Doctor of Philosophy (Ph.D.)
Degree Granting Department
David J. Merkler, Ph.D.
Nicole A. Horenstein, Ph.D.
H. Lee Woodcock, Ph.D.
Stanley M. Stevens, Ph.D.
Jianfeng Cai, Ph.D.
arylalkylamine N-acetyltransferase, Drosophila melanogaster, N-acyldopamines, N-acylserotonins
Fatty acid amides are an emerging family of bioactive lipids that consists of N-acylethanolamines, N-acylarylalkylamides, N-acylglycines, N-acyl amino acids, N-monoacylpolyamides, and primary fatty acid amides. Short chain fatty acid amides are products of inactivated biogenic amines such as dopamine, histamine, octopamine, and serotonin, whereas long chain fatty acid amides have been implicated in a number of physiological process such as the perception and inhibition of chronic pain through binding to their specific receptors. The most famous; therefore, the most studied long chain fatty acid amide is anandamide or also known as N-arachidonylethanolamine. The biosynthesis of anandamide is well defined; however, other long-chain fatty acid amides, such as the N-acyldopamines, N-acylserotonins, N-acylglycines, N-acyl amino acids, and primary fatty acid amides have remained elusive to date. Understanding the complete biosynthetic pathway for these cell signaling lipids, may yield new exciting molecular targets for human health and disease. Discovery of the long-chain fatty acid amide biosynthetic enzymes has proven to be challenging due to the low biologic abundance of the respective metabolites found in organisms, the interconnection of the pathways, and expense of using mammalian cells and/or organisms. This led to the transition of studying these metabolites and their respective biosynthetic enzymes in Drosophila melanogaster. D. melanogaster is an ideal system to study fatty acid amide biosynthesis because the respective metabolites have been identified, the cost of maintaining the organism is relatively low, and genetic manipulation (RNAi) is universally available.
This dissertation is dedicated to defining enzymes involved in D. melanogaster N-acylarylalkyamide biosynthesis. The biologically relevant long-chain N-acylarylalkylamides are comprised of long-chain N-acyldopamines and N-acylserotonins. Very little is known for how these potent cell signaling lipids are biosynthesized in the cell. One possible route is the N -acylation of the respective biogenic amine by an N-acyltransferase enzyme. An enzyme known to catalyze this chemistry is arylalkylamine N-acetyltransferase (AANAT), which catalyzes the formation of N-acetylarylalkylamides from acetyl CoA and the corresponding arylalkylamide. The N-acetylation of biogenic amines is a critical step in Drosophila melanogaster for the inactivation of amine neurotransmitters, sclerotization of the cuticle, and to serve as the penultimate intermediate in the biosynthesis of melatonin. Two AANAT(L) enzymes has been previously evaluated in D. melanogaster and six other putative AANATL enzymes have identified in the fly genome. One AANAT is expressed as two biologically relevant isoforms, AANAT variant A (AANATA) and AANAT variant B (AANATB), where AANATA differs from AANATB by the truncation of 35 amino acids on the N-terminus. The other AANATL enzyme to be previously studied is AANATL2, which was found to catalyze the formation of N-acetyltryptamine from acetyl CoA and tryptamine. Herein, we expressed six AANAT(L) enzymes (AANATA and AANATB, AANATL2, AANATL3, AANATL7, and AANATL8) and sought to define the acyl-CoA and amine substrates for each enzyme. To accomplish this, we developed an activity based screening assay to define acyl-CoA and amine substrates for AANATL2, AANATL3, AANATL7, and AANATL8. Following this work, we defined the acyl-CoA and amine substrate specificity for AANATA, AANATL2, AANATL3, and AANATL7. We have identified acetyl CoA and arylalkylamines as substrates for AANATA, AANATL2, and AANATL3; whereas AANATL7 acetylates histamine and arylalkylamines. AANATL2 was additionally shown to catalyze the formation of long-chain N-acyldopamines and N-acylserotonins. Following these important set of results, we solved the kinetic mechanism for AANATA, AANATL2, and AANATL7 in which these enzymes were shown to catalyze the formation of N-acylarylalkylamides by an ordered sequential mechanism where the acyl-CoA substrate binds first followed by the corresponding amine substrate. Finally, we evaluated the function of structural amino acids on regulating catalysis, structural features of substrates that effect binding and/or catalysis, and generated data leading to a proposed chemical mechanism by means of pH-activity profiles and site-directed mutagenesis of prospective catalytic residues.
Scholar Commons Citation
Dempsey, Daniel Robert, "Identification and Characterization of N-acyltransferase Enzymes that are Involved in the Biosynthesis of Fatty Acid Amides" (2015). Graduate Theses and Dissertations.