Crystal Structure of Dopamine N-Acetyltransferase ternary Complex provides insights into the catalyt

• 作 者: Kuo-Chang Cheng Shin-Ju Lin Jhen-Ni Liao Ping-Chiang Lvu
• 机 构: InstituteofBioinformaticsandStructuralBiology,NationalTsingHuaUniversity,Hsinchu,30013,Taiwan
• 出 处: 中国化学会第三届全国生物物理化学会议暨国际华人生物物理化学发展论坛
• 发表日期: 2014年7期

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 ternary complex for D.melanogaster Dat/tryptamine/acetyl coenzyme A (AcCoA) 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 indicated that Dat contained a novel AANAT catalytic triad.Site-directed mutagenesis and kinetic study confirmed that Glu47,Ser182,and Ser186 were critical for catalysis.These results suggest that Dat possesses a specialized active site structure dedicated to a catalytic mechanism.According to the ternary complex structure,we also proposed that three aromatic residues (F43,Y64,and F114) in a hydrophobic substrate-binding pocket of Dat may play key roles in substrate specificity.This study provides the structural insights into the enzyme activity and the substrate binding selectivity in Drosophila AANAT.