dependent on uptake of exogenous polyamines by high affinity transporters, synthesis of trypanothione may be circumstantially limited by lack of spermidine. Here, we show that the parasite is able to circumvent the potential shortage of spermidine by conjugating glutathione with other physiological polyamine substrates from exogenous sources (spermine, N⁸-acetylspermidine, and N-acetylspermine). Novel thiols were purified from epimastigotes, and structures were determined by matrix-assisted laser desorption ionization time-of-flight analysis to be N¹,N¹²-bis(glutathionyl)spermine, N¹-glutathionyl-N⁸-acetylspermidine, and N¹-glutathionyl-N¹²-acetylspermine, respectively. Structures were confirmed by enzymatic synthesis with recombinant TcTryS, which catalyzes formation of these compounds with kinetic parameters equivalent to or better than those of spermidine. Despite containing similar amounts of spermine and spermidine, the epimastigotes, trypomastigotes, and amastigotes of T. cruzi preferentially synthesized trypanothione. Bis(glutathionyl)spermine disulfide is a physiological substrate of recombinant trypanothione reductase, comparable to trypanothione and homotrypanothione disulfides. The broad substrate specificity of TcTryS could be exploited in the design of polyamine-based inhibitors of trypanothione metabolism.

Received for publication, March 18, 2003 , and in revised form, May 14, 2003.

^* This work was supported by the Wellcome Trust. 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.

To whom correspondence should be addressed. Tel.: 44-1382-345155; Fax: 44-1382-345542; E-mail: a.h.fairlamb{at}dundee.ac.uk.

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