title: Mechanistic and structural analysis of Drosophila melanogaster arylalkylamine N-acetyltransferases.
abstract: 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-activity 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 pKa of 7.0. Using the data generated for the kinetic mechanism, structure-function relationships, pH-rate profiles, and site-directed mutagenesis, we propose a chemical mechanism for AANATA.
authors.affiliation: Department of Chemistry, University of South Florida , Tampa, Florida 33620, United States.
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Dempsey
authors.forename: Kristen A
authors.initials: KA
authors.lastname: Jeffries
authors.forename: Jason D
authors.initials: JD
authors.lastname: Bond
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Carpenter
authors.forename: Santiago
authors.initials: S
authors.lastname: Rodriguez-Ospina
authors.forename: Leonid
authors.initials: L
authors.lastname: Breydo
authors.forename: K Kenneth
authors.initials: KK
authors.lastname: Caswell
authors.forename: David J
authors.initials: DJ
authors.lastname: Merkler
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism; D000107:Acetylation / Q000187:drug effects; D019943:Amino Acid Substitution; D000818:Animals; D047068:Arylalkylamine N-Acetyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis / Q000187:drug effects; D020134:Catalytic Domain; D029721:Drosophila Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018698:Glutamic Acid / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D013379:Substrate Specificity; D014439:Tyramine / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D029721:Drosophila Proteins; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D008024:Ligands; D050505:Mutant Proteins; D018698:Glutamic Acid; D000105:Acetyl Coenzyme A; C054479:N-acetyltyramine; D047068:Arylalkylamine N-Acetyltransferase; D014439:Tyramine