title: Probing the chemical mechanism and critical regulatory amino acid residues of Drosophila melanogaster arylalkylamine N-acyltransferase like 2.
abstract: Arylalkylamine N-acyltransferase like 2 (AANATL2) catalyzes the formation of N-acylarylalkylamides from the corresponding acyl-CoA and arylalkylamine. The N-acylation of biogenic amines in Drosophila melanogaster is a critical step for the inactivation of neurotransmitters, cuticle sclerotization, and melatonin biosynthesis. In addition, D. melanogaster has been used as a model system to evaluate the biosynthesis of fatty acid amides: a family of potent cell signaling lipids. We have previously showed that AANATL2 catalyzes the formation of N-acylarylakylamides, including long-chain N-acylserotonins and N-acyldopamines. Herein, we define the kinetic mechanism for AANATL2 as an ordered sequential mechanism with acetyl-CoA binding first followed by tyramine to generate the ternary complex prior to catalysis. Bell shaped kcat,app - acetyl-CoA and (kcat/Km)app - acetyl-CoA pH-rate profiles identified two apparent pKa,app values of ∼7.4 and ∼8.9 that are critical to catalysis, suggesting the AANATL2-catalyzed formation of N-acetyltyramine occurs through an acid/base chemical mechanism. Site-directed mutagenesis of a conserved glutamate that corresponds to the catalytic base for other D. melanogaster AANATL enzymes did not produce a substantial depression in the kcat,app value nor did it abolish the pKa,app value attributed to the general base in catalysis (pKa ∼7.4). These data suggest that AANATL2 catalyzes the formation of N-acylarylalkylamides using either different catalytic residues or a different chemical mechanism relative to other D. melanogaster AANATL enzymes. In addition, we constructed other site-directed mutants of AANATL2 to help define the role of targeted amino acids in substrate binding and/or enzyme catalysis.
authors.affiliation: Department of Chemistry, University of South Florida, Tampa, FL, 33620, USA.
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Dempsey
authors.affiliation: Department of Chemistry, University of South Florida, Tampa, FL, 33620, USA.
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Carpenter
authors.affiliation: Department of Chemistry, University of South Florida, Tampa, FL, 33620, USA.
authors.forename: Santiago Rodriguez
authors.initials: SR
authors.lastname: Ospina
authors.affiliation: Department of Chemistry, University of South Florida, Tampa, FL, 33620, USA. Electronic address: merkler@usf.edu.
authors.forename: David J
authors.initials: DJ
authors.lastname: Merkler
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism; D000217:Acyltransferases / Q000378:metabolism*; D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D002384:Catalysis; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster / Q000201:enzymology*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D014439:Tyramine / Q000378:metabolism
chemical_list: D000596:Amino Acids; D029721:Drosophila Proteins; D000105:Acetyl Coenzyme A; D000217:Acyltransferases; C000613263:AANATL2 protein, Drosophila; D014439:Tyramine
keywords: AANATL2; Chemical mechanism; N-acyldopamines; N-acylserotonins; Structure-function relationships