Mechanistic and Structural Analysis of a <i>Drosophila melanogaster</i> Enzyme, Arylalkylamine <i>N</i>‑Acetyltransferase Like 7, an Enzyme That Catalyzes the Formation of <i>N</i>‑Acetylarylalkylamides and <i>N</i>‑Acetylhistamine

Arylalkylamine <i>N</i>-acetyltransferase like 7 (AANATL7) catalyzes the formation of <i>N</i>-acetylarylalkylamides and <i>N</i>-acetylhistamine from acetyl-CoA and the corresponding amine substrate. AANATL7 is a member of the GNAT superfamily of >10000 GCN5-related <i>N</i>-acetyltransferases, many members being linked to important roles in both human metabolism and disease. <i>Drosophila melanogaster</i> utilizes the N-acetylation of biogenic amines for the inactivation of neurotransmitters, the biosynthesis of melatonin, and the sclerotization of the cuticle. We have expressed and purified <i>D. melanogaster</i> AANATL7 in <i>Escherichia coli</i> and used the purified enzyme to define the substrate specificity for acyl-CoA and amine substrates. Information about the substrate specificity provides insight into the potential contribution made by AANATL7 to fatty acid amide biosynthesis because <i>D. melanogaster</i> has emerged as an important model system contributing to our understanding of fatty acid amide metabolism. Characterization of the kinetic mechanism of AANATL7 identified an ordered sequential mechanism, with acetyl-CoA binding first followed by histamine to generate an AANATL7·acetyl-CoA·histamine ternary complex prior to catalysis. Successive pH–activity profiling and site-directed mutagenesis experiments identified two ionizable groups: one with a p<i>K</i>_a of 7.1 that is assigned to Glu-26 as a general base and a second p<i>K</i>_a of 9.5 that is assigned to the protonation of the thiolate of the coenzyme A product. Using the data generated herein, we propose a chemical mechanism for AANATL7 and define functions for other important amino acid residues involved in substrate binding and regulation of catalysis