Structural Basis for the Allosteric Activation of Trypanosoma Brucei S-adenosylmethionine Decarboxylase by a Catalytically Dead Homolog

Human African Trypanosomiasis (HAT) is caused by single-celled parasites, Trypanosoma brucei, which are transmitted to humans by infected tsetse flies. Trypanosomiasis has a profound impact on the health of a large number of people in sub-Saharan Africa and it is fatal when untreated. Unfortunately, current drug therapy is limited mostly because of toxic effects on the patients. The polyamine biosynthetic pathway is a validated target for the development of drugs. Enzymes involved in polyamine biosynthesis exhibit features that differ significantly between the parasites and the human host. Therefore, exploitation of such differences can lead to the design of new inhibitors that can selectively kill the parasites. My work is focused on S-adenosylmethionine decarboxylase (AdoMetDC), which in the trypanosomatids is regulated by a unique mechanism, heterodimer formation with a catalytically dead homolog. This protein, designated prozyme, forms a high-affinity heterodimer with AdoMetDC and increases its activity by >1000-fold. The heterodimer is confirmed to be the functional enzyme in vivo. Therefore, understanding the mechanisms that regulate T. brucei AdoMetDC activation by prozyme can provide essential information for more effective inhibitory strategies. The role of specific residues involved in the process was studied by deletion and site-directed mutagenesis. Results indicate that 12 key amino acids at the N-terminal portion of the enzyme, which are fully conserved in the trypanosomatids but absent from other eukaryotic homologs, play a crucial role since there is more than 50 percent less activation by prozyme when they are either removed or mutated to alanine. AdoMetDC L8 and L10 seem to be the strongest determinants for stimulation by prozyme in this region. Analytical ultracentrigugation analyses in the sedimentation velocity mode indicated that dimerization is not impaired when these essential residues are removed, since binding affinities between wildtype and mutant heterodimers remain similar (Kd= <0.5 and 1μM, respectively). Thus, these results imply that key residues in the area must be acting through an allosteric regulatory mechanism. I have also characterized the activity of the L. major AdoMetDC/prozyme complex, the catalytic efficency from which increases by 170-fold upon binding of the homolog. Swapped complexes containing AdoMetDC and prozyme from different trypanosomatids (T. brucei, T. cruzi and L.major) are functional, supporting the idea that amino acid residues essential for the activation mechanism are conserved in all species.