Mechanistic and Structural Analysis of Drosophila melanogaster Arylalkylamine N-Acetyltransferases

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• 作者：Daniel R. Dempsey ; Kristen A. Jeffries ; Jason D. Bond ; Anne-Marie Carpenter ; Santiago Rodriguez-Ospina ; Leonid Breydo ; K. Kenneth Caswell ; David J. Merkler
• 刊名：Biochemistry
• 出版年：2014
• 出版时间：December 16, 2014
• 年：2014
• 卷：53
• 期：49
• 页码：7777-7793
• 全文大小：748K
• ISSN：1520-4995

文摘

Arylalkylamine N-acetyltransferase (AANAT) catalyzes the penultimate step in the biosynthesis of melatonin and other N-acetylarylalkylamides from the corresponding arylalkylamine and acetyl-CoA. The N-acetylation of arylalkylamines is a critical step in Drosophila melanogaster for the inactivation of the bioactive amines and the sclerotization of the cuticle. Two AANAT variants (AANATA and AANATB) have been identified in D. melanogaster, in which AANATA differs from AANATB by the truncation of 35 amino acids from the N-terminus. We have expressed and purified both D. melanogaster AANAT variants (AANATA and AANATB) in Escherichia coli and used the purified enzymes to demonstrate that this N-terminal truncation does not affect the activity of the enzyme. Subsequent characterization of the kinetic and chemical mechanism of AANATA identified an ordered sequential mechanism, with acetyl-CoA binding first, followed by tyramine. We used a combination of pH鈥揳ctivity profiling and site-directed mutagenesis to study prospective residues believed to function in AANATA catalysis. These data led to an assignment of Glu-47 as the general base in catalysis with an apparent pK_a of 7.0. Using the data generated for the kinetic mechanism, structure鈥揻unction relationships, pH鈥搑ate profiles, and site-directed mutagenesis, we propose a chemical mechanism for AANATA.