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Upon a stimulus of light, histamine is released from Drosophila photoreceptor axonal endings. It is taken up into glia where Ebony converts it into beta-alanyl-histamine (carcinine). Carcinine moves into photoreceptor cells and is there cleaved into beta-alanine and histamine by Tan activity. Tan thus provides a key function in the recycling pathway of the neurotransmitter histamine. It is also involved in the process of cuticle formation. There, it cleaves beta-alanyl-dopamine, a major component in cuticle sclerotization. Active Tan enzyme is generated by a self-processing proteolytic cleavage from a pre-protein at a conserved Gly-Cys sequence motif. We confirmed the dependence on the Gly-Cys motif by in vitro mutagenesis. Processing time delays the rise to full Tan activity up to 3 h behind its putative circadian RNA expression in head. To investigate its pleiotropic functions, we have expressed Tan as a His(6) fusion protein in Escherichia coli and have purified it to homogeneity. We found wild type and mutant His(6)-Tan protein co-migrating in size exclusion chromatography with a molecular weight compatible with homodimer formation. We conclude that dimer formation is preceding pre-protein processing. Drosophila tan(1) null mutant analysis revealed that amino acid Arg(217) is absolutely required for processing. Substitution of Met(256) in tan(5), on the contrary, does not affect processing extensively but renders it prone to degradation. This also leads to a strong tan phenotype although His(6)-Tan(5) retains activity. Kinetic parameters of Tan reveal characteristic differences in K(m) and k(cat) values of carcinine and beta-alanyl-dopamine cleavage, which conclusively illustrate the divergent tasks met by Tan.

Acetate:succinate CoA-transferases (ASCT) are acetate-producing enzymes in hydrogenosomes, anaerobically functioning mitochondria and in the aerobically functioning mitochondria of trypanosomatids. Although acetate is produced in the hydrogenosomes of a number of anaerobic microbial eukaryotes such as Trichomonas vaginalis, no acetate producing enzyme has ever been identified in these organelles. Acetate production is the last unidentified enzymatic reaction of hydrogenosomal carbohydrate metabolism. We identified a gene encoding an enzyme for acetate production in the genome of the hydrogenosome-containing protozoan parasite T. vaginalis. This gene shows high similarity to Saccharomyces cerevisiae acetyl-CoA hydrolase and Clostridium kluyveri succinyl-CoA:CoA-transferase. Here we demonstrate that this protein is expressed and is present in the hydrogenosomes where it functions as the T. vaginalis acetate:succinate CoA-transferase (TvASCT). Heterologous expression of TvASCT in CHO cells resulted in the expression of an active ASCT. Furthermore, homologous overexpression of the TvASCT gene in T. vaginalis resulted in an equivalent increase in ASCT activity. It was shown that the CoA transferase activity is succinate-dependent. These results demonstrate that this acetyl-CoA hydrolase/transferase homolog functions as the hydrogenosomal ASCT of T. vaginalis. This is the first hydrogenosomal acetate-producing enzyme to be identified. Interestingly, TvASCT does not share any similarity with the mitochondrial ASCT from Trypanosoma brucei, the only other eukaryotic succinate-dependent acetyl-CoA-transferase identified so far. The trichomonad enzyme clearly belongs to a distinct class of acetate:succinate CoA-transferases. Apparently, two completely different enzymes for succinate-dependent acetate production have evolved independently in ATP-generating organelles.