Crystal structure of the dopamine N-acetyltransferase-acetyl-CoA complex provides insights into the catalytic mechanism.
The daily cycle of melatonin biosynthesis in mammals is regulated by AANAT (arylalkylamine N-acetyltransferase; EC 2.3.1.87), making it an attractive target for therapeutic control of abnormal melatonin production in mood and sleep disorders. Drosophila melanogaster Dat (dopamine N-acetyltransferase) is an AANAT. Until the present study, no insect Dat structure had been solved, and, consequently, the structural basis for its acetyl-transfer activity was not well understood. We report in the present paper the high-resolution crystal structure for a D. melanogaster Dat-AcCoA (acetyl-CoA) complex obtained using one-edge (selenium) single-wavelength anomalous diffraction. A binding study using 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. Site-directed mutagenesis, kinetic studies and pH-rate profiles confirmed that Glu(47), Ser(182) and Ser(186) were critical for catalysis. Collectively, the results of the present study suggest that Dat possesses a specialized active site structure dedicated to a catalytic mechanism.
Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 30013, Taiwan.
Kuo-Chang
KC
Cheng
Jhen-Ni
JN
Liao
Ping-Chiang
PC
Lyu
D000105:Acetyl Coenzyme A / Q000737:chemistry* / Q000378:metabolism; D000123:Acetyltransferases / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D029721:Drosophila Proteins / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D016415:Sequence Alignment
D029721:Drosophila Proteins; D000105:Acetyl Coenzyme A; D000123:Acetyltransferases; C493213:AANAT1 protein, Drosophila