Structural and Mechanistic Analysis of Drosophila melanogaster Agmatine N-Acetyltransferase, an Enzyme that Catalyzes the Formation of N-Acetylagmatine (original) (raw)

Abstract

Agmatine N-acetyltransferase (AgmNAT) catalyzes the formation of N-acetylagmatine from acetyl-CoA and agmatine. Herein, we provide evidence that Drosophila melanogaster AgmNAT (CG15766) catalyzes the formation of N-acetylagmatine using an ordered sequential mechanism; acetyl-CoA binds prior to agmatine to generate an AgmNAT•acetyl-CoA•agmatine ternary complex prior to catalysis. Additionally, we solved a crystal structure for the apo form of AgmNAT with an atomic resolution of 2.3 Å, which points towards specific amino acids that may function in catalysis or active site formation. Using the crystal structure, primary sequence alignment, pH-activity profiles, and site-directed mutagenesis, we evaluated a series of active site amino acids in order to assign their functional roles in AgmNAT. More specifically, pH-activity profiles identified at least one catalytically important, ionizable group with an apparent pK a of ~7.5, which corresponds to the general base in catalysis, Glu-34. Moreover, these data led to a proposed chemical mechanism, which is consistent with the structure and our biochemical analysis of AgmNAT. The discovery and characterization of enzymes involved in fatty acid amide biosynthesis has been a longstanding focus of our research 1. One possible biosynthetic route for the fatty acid amides would be the reaction between an amine and a fatty acyl-CoA: R 1-NH 2 + R 2-COS -CoA → R 2-CO-NH-R 1 + CoA-SH. Enzymes of the GCN5-related N-acetyltransferase family (GNAT) catalyze a similar reaction using acetyl-CoA as a substrate to generate N-acetylamides 2. Acetyl-CoA-dependent N-acetylation by N-acetyltransferases is known for a diversity of amines 3-5 in a broad range of organisms 2,5-8. We have long suspected that enzymes identified as N-acetyltransferases might accept longer-chain fatty acyl-CoA thioesters as substrates or that novel N-acetyltransferase-like enzymes exist that utilize fatty acyl-CoA thioesters as substrates. Drosophila melanogaster is an excellent model organism to study fatty acid amide biosynthesis. These insects are known to produce fatty acid amides 9,10 , its genome has been sequenced 11 , these organisms can be manipulated genetically 12 , and are inexpensive to maintain. In addition, two N-acetyltransferases had been identified from D. melanogaster, arylalkylamine N-acetyltransferase variant A (AANATA, also called dopamine N-acetyltransferase) 13 and arylalkylamine N-acetyltransferase-like 2 (AANATL2) 14. Both enzymes catalyze the N-acetylation of arylalkylamines, but their respective substrate specificities, kinetic mechanisms, and chemical

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