Assignment of endogenous substrates to enzymes by global metabolite profiling

Enzymes regulate biological processes through the conversion of specific substrates to products. Therefore, of fundamental interest for every enzyme is the elucidation of its natural substrates. Here, we describe a general strategy for identifying endogenous substrates of enzymes by untargeted liquid chromatography-mass spectrometry (LC-MS) analysis of tissue metabolomes from wild-type and enzyme-inactivated organisms. We use this method to discover several brain lipids regulated by the mammalian enzyme fatty acid amide hydrolase (FAAH) in vivo, including known signaling molecules (e.g., the endogenous cannabinoid anandamide) and a novel family of nervous system-enriched natural products, the taurine-conjugated fatty acids. Remarkably, the relative hydrolytic activity that FAAH exhibited for lipid metabolites in vitro was not predictive of the identity of specific FAAH substrates in vivo. Thus, global metabolite profiling establishes unanticipated connections between the proteome and metabolome that enable assignment of an enzyme's unique biochemical functions in vivo.     

The enzymatic inactivation of the fatty acid amide class of signaling lipids

The fatty acid amide (FAA) class of signaling lipids modulates a number of neurobehavioral processes in mammals, including pain, sleep, feeding, and locomotor activity. Representative FAAs include the endogenous cannabinoid anandamide and the sleep-inducing lipid oleamide. Despite activating several neuroreceptor systems in vitro, most FAAs produce only weak and transient behavioral effects in vivo, presumably due to their expeditious catabolism. This review focuses on one enzyme, fatty acid amide hydrolase (FAAH) that appears to play a major role in regulating the amplitude and duration of FAA signals in vivo. In particular, we will highlight a series of recent papers that have investigated the physiological functions of the mouse and human FAAH enzymes. Collectively, these studies promote FAAH as a central component of FAA signaling pathways, especially those mediated by the endocannabinoid anandamide, and suggest that this enzyme may represent an attractive pharmaceutical target for the treatment of pain and related neurophysiological disorders.     

High-throughput screening for the discovery of inhibitors of fatty acid amide hydrolase using a microsome-based fluorescent assay

Fatty acid amide hydrolase (FAAH) is a membrane-associated enzyme that catalyzes the hydrolysis of several endogenous bioactive lipids, including anandamide (AEA), N-palmitoylethanolamine (PEA), oleamide, and N-oleoylethanolamine (OEA). These fatty acid amides participate in many physiological activities such as analgesia, anxiety, sleep modulation, anti inflammatory responses, and appetite suppression. Because FAAH plays an essential role in controlling the tone and activity of these endogenous bioactive lipids, this enzyme has been implicated to be a drug target for the therapeutic management of pain, anxiety, and other disorders. In an effort to discover FAAH inhibitors, the authors have previously reported the development of a novel fluorescent assay using purified FAAH microsomes as an enzyme source and a fluorogenic substrate, arachidonyl 7-amino, 4-methyl coumarin amide (AAMCA). Herein, the authors have adapted this assay to a high-throughput format and have screened a large library of small organic compounds, identifying a number of novel FAAH inhibitors. These data further verify that this fluorescent assay is sufficiently robust, efficient, and low-cost for the identification of FAAH inhibitory molecules and open this class of enzymes for therapeutic exploration.     

Characterization and manipulation of the acyl chain selectivity of fatty acid amide hydrolase

Fatty acid amide hydrolase (FAAH) is a mammalian integral membrane enzyme that catabolizes several neuromodulatory fatty acid amides, including the endogenous cannabinoid anandamide and the sleep-inducing lipid oleamide. FAAH belongs to a large group of hydrolytic enzymes termed the amidase signature (AS) family that is defined by a conserved, linear AS sequence of approximately 130 amino acids. Members of the AS family display strikingly different substrate selectivities, yet the primary structural regions responsible for defining substrate recognition in these enzymes remain unknown. In this study, a series of unbranched p-nitroanilide (pNA) substrates ranging from 6 to 20 carbons in length was used to probe the acyl chain binding specificity of FAAH, revealing that this enzyme exhibits a strong preference for acyl chains 9 carbons in length or longer. A fluorophosphonate inhibitor of FAAH containing a photoactivatable benzophenone group was synthesized and used to locate a region of the enzyme implicated in substrate binding. Protease digestion and mass spectrometry analysis of FAAH-inhibitor conjugates identified the major site of cross-linking as residues 487-493. Site-directed mutagenesis revealed that a single residue in this region, I491, strongly influenced substrate specificity of FAAH. For example, an I491A mutant displayed a greatly reduced binding affinity for medium-chain pNA substrates (7-12 carbons) but maintained nearly wild-type binding and catalytic constants for longer chain substrates (14-20 carbons). Mutation of I491 to aromatic or more polar residues generated enzymes with relative hydrolytic efficiencies for medium- versus long-chain pNAs that varied up to 90-fold. Collectively, these studies indicate that I491 participates in hydrophobic binding interactions with medium-chain FAAH substrates. Additionally, the significant changes in substrate selectivity achieved by single amino acid changes suggest that FAAH possesses a rather malleable substrate binding domain and may serve, along with other AS enzymes, as a template for the engineering of amidases with novel and/or tailored specificities.     

A FAAH-regulated class of N-acyl taurines that activates TRP ion channels

Fatty acid amide hydrolase (FAAH) is an integral membrane enzyme that catabolizes several bioactive lipids in vivo. Most of the physiological substrates of FAAH characterized to date belong to the N-acyl ethanolamine (NAE) class of fatty acid amides, including the endocannabinoid anandamide, the anti-inflammatory lipid N-palmitoyl ethanolamine, and the satiating factor N-oleoyl ethanolamine. We recently identified a second structural class of fatty acid amides regulated by FAAH in vivo: the N-acyl taurines (NATs). Global metabolite profiling revealed high concentrations of long chain (> or = C20) saturated NATs in the central nervous system (CNS) of FAAH(-/-) mice. Here, we use metabolite profiling to characterize the FAAH-NAT system in peripheral mouse tissues. Livers and kidneys of FAAH(-/-) mice possessed dramatic elevations in NATs, which, in contrast to those detected in the CNS, were enriched in polyunsaturated acyl chains (e.g., C20:4, C22:6). Peripheral NATs rose more than 10-fold within 1 h following pharmacological inactivation of FAAH and reached levels up to approximately 5000 pmol/g tissue (C22:6 in kidney), implicating a constitutive and highly active pathway for NAT metabolism in which FAAH plays an integral part. Interestingly, NATs were found to activate multiple members of the transient receptor potential (TRP) family of calcium channels, including TRPV1 and TRPV4, which are both expressed in kidney. The dramatic elevation in endogenous levels of NATs following acute or chronic inactivation of FAAH, in conjunction with the pharmacological effects of these lipids on TRP channels, suggests the existence of a second major lipid signaling system regulated by FAAH in vivo.     

The fatty acid amide hydrolase (FAAH)

The topic of this review is fatty acid amide hydrolase (FAAH), one of the best-characterized enzymes involved in the hydrolysis of bioactive lipids such as anandamide, 2-arachidonoylglycerol (2-AG), and oleamide. Herein, we discuss the nomenclature, the various assays that have been developed, the relative activity of the various substrates and the reversibility of the enzyme reactions catalyzed by FAAH. We also describe the cloning of the enzyme from rat and subsequent cDNA isolation from mouse, human, and pig. The proteins and the mRNAs from different species are compared. Cloning the enzyme permitted the purification and characterization of recombinant FAAH. The conserved regions of FAAH are described in terms of sequence and function, including the amidase domain which contains the serine catalytic nucleophile, the hydrophobic domain important for self association, and the proline rich domain region, which may be important for subcellular localization. The distribution of FAAH in the major organs of the body is described as well as regional distribution in the brain and its correlation with cannabinoid receptors. Since FAAH is recognized as a drug target, a large number of inhibitors have been synthesized and tested since 1994 and these are reviewed in terms of reversibility, potency, and specificity for FAAH and cannabinoid receptors.     

A second fatty acid amide hydrolase with variable distribution among placental mammals

Fatty acid amides constitute a large and diverse class of lipid transmitters that includes the endogenous cannabinoid anandamide and the sleep-inducing substance oleamide. The magnitude and duration of fatty acid amide signaling are controlled by enzymatic hydrolysis in vivo. Fatty acid amide hydrolase (FAAH) activity in mammals has been primarily attributed to a single integral membrane enzyme of the amidase signature (AS) family. Here, we report the functional proteomic discovery of a second membrane-associated AS enzyme in humans that displays FAAH activity. The gene that encodes this second FAAH enzyme was found in multiple primate genomes, marsupials, and more distantly related vertebrates, but, remarkably, not in a number of lower placental mammals, including mouse and rat. The two human FAAH enzymes, which share 20% sequence identity and are referred to hereafter as FAAH-1 and FAAH-2, hydrolyzed primary fatty acid amide substrates (e.g. oleamide) at equivalent rates, whereas FAAH-1 exhibited much greater activity with N-acyl ethanolamines (e.g. anandamide) and N-acyl taurines. Both enzymes were sensitive to the principal classes of FAAH inhibitors synthesized to date, including O-aryl carbamates and alpha-keto heterocycles. These data coupled with the overlapping, but distinct tissue distributions of FAAH-1 and FAAH-2 suggest that these proteins may collaborate to control fatty acid amide catabolism in primates. The apparent loss of the FAAH-2 gene in some lower mammals should be taken into consideration when extrapolating genetic or pharmacological findings on the fatty acid amide signaling system across species.     

Structure-based design of a FAAH variant that discriminates between the N-acyl ethanolamine and taurine families of signaling lipids

Fatty acid amide hydrolase (FAAH) inactivates a large and diverse class of endogenous signaling lipids termed fatty acid amides. Representative fatty acid amides include the N-acyl ethanolamines (NAEs) anandamide, which serves as an endogenous ligand for cannabinoid receptors, and N-oleoyl and N-palmitoyl ethanolamine, which produce satiety and anti-inflammatory effects, respectively. Global metabolite profiling studies of FAAH (-/-) mice have recently identified a second class of endogenous FAAH substrates: the N-acyl taurines (NATs). To determine the metabolic and signaling functions performed by NAEs and NATs in vivo, a FAAH variant that discriminates between these two substrate classes would be of value. Here, we report the structure-guided design of a point mutant in the active site of FAAH that selectively disrupts interactions with NATs. This glycine-to-aspartate (G268D) mutant was found to exhibit wild-type kinetic parameters with NAEs, but more than a 100-fold reduction in activity with NATs attributable to combined effects on Km and kcat values. These in vitro properties were also observed in living cells, where WT-FAAH and the G268D mutant displayed equivalent hydrolytic activity with NAEs, but the latter enzyme was severely impaired in its ability to catabolize NATs. The G268D FAAH mutant may thus serve as a valuable research tool to illuminate the unique roles played by the NAE and NAT classes of signaling lipids in vivo.     

Keys to Lipid Selection in Fatty Acid Amide Hydrolase Catalysis: Structural Flexibility,Gating Residues and Multiple Binding Pockets

The fatty acid amide hydrolase (FAAH) regulates the endocannabinoid system cleaving primarily the lipid messenger anandamide. FAAH has been well characterized over the years and, importantly, it represents a promising drug target to treat several diseases, including inflammatory-related diseases and cancer. But its enzymatic mechanism for lipid selection to specifically hydrolyze anandamide, rather than similar bioactive lipids, remains elusive. Here, we clarify this mechanism in FAAH, examining the role of the dynamic paddle, which is formed by the gating residues Phe432 and Trp531 at the boundary between two cavities that form the FAAH catalytic site (the “membrane-access” and the “acyl chain-binding” pockets). We integrate microsecond-long MD simulations of wild type and double mutant model systems (Phe432Ala and Trp531Ala) of FAAH, embedded in a realistic membrane/water environment, with mutagenesis and kinetic experiments. We comparatively analyze three fatty acid substrates with different hydrolysis rates (anandamide > oleamide > palmitoylethanolamide). Our findings identify FAAH’s mechanism to selectively accommodate anandamide into a multi-pocket binding site, and to properly orient the substrate in pre-reactive conformations for efficient hydrolysis that is interceded by the dynamic paddle. Our findings therefore endorse a structural framework for a lipid selection mechanism mediated by structural flexibility and gating residues between multiple binding cavities, as found in FAAH. Based on the available structural data, this exquisite catalytic strategy for substrate specificity seems to be shared by other lipid-degrading enzymes with similar enzymatic architecture. The mechanistic insights for lipid selection might assist de-novo enzyme design or drug discovery efforts.     

Hydrolysis of prostaglandin glycerol esters by the endocannabinoid-hydrolyzing enzymes, monoacylglycerol lipase and fatty acid amide hydrolase

Cyclooxygenase-2 (COX-2) can oxygenate the endocannabinoids, arachidonyl ethanolamide (AEA) and 2-arachidonylglycerol (2-AG), to prostaglandin-H2-ethanolamide (PGH2-EA) and -glycerol ester (PGH2-G), respectively. Further metabolism of PGH2-EA and PGH2-G by prostaglandin synthases produces a variety of prostaglandin-EA's and prostaglandin-G's nearly as diverse as those derived from arachidonic acid. Thus, COX-2 may regulate endocannabinoid levels in neurons during retrograde signaling or produce novel endocannabinoid metabolites for receptor activation. Endocannabinoid-metabolizing enzymes are important regulators of their action, so we tested whether PG-G levels may be regulated by monoacylglycerol lipase (MGL) and fatty acid amide hydrolase (FAAH). We found that PG-Gs are poor substrates for purified MGL and FAAH compared to 2-AG and/or AEA. Determination of substrate specificity demonstrates a 30-100- and 150-200-fold preference of MGL and FAAH for 2-AG over PG-Gs, respectively. The substrate specificity of AEA compared to those of PG-Gs was approximately 200-300 fold higher for FAAH. Thus, PG-Gs are poor substrates for the major endocannabinoid-degrading enzymes, MGL and FAAH.     

Lipoxygenase-mediated oxidation of polyunsaturated N-acylethanolamines in Arabidopsis

N-acylethanolamines (NAEs) are bioactive fatty acid derivatives that occur in all eukaryotes. In plants, NAEs have potent negative growth-regulating properties, and fatty acid amide hydrolase (FAAH)-mediated hydrolysis is a primary catabolic pathway that operates during seedling establishment to deplete these compounds. Alternatively, polyunsaturated (PU)-NAEs may serve as substrates for lipid oxidation. In Arabidopsis, PU-NAEs (NAE 18:2 and NAE 18:3) were the most abundant NAE species in seeds, and their levels were depleted during seedling growth even in FAAH tDNA knock-out plants. Therefore, we hypothesized that lipoxygenase (LOX) participated in the metabolism of PU-NAEs through the formation of NAE-oxylipins. Comprehensive chromatographic and mass spectrometric methods were developed to identify NAE hydroperoxides and -hydroxides. Recombinant Arabidopsis LOX enzymes expressed in Escherichia coli utilized NAE 18:2 and NAE 18:3 as substrates with AtLOX1 and AtLOX5 exhibiting 9-LOX activity and AtLOX2, AtLOX3, AtLOX4, and AtLOX6 showing predominantly 13-LOX activity. Feeding experiments with exogenous PU-NAEs showed they were converted to hydroxide metabolites indicating that indeed Arabidopsis seedlings had the capacity for LOX-mediated metabolism of PU-NAEs in planta. Detectable levels of endogenous NAE-oxylipin metabolites were identified in FAAH fatty acid amide hydrolase seedlings but not in wild-type or FAAH overexpressors, suggesting that NAE hydroxide pools normally do not accumulate unless flux through hydrolysis is substantially reduced. These data suggest that Arabidopsis LOXs indeed compete with FAAH to metabolize PU-NAEs during seedling establishment. Identification of endogenous amide-conjugated oxylipins suggests potential significance of these metabolites in vivo, and FAAH mutants may offer opportunities to address this in the future.     

Expression and secretion of N-acylethanolamine-hydrolysing acid amidase in human prostate cancer cells

N-acylethanolamines (NAEs) are a class of bioactive lipid molecules in animal tissues, including the endocannabinoid anandamide and the anti-inflammatory substance N-palmitoylethanolamine. Enzymatic hydrolysis of NAEs is considered to be an important step to regulate their endogenous levels. Lysosomal NAE-hydrolysing acid amidase (NAAA) as well as fatty acid amide hydrolase (FAAH) is responsible for this reaction. Here, we report relatively high expression of NAAA in human prostate cancer cells (PC-3, DU-145 and LNCaP) and prostate epithelial cells (PrEC), with the highest mRNA level in LNCaP cells. FAAH and the NAE-forming enzyme N-acylphosphatidylethanolamine-hydrolysing phospholipase D (NAPE-PLD) were also detected in these cells. NAAA activity in LNCaP cells could be distinguished from coexisting FAAH activity, based on their different pH dependency profiles and specific inhibition of FAAH activity by URB597. These results showed that both the enzymes were functionally active. We also found that NAAA was partly secreted from LNCaP cells, which underlined possible usefulness of this enzyme as a biomarker of prostate cancer.     

Chemical and mutagenic investigations of fatty acid amide hydrolase: evidence for a family of serine hydrolases with distinct catalytic properties.

Fatty acid amide hydrolase (FAAH) is a membrane-bound enzyme responsible for the catabolism of neuromodulatory fatty acid amides, including anandamide and oleamide. FAAH's primary structure identifies this enzyme as a member of a diverse group of alkyl amidases, known collectively as the "amidase signature family". At present, this enzyme family's catalytic mechanism remains poorly understood. In this study, we investigated the catalytic features of FAAH through mutagenesis, affinity labeling, and steady-state kinetic methods. In particular, we focused on the respective roles of three serine residues that are conserved in all amidase signature enzymes (S217, S218, and S241 in FAAH). Mutation of each of these serines to alanine resulted in a FAAH enzyme bearing significant catalytic defects, with the S217A and S218A mutants showing 2300- and 95-fold reductions in k(cat), respectively, and the S241A mutant exhibiting no detectable catalytic activity. The double S217A:S218A FAAH mutant displayed a 230 000-fold decrease in k(cat), supporting independent catalytic functions for these serine residues. Affinity labeling of FAAH with a specific nucleophile reactive inhibitor, ethoxy oleoyl fluorophosphonate, identified S241 as the enzyme's catalytic nucleophile. The pH dependence of FAAH's k(cat) and k(cat)/K(m) implicated a base involved in catalysis with a pK(a) of 7.9. Interestingly, mutation of each of FAAH's conserved histidines (H184, H358, and H449) generated active enzymes, indicating that FAAH does not contain a Ser-His-Asp catalytic triad commonly found in other mammalian serine hydrolytic enzymes. The unusual properties of FAAH identified here suggest that this enzyme, and possibly the amidase signature family as a whole, may hydrolyze amides by a novel catalytic mechanism.     

Endocannabinoid metabolism in the absence of fatty acid amide hydrolase (FAAH): discovery of phosphorylcholine derivatives of N-acyl ethanolamines

Lipid transmitters are tightly regulated by a balance of biosynthetic and degradative enzymes. Termination of the activity of the N-acyl ethanolamine (NAE) class of lipid-signaling molecules, including the endocannabinoid anandamide (AEA), is principally mediated by the integral membrane enzyme fatty acid amide hydrolase (FAAH) in vivo. FAAH(-/-) mice are highly sensitized to the pharmacological effects of AEA; however, these animals eventually recover from AEA treatment, implying the existence of alternative routes for NAE metabolism. Here, we have pursued the characterization of these pathways by profiling the metabolome of FAAH(-/-) mice treated with AEA. Multiple AEA-induced metabolites were observed in brains from FAAH(-/-) mice, including a major product with a mass shift of +165 Da (m/z 513). The structure of this product was determined to be O-phosphorylcholine (PC)-AEA. Analysis of untreated mice identified PC-NAEs as endogenous constituents of the central nervous system (CNS) that were highly elevated in FAAH(-/-) animals. PC-NAEs were very poor substrates for FAAH; however, a vanadate-sensitive enzymatic activity was detected in brain membranes that converted PC-NAEs back to their parent NAEs. The choline-specific phosphodiesterase NPP6 was identified as a candidate enzyme responsible for this activity. These data indicate the presence of a complete metabolic pathway for the production and degradation of PC-NAEs in the CNS that constitutes an alternative route for endocannabinoid metabolism.     

Fatty acid amide hydrolase substrate specificity

Fatty acid amide hydrolase (FAAH), also referred to as oleamide hydrolase and anandamide amidohydrolase, is a serine hydrolase responsible for the degradation of endogenous oleamide and anandamide, fatty acid amides that function as chemical messengers. FAAH hydrolyzes a range of fatty acid amides, and the present study examines the relative rates of hydrolysis of a variety of natural and unnatural fatty acid primary amide substrates using pure recombinant rat FAAH.     

Development of highly sensitive fluorescent assays for fatty acid amide hydrolase

Fatty acid amide hydrolase (FAAH) is a pharmaceutical target whose inhibition may lead to valuable therapeutics. Sensitive substrates for high-throughput assays are crucial for the rapid-screening FAAH inhibitors. Here we describe the development of novel and highly sensitive fluorescent assays for FAAH based on substituted aminopyridines. Examining the relationship between the structure and the fluorescence of substituted aminopyridines suggested that a methoxy group in the para position relative to the amino group in aminopyridines greatly increased the fluorescence (i.e., quantum yields approach unity). These novel fluorescent reporters had a high Stokes' shift of 94 nm, and their fluorescence in buffer systems increased with pH values from neutral to basic. Fluorescent substrates with these reporters displayed a very low fluorescent background and high aqueous solubility. Most importantly, fluorescent assays for FAAH based on these substrates were at least 25 times more sensitive than assays using related compounds with published colorimetric or fluorescent reporters. This property results in shorter assay times and decreased protein concentrations in the assays. Such sensitive assays will facilitate distinguishing the relative potency of powerful inhibitors of FAAH. When these fluorescent substrates were applied to human liver microsomes, results suggested that there was at least one amide hydrolase in addition to FAAH that could hydrolyze long-chain fatty acid amides. These results show that these fluorescent substrates are very valuable tools in FAAH activity assays including screening inhibitors by high-throughput assays instead of using the costly and labor-intensive radioactive ligands. Potential applications of novel fluorescent reporters are discussed.     

Evidence for distinct roles in catalysis for residues of the serine-serine-lysine catalytic triad of fatty acid amide hydrolase

Fatty acid amide hydrolase (FAAH) is a mammalian amidase signature enzyme that inactivates neuromodulatory fatty acid amides, including the endogenous cannabinoid anandamide and the sleep-inducing substance oleamide. The recent determination of the three-dimensional structures of FAAH and two distantly related bacterial amidase signature enzymes indicates that these enzymes employ an unusual serine-serine-lysine triad for catalysis (Ser-241/Ser-217/Lys-142 in FAAH). Mutagenesis of each of the triad residues in FAAH has been shown to severely reduce amidase activity; however, how these residues contribute, both individually and in cooperation, to catalysis remains unclear. Here, through a combination of site-directed mutagenesis, enzyme kinetics, and chemical labeling experiments, we provide evidence that each FAAH triad residue plays a distinct role in catalysis. In particular, the mutation of Lys-142 to alanine indicates that this residue functions as both a base involved in the activation of the Ser-241 nucleophile and an acid that participates in the protonation of the substrate leaving group. This latter property appears to support the unusual ability of FAAH to hydrolyze amides and esters at equivalent rates. Interestingly, although structural evidence indicates that the impact of Lys-142 on catalysis probably occurs through the bridging Ser-217, the mutation of this latter residue to alanine impaired catalytic activity but left the amide/ester hydrolysis ratios of FAAH intact. Collectively, these findings suggest that FAAH possesses a specialized active site structure dedicated to a mechanism for competitive amide and ester hydrolysis where nucleophile attack and leaving group protonation occur in a coordinated manner dependent on Lys-142.     

Thiadiazolopiperazinyl ureas as inhibitors of fatty acid amide hydrolase

A series of thiadiazolopiperazinyl aryl urea fatty acid amide hydrolase (FAAH) inhibitors is described. The molecules were found to inhibit the enzyme by acting as mechanism-based substrates, forming a covalent bond with Ser241. SAR and PK properties are presented.     

Lipidomic analysis of N-acylphosphatidylethanolamine molecular species in Arabidopsis suggests feedback regulation by N-acylethanolamines

N-Acylphosphatidylethanolamine (NAPE) and its hydrolysis product, N-acylethanolamine (NAE), are minor but ubiquitous lipids in multicellular eukaryotes. Various physiological processes are severely affected by altering the expression of fatty acid amide hydrolase (FAAH), an NAE-hydrolyzing enzyme. To determine the effect of altered FAAH activity on NAPE molecular species composition, NAE metabolism, and general membrane lipid metabolism, quantitative profiles of NAPEs, NAEs, galactolipids, and major and minor phospholipids for FAAH mutants of Arabidopsis were determined. The NAPE molecular species content was dramatically affected by reduced FAAH activity and elevated NAE content in faah knockouts, increasing by as much as 36-fold, far more than the NAE content, suggesting negative feedback regulation of phospholipase D-mediated NAPE hydrolysis by NAE. The N-acyl composition of NAPE remained similar to that of NAE, suggesting that the NAPE precursor pool largely determines NAE composition. Exogenous NAE 12:0 treatment elevated endogenous polyunsaturated NAE and NAPE levels in seedlings; NAE levels were increased more in faah knockouts than in wild-type or FAAH overexpressors. Treated seedlings with elevated NAE and NAPE levels showed impaired growth and reduced galactolipid synthesis by the "prokaryotic" (i.e., plastidic), but not the "eukaryotic" (i.e., extraplastidic), pathway. Overall, our data provide new insights into the regulation of NAPE-NAE metabolism and coordination of membrane lipid metabolism and seedling development.