Fatty Acid Amide Biosynthesis: A Possible New Role for Peptidylglycine α-Amidating Enzyme and Acyl-Coenzyme A:GlycineN-Acyltransferase

doi:10.1006/abbi.1996.0272

Archives of Biochemistry and Biophysics

Volume 330, Issue 2, 15 June 1996, Pages 430-434

https://doi.org/10.1006/abbi.1996.0272 Get rights and content

Abstract

Fatty acid primary amides have recently been recognized as mammalian hormones [Cravattet al.(1995)Science268, 1506–1509]. The route to their biosynthesis is unknown. Many mammalian peptide hormones also possess a C-terminal α-amide moiety that arises from the posttranslational oxidative cleavage of a C-terminal glycine-extended precursor. The enzyme that catalyzes this reaction is peptidylglycine α-amidating enzyme, which is known to preferentially amidate peptide substrates containing a penultimate, hydrophobic amino acid [Tamburiniet al.(1990)Int. J. Pept. Protein Res.35, 153–156]. We show thatN-myristoylglycine is a substrate for peptidylglycine α-amidating enzyme with a (V/K)_appthat is 55 ± 4% of the value measured for D-Tyr-Val-Gly.N-Fatty acylglycines are enzymatically produced in mammals from fatty acyl-coenzyme A (CoAs) and glycine by acyl-CoA:glycineN-acyltransferase. The sequential actions of acyl-CoA:glycineN-acyltransferase and peptidylglycine α-amidating enzyme would lead to the biosynthesis of fatty acid amides.

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Cited by (63)

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A critical and as-yet unmet need in Alzheimer's disease (AD) is the discovery of peripheral small molecule biomarkers. Given that brain pathology precedes clinical symptom onset, we set out to test whether metabolites in blood associated with pathology as indexed by cerebrospinal fluid (CSF) AD biomarkers.
This study analyzed 593 plasma samples selected from the European Medical Information Framework for Alzheimer's Disease Multimodal Biomarker Discovery study, of individuals who were cognitively healthy (n = 242), had mild cognitive impairment (n = 236), or had AD-type dementia (n = 115). Logistic regressions were carried out between plasma metabolites (n = 883) and CSF markers, magnetic resonance imaging, cognition, and clinical diagnosis.
Eight metabolites were associated with amyloid β and one with t-tau in CSF, these were primary fatty acid amides (PFAMs), lipokines, and amino acids. From these, PFAMs, glutamate, and aspartate also associated with hippocampal volume and memory.
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Bm-iAANAT and its potential role in fatty acid amide biosynthesis in Bombyx mori
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One focus of our research has been on the identification and characterization of enzymes involved in fatty acid amide biosynthesis and melding in vitro substrate specificity data with metabolomic data [23,24]. We have proposed N-acyltransferases operate in fatty acid amide biosynthesis: acyl-CoA + amine → N-acylamide + CoA-SH (Fig. 1) [25]. Such N-acyltransferases are likely members of the GCN5-related superfamily of N-acetyltransferases (GNATs) [26,27], which would accept long-chain acyl-CoA thioesters as substrates.
The purpose of this research is to unravel the substrate specificity and kinetic properties of an insect arylalkylamine N-acyltransferase from Bombyx mori (Bm-iAANAT) and to determine if this enzyme will catalyze the formation of long chain N-acylarylalkylamides in vitro. However, the determination of substrates and products for Bm-iAANAT in vitro is no guarantee that these same molecules are substrates and products for the enzyme in the organism. Therefore, RT-PCR was performed to detect the Bm-iAANAT transcripts and liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QToF-MS) analysis was performed on purified lipid extracts from B. mori larvae (fourth instar, Bmi4) to determine if long chain fatty acid amides are produced in B. mori. Ultimately, we found that recombinant Bm-iAANAT will utilize long-chain acyl-CoA thioesters as substrates and identified Bm-iAANAT transcripts and long-chain fatty acid amides in Bmi4. Together, these data show Bm-iAANAT will catalyze the formation of long-chain N-acylarylalkylamides in vitro and provide evidence demonstrating that Bm-iAANAT has a role in fatty acid amide biosynthesis in B. mori, as well.
Glycine N-acyltransferase-like 3 is responsible for long-chain N-acylglycine formation in N18TG2 cells
2016, Journal of Lipid Research
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 (PFAMs) 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 PFAMs. 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 PFAMs. This is the first report of an enzyme responsible for long-chain N-acylglycine production in cellula. The production of the PFAMs in N₁₈TG₂ 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 were grown in the presence of [¹³C₁₈]oleic acid. The addition of [1-¹³C]palmitate to the N₁₈TG₂ cell growth media led to the production of a family of [1-¹³C]palmitoylated fatty acid amides, consistent with the biosynthetic pathways detailed herein.
Probing the chemical mechanism and critical regulatory amino acid residues of Drosophila melanogaster arylalkylamine N-acyltransferase like 2
2015, Insect Biochemistry and Molecular Biology
Citation Excerpt :
One important step in better understanding the various AANATs is a careful and thorough characterization of these enzymes individually, particularly in consideration of their potential involvement in fatty acid amide biosynthesis. We have long thought that an AANAT or AANAT-like (AANATL) enzyme would be involved in biosynthesis of long-chain N-fatty acid amides (Merkler et al., 1996), a diverse family of cell signaling lipids (Farrell and Merkler, 2008; Fezza et al., 2014). The identification of glycine N-acyltransferase-like enzymes that catalyze the formation of N-fatty acylglycines (Waluk et al., 2010) and our discovery that AANATL2 will catalyze the formation of N-fatty acylserotonins and N-fatty acyldopamines support our hypothesis.
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 k_{cat,app – acetyl-CoA} and (k_cat/K_m)_{app – acetyl-CoA} pH-rate profiles identified two apparent pK_a,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 k_cat,app value nor did it abolish the pK_a,app value attributed to the general base in catalysis (pK_a ∼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.
Mammalian Fatty Acid Amides of the Brain and CNS
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Fatty acid amides have emerged as an intriguing family of diverse, mammalian neuroactive lipids. Herein, we review the current state of our knowledge about the individual classes of fatty acid amides: which ones have been identified and characterized from mammals, their receptors, their functions, and the pathways for their biosynthesis and degradation. Much remains to be elucidated regarding this family of molecules and we hope that our review stimulates additional research in the fatty acid amide field. We also show that there are extensive metabolic connections between the different classess of fatty acid amides. Such metabolic connections hint at novel modes of regulation as one fatty acid amide is converted to another and suggests that the enzymes involved in fatty acid amide metabolism are not simply enzymes of biosynthesis or degradation, but also serve important regulatory functions.
C-terminal modification of monoclonal antibody drugs: Amidated species as a general product-related substance
2013, International Journal of Biological Macromolecules
Citation Excerpt :
Neuroendocrine hormones and neurotransmitters such as gonadotropin-releasing hormone, adrenomedullin, neuropeptide Y, and cholecystokinin are known as PAM substrates, and all of them possess Gly at their C-termini [14,16–18]. PAM is also known to amidate fatty acid primary amides [19] and bile acid Gly conjugates [20]. However, large proteins such as immunoglobulins, which also often possess Gly at the C-termini of their heavy chains, have not been shown to be substrates for PAM.
Twelve therapeutic mAbs, comprising 10 IgG1s and 2 IgG4s, were analyzed by a peptide mapping technique to detect and quantify C-terminal modifications. C-terminal amidated structures were found in 8 out of the 12 mAbs. An in vitro study using a commercially available peptidylglycine alpha-amidating monooxygenase (PAM) revealed that both IgG1 and IgG4 can be substrates for PAM. This study showed that C-terminal amidation is a general C-terminal modification on the heavy chains of therapeutic mAbs and that C-terminal amidation of mAbs can be catalyzed by a certain PAM(s) in the Chinese hamster ovary (CHO) cells that are widely used for manufacturing therapeutic mAbs.

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Research ReportFatty Acid Amide Biosynthesis: A Possible New Role for Peptidylglycine α-Amidating Enzyme and Acyl-Coenzyme A:GlycineN-Acyltransferase

Abstract

Research Report
Fatty Acid Amide Biosynthesis: A Possible New Role for Peptidylglycine α-Amidating Enzyme and Acyl-Coenzyme A:GlycineN-Acyltransferase