期刊
BIOCHEMICAL JOURNAL
卷 446, 期 -, 页码 395-404出版社
PORTLAND PRESS LTD
DOI: 10.1042/BJ20120520
关键词
arylalkylamine N-acetyltransferase (AANAT); catalytic triad; dopamine N-acetyltransferase (Dat); melatonin; single-wavelength anomalous diffraction
资金
- National Science Council, Taiwan [NSC 101-2311-B-007-003, NSC 101-2319-B-400-001, NSC 100-2627-B-007-005]
- National Science Council of Taiwan, ROC.
- National Core Facility Program for Biotechnology
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.
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