Serotonin synthesis by two distinct enzymes in Drosophila melanogaster.

Annotation of the sequenced Drosophila genome suggested the presence of an additional enzyme with extensive homology to mammalian tryptophan hydroxylase, which we have termed DTRH. In this work, we show that enzymatic analyses of the putative DTRH enzyme expressed in Escherichia coli confirm that it acts as a tryptophan hydroxylase but can also hydroxylate phenylalanine, in vitro. Building upon the knowledge gained from the work in mice and zebrafish, it is possible to hypothesize that DTRH may be primarily neuronal in function and expression, and DTPH, which has been previously shown to have phenylalanine hydroxylation as its primary role, may be the peripheral tryptophan hydroxylase in Drosophila. The experiments presented in this report also show that DTRH is similar to DTPH in that it exhibits differential hydroxylase activity based on substrate. When DTRH uses tryptophan as a substrate, substrate inhibition, catecholamine inhibition, and decreased tryptophan hydroxylase activity in the presence of serotonin synthesis inhibitors are observed. When DTRH uses phenylalanine as a substrate, end product inhibition, increased phenylalanine hydroxylase activity after phosphorylation by cAMP-dependent protein kinase, and a decrease in phenylalanine hydroxylase activity in the presence of the serotonin synthesis inhibitor, alpha-methyl-(DL)-tryptophan are observed. These experiments suggest that the presence of distinct tryptophan hydroxylase enzymes may be evolutionarily conserved and serve as an ancient mechanism to appropriately regulate the production of serotonin in its target tissues.

Substrate regulation of serotonin and dopamine synthesis in Drosophila.

In Drosophila melanogaster, serotonin (5-hydroxytryptamine, 5-HT) is required for both very early non-neuronal developmental events, and in the CNS as a neurotransmitter to modulate behavior. 5-HT is synthesized, at least in part, by the actions of Drosophila tryptophan-phenylalanine hydroxylase (DTPH), a dual function enzyme that hydroxylates both phenylalanine and tryptophan. DTPH is expressed in numerous tissues as well as dopaminergic and serotonergic neurons, but it does not necessarily function as both enzymes in these tissues. Deficiencies in DTPH could affect the production of dopamine and serotonin, and thus dopaminergic and serotonergic signaling pathways. In this paper, we show that DTPH exhibits differential hydroxylase activity based solely on substrate. When DTPH uses phenylalanine as a substrate, regulatory control (end product inhibition, decreased PAH activity following phosphorylation, catecholamine inhibition) is observed that is not seen when the enzyme uses tryptophan as a substrate. These studies suggest that regulation of DTPH enzymatic activity occurs, at least in part, through the actions of its substrate.