The Role of S-Adenosylmethionine Decarboxylase on Regulation of Polyamine and Trypanothione Metabolism in Trypanosoma Brucei

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2008-05-13

Authors

Willert, Erin Kathleen

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Abstract

Trypanosoma brucei is the causative agent of Human African Trypanosomiasis (HAT), a fatal and neglected disease affecting Sub-Saharan Africa. Current therapeutics are limited for several reasons, underscoring the need for new and improved drugs. The polyamine/trypanothione pathway is essential for T. brucei, and the biosynthetic enzymes in this pathway are potential drug targets. We have characterized T. brucei S-adenosylmethionine decarboxylase (AdoMetDC), a key enzyme required for the synthesis of spermidine and trypanothione, and examined the role of AdoMetDC on the regulation of polyamine and trypanothione metabolism. The recombinant T. brucei AdoMetDC enzyme displays low catalytic efficiency as compared to the human enzyme (1000 fold lower). Also, the specific activity in trypanosome cell lysates is about 400 fold higher than that of the recombinant enzyme. We have discovered that the product of a second gene, which we have named prozyme, is required for full activity. Prozyme arose through gene duplication and mutational drift, and has no intrinsic decarboxylase activity. AdoMetDC and prozyme form a tight heterodimer, and have a catalytic efficiency that is 1,200 fold higher than AdoMetDC alone. The heterodimeric organization may be a means for polyamine regulation in T. brucei, and the differences between host and parasite enzymes suggest that AdoMetDC is an intriguing drug target. In order to better understand the role of AdoMetDC, we created a stable T. brucei cell line that can be induced to knockdown AdoMetDC expression by RNAi. AdoMetDC knockdown cells are auxotrophic for spermidine. In these cells, putrescine, the precursor of spermidine, is increased five fold, and spermidine levels drop to about 50% of uninduced cells. Levels of glutathionyl-spermidine and trypanothione are almost completely abolished, indicating that the trypanosomes are maintaining spermidine levels at the expense of trypanothione. Protein levels of prozyme, ornithine decarboxylase and trypanothione synthetase are increased during AdoMetDC knockdown. Therefore AdoMetDC has a central role in the biosynthesis and metabolism of polyamines and trypanothione.

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