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Biochem J. 2012 Sep 15;446(3):395-404. doi: 10.1042/BJ20120520.

Crystal structure of the dopamine N-acetyltransferase-acetyl-CoA complex provides insights into the catalytic mechanism.

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1
Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 30013, Taiwan.

Abstract

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.

PMID:
22716280
PMCID:
PMC3429931
DOI:
10.1042/BJ20120520
[Indexed for MEDLINE]
Free PMC Article
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