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.

Arylalkylamine N-acyltransferase-like 22 (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.

Ceramides are important participants of signal transduction, regulating fundamental cellular processes. Here we report the mechanism for activation of p53 tumor suppressor by C16-ceramide. C16-ceramide tightly binds within the p53 DNA-binding domain (Kd ~ 60 nM), in close vicinity to the Box V motif. This interaction is highly selective toward the ceramide acyl chain length with its C10 atom being proximal to Ser240 and Ser241. Ceramide binding stabilizes p53 and disrupts its complex with E3 ligase MDM2 leading to the p53 accumulation, nuclear translocation and activation of the downstream targets. This mechanism of p53 activation is fundamentally different from the canonical p53 regulation through protein–protein interactions or posttranslational modifications. The discovered mechanism is triggered by serum or folate deprivation implicating it in the cellular response to nutrient/metabolic stress. Our study establishes C16-ceramide as a natural small molecule activating p53 through the direct binding.

Long-chain fatty acid amides are cell-signaling lipids identified in mammals and, recently, in invertebrates, as well. Many details regarding fatty acid amide metabolism remain unclear. Herein, we demonstrate that Drosophila melanogaster is an excellent model system for the study long-chain fatty acid amide metabolism as we have quantified the endogenous levels of N-acylglycines, N-acyldopamines, N-acylethanolamines, and primary fatty acid amides by LC/QTOF-MS. Growth of Drosophila melanogaster on media supplemented with [1-13C]-palmitate lead to a family of 13C-palmitate-labeled fatty acid amides in the fly heads. The [1-13C]-palmitate feeding studies provide insight into the biosynthesis of the fatty acid amides.

ceramides and sphingolipids ( 1, 2 ). Primary fatty acid amides (PFAM), R-CO-NH 2 with R being a long-chain fatty acid, were fi rst identifi ed from a biological source in 1989 with the identifi cation of palmitamide, palmitoleamide, oleamide, elaidamide, and linoleamide in luteal phase plasma ( 3 ). At the time of their discovery, the biological function of these mammalian PFAMs was unknown. Interest in the PFAMs dramatically intensifi ed with the discoveries that oleamide accumulated in the cerebrospinal fl uid (CSF) of sleep-deprived cats, that it is a natural component of the CSF in the cat, rat, and human, and that the administration of synthetic oleamide induced physiological sleep in rats ( 4 ). Intriguingly, later studies found that oleamide levels in the brain of the ground squirrel were ‫ف‬ 2.5-fold higher in hibernating animals relative to those found in nonhibernating animals ( 5 ). Other functions ascribed to oleamide, since its discovery as a sleep-inducing PFAM, include the ability to modulate gap junction communication in glial cells ( 6, 7 ), tracheal epithelial cells ( 8 ), seminiferous tubule cells ( 9 ), and fi broblasts ( 10 ); to allosterically activate the GABA A receptors and specifi c subtypes of the serotonin receptor ( 11-13 ); to affect memory processes ( 14 ); to increase food intake ( 15 ); to reduce anxiety and pain ( 16,17 ); to depress body temperature and locomotor activity ( 17,18 ); to stimulate Ca(II) release ( 19 ); and to relax blood vessels ( 20,21 ).Although much of the research regarding the PFAMs has focused on oleamide, studies show that some of the other known PFAMs are bioactive. Palmitamide is weakly anticonvulsant ( 22 ); linoleamide regulates Ca(II) fl ux ( 23 ) and inhibits the erg current ( 24 ); and erucamide stimulates Abstract 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 signifi cantly 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 fi ve 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 fl ux throug...

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.

The long-chain fatty acid amides are an emerging family of bioactive lipids with members that include N-acyl amino acids, primary fatty acid amides (PFAMs), N-acylarylalkylamides, N-acylethanolamines, and N-monoacylpolyamines. Fatty acid amides were first identified from biological sources over 50 years ago with the isolation and identification of N-palmitoylethanolamine from egg yolk in 1957 (1) and, in 1965, N-palmitoylethanolamine and N-stearoylethanolamine in several tissues from rat and guinea pig (2). The discovery of N-arachidonoylethanolamine (anandamide) as the endogenous ligand for the mammalian brain cannabinoid receptor CB 1 sparked a dramatic interest in the fatty acid amides (3). Since these early discoveries, a diversity of long-chain fatty acid amides have been identified in mammals and, more recently, in invertebrates as well (4)(5)(6)(7)(8).The N-fatty acylglycines, a subclass of the N-fatty acyl amino acids, are an important class of cell signaling lipids that are distributed throughout the central nervous system and the rest of the body (6, 7, 9). Identification of glycine conjugates dates back to the 1820s with the identification of N-benzoylglycine (hippurate) as a mammalian metabolite (10). N-arachidonoylglycine was the first longchain N-acylglycine identified from a mammalian source and was determined to have anti-nociceptive and antiinflammatory effects in rat models of pain (11). Since

Our previous study suggested that ceramide synthase 6 (CerS6), an enzyme in sphingolipid biosynthesis, is regulated by p53: CerS6 was elevated in several cell lines in response to transient expression of p53 or in response to folate stress, which is known to activate p53. It was not clear, however, whether CerS6 gene is a direct transcriptional target of p53 or whether this was an indirect effect through additional regulatory factors. In the present study, we have shown that the CerS6 promoter is activated by p53 in luciferase assays, whereas transcriptionally inactive R175H p53 mutant failed to induce the luciferase expression from this promoter.