Crystal Structure of Dopamine {Less than}i{Greater than}N{Less than}/i{Greater than}-Acetyltransferase/Acetyl Coenzyme A Complex Provide Insights into the Catalytic Mechanism

The daily cycle of melatonin biosynthesis in mammals is regulated by arylalkylamine N-acetyltransferase (EC 2.3.1.87, AANAT), making it an attractive target for therapeutic control of abnormal melatonin production in mood and sleep disorders. Drosophila melanogaster dopamine N-acetyltransferase (Dat) is an AANAT. Until this report, no insect Dat structure had been solved, and consequently, the structural basis for its acetyl-transfer activity was not well understood. We report herein the high-resolution crystal structure for a D. melanogaster Dat/acetyl coenzyme A (AcCoA) complex obtained using one-edge (Selenium) single-wavelength anomalous diffraction. The binding study by isothermal titration calorimetry suggested that the cofactor bound to Dat first before substrate. Examination of the complex structure and a substrate-docked model indicated that Dat contains a novel AANAT catalytic triad. A site-directed mutagenesis, a kinetic study and pH­­-rate profiles confirmed that Glu47, Ser182, and Ser186 were critical for catalysis. Collectively, our results suggest that Dat possesses a specialized active site structure dedicated to a catalytic mechanism.