Abstract
Abstract Drosophila melanogaster dopamine N-acetyltransferase (Dat, EC 2.3.1.87) is an arylalkylamine N-acetyltransferase (AANAT) which catalyzes the synthesis of the hormonal precursor (melatonin). The structures of Dat both in the apo form and in complexed with acetyl coenzyme A and the catalytic mechanism have been solved in our laboratory. In this study, we report the crystal structure of Dat ternary complex with coenzyme A and serotonin at 1.20 Å resolution. According to our structure, we proposed that three aromatic residues (F43, Y64, and F114) in a hydrophobic substrate-binding pocket of Dat may play key roles in substrate specificity. These three residues F43, Y64 and F114 were individually replaced with tryptophan to estimate the effect of an indole ring in the substrate selectivity. Enzyme kinetic studies demonstrated that mutant F43W apparently decreased the substrate binding and the activity of wild type, indicating this residue is critical for enzyme activity. On the contrary, F114W demonstrated no relationship with substrate selectivity or substrate binding. According to the previous study, wild type has substrate specificity with phenylalkylamine. However, the kinetic results combined with ITC data showed that the Y64W had a broad spectrum substrate selectivity slightly enhanced the enzyme activity toward indoalkylamine. The result indicated that residue Y64 may modulate the pi-pi interaction between Dat and substrates. Our study contributes a ternary complex structural that may help to understand enzyme activity and the substrate binding selectivity in Drosophila AANAT.