Long-chain fatty acid amides are signaling lipids found in mammals and other organisms; however, details of the metabolic pathways for the N-acylglycines and primary fatty acid amides have remained elusive. Heavy-labeled precursor and subtraction lipidomic experiments in mouse neuroblastoma N₁₈TG₂ cells, a model cell line for the study of fatty acid amide metabolism, establish the biosynthetic pathways for the N-acylglycines and the primary fatty acid amides. We provide evidence that the N-acylglycines are formed by a long-chain specific glycine-conjugating enzyme, glycine N-acyltransferase like 3 (GLYATL3). siRNA knockdown of GLYATL3 in the N₁₈TG₂ cells resulted in a decrease in the levels of the N-acylglycines and the primary fatty acid amides. This is the first report of an enzyme responsible for long-chain N-acylglycine production in cellula. The production of the primary fatty acid amides in N₁₈ TG₂ cells was reported to occur by the oxidative cleavage of the N-acylglycines, as catalyzed by peptidylglycine α-amidating monooxygenase (PAM). siRNA knockdown of PAM resulted in an accumulation of [¹³C₁₈]-N-oleoylglycine and decreased levels of [¹³C₁₈]-oleamide when the N₁₈TG₂ cells are grown in the presence of [¹³C₁₈]-oleic acid. The addition of [1-¹³C]-palmitate to the N₁₈TG₂ cell growth media leads to the production of a family of [1-¹³C]-palmitoylated fatty acid amides, consistent with the biosynthetic pathways detailed herein.

Primary fatty acid amides (PFAM) are important signaling molecules in the mammalian nervous system, binding to many drug receptors and demonstrating control over sleep, locomotion, angiogenesis, and many other processes. Oleamide is the best-studied of the primary fatty acid amides, whereas the other known PFAMs are significantly less studied. Herein, quantitative assays were used to examine the endogenous amounts of a panel of PFAMs, as well as the amounts produced after incubation of mouse neuroblastoma N(18)TG(2) and sheep choroid plexus (SCP) cells with the corresponding fatty acids or N-tridecanoylethanolamine. Although five endogenous primary amides were discovered in the N(18)TG(2) and SCP cells, a different pattern of relative amounts were found between the two cell lines. Higher amounts of primary amides were found in SCP cells, and the conversion of N-tridecanoylethanolamine to tridecanamide was observed in the two cell lines. The data reported here show that the N(18)TG(2) and SCP cells are excellent model systems for the study of PFAM metabolism. Furthermore, the data support a role for the N-acylethanolamines as precursors for the PFAMs and provide valuable new kinetic results useful in modeling the metabolic flux through the pathways for PFAM biosynthesis and degradation.

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 pK_a 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.

Arylalkylamine N‐acyltransferase‐like 2 ² (AANATL2) from Drosophila melanogaster was expressed and shown to catalyze the formation of long‐chain N‐acylserotonins and N‐acydopamines. Subsequent identification of endogenous amounts of N‐acylserotonins and colocalization of these fatty acid amides and AANATL2 transcripts gives supporting evidence that AANATL2 has a role in the biosynthetic formation of these important cell signalling lipids.