title: Mechanistic and Structural Analysis of a Drosophila melanogaster Enzyme, Arylalkylamine N-Acetyltransferase Like 7, an Enzyme That Catalyzes the Formation of N-Acetylarylalkylamides and N-Acetylhistamine.
abstract: Arylalkylamine N-acetyltransferase like 7 (AANATL7) catalyzes the formation of N-acetylarylalkylamides and N-acetylhistamine from acetyl-CoA and the corresponding amine substrate. AANATL7 is a member of the GNAT superfamily of >10000 GCN5-related N-acetyltransferases, many members being linked to important roles in both human metabolism and disease. Drosophila melanogaster 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 D. melanogaster AANATL7 in Escherichia coli 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 D. melanogaster 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 pKa of 7.1 that is assigned to Glu-26 as a general base and a second pKa 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.
authors: †Department of Chemistry, University of South Florida, Tampa, Florida 33620, United States.
authors: Daniel R
authors: DR
authors: Dempsey
authors: †Department of Chemistry, University of South Florida, Tampa, Florida 33620, United States.
authors: Kristen A
authors: KA
authors: Jeffries
authors: †Department of Chemistry, University of South Florida, Tampa, Florida 33620, United States.
authors: Sumit
authors: S
authors: Handa
authors: †Department of Chemistry, University of South Florida, Tampa, Florida 33620, United States.
authors: Anne-Marie
authors: AM
authors: Carpenter
authors: †Department of Chemistry, University of South Florida, Tampa, Florida 33620, United States.
authors: Santiago
authors: S
authors: Rodriguez-Ospina
authors: Leonid
authors: L
authors: Breydo
authors: †Department of Chemistry, University of South Florida, Tampa, Florida 33620, United States.
authors: David J
authors: DJ
authors: Merkler
mesh_terms: D000214:Acyl Coenzyme A / Q000737:chemistry / Q000378:metabolism; D000577:Amides / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D047068:Arylalkylamine N-Acetyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster; D005227:Fatty Acids / Q000737:chemistry / Q000378:metabolism; D006632:Histamine / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011994:Recombinant Proteins
chemical_list: D000214:Acyl Coenzyme A; D000577:Amides; D029721:Drosophila Proteins; D005227:Fatty Acids; D011994:Recombinant Proteins; C021606:N-acetylhistamine; D006632:Histamine; D047068:Arylalkylamine N-Acetyltransferase