Alternative Tasks of Drosophila Tan in Neurotransmitter Recycling Versus Cuticle Sclerotization Disclosed by Kinetic Properties

Upon a stimulus of light, histamine is released from Drosophila photoreceptor axonal endings. It is taken up into glia where Ebony converts it into β-alanyl-histamine (carcinine). Carcinine moves into photoreceptor cells and is there cleaved into β-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 β-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 His6 fusion protein in Escherichia coli and have purified it to homogeneity. We found wild type and mutant His6-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 tan1 null mutant analysis revealed that amino acid Arg217 is absolutely required for processing. Substitution of Met256 in tan5, on the contrary, does not affect processing extensively but renders it prone to degradation. This also leads to a strong tan phenotype although His6-Tan5 retains activity. Kinetic parameters of Tan reveal characteristic differences in Km and kcat values of carcinine and β-alanyl-dopamine cleavage, which conclusively illustrate the divergent tasks met by Tan.