Putative N-Acylphosphatidylethanolamine Synthase from Arabidopsis thaliana Is a Lysoglycerophospholipid Acyltransferase

Abstract

AT1G78690, a gene found in Arabidopsis thaliana, has been reported to encode a N-acyltransferase that transfers an acyl chain from acyl-CoA to the headgroup of phosphatidylethanolamine (PE) to form N-acylphosphatidylethanolamine (N-acyl-PE). Our investigation suggests that At1g78690p is not a PE-dependent N-acyltransferase but is instead a lysoglycerophospholipid O-acyltransferase. We overexpressed AT1G78690 in Escherichia coli, extracted the cellular lipids, and identified the accumulating glycerophospholipid as acylphos- phatidylglycerol (acyl-PG). Electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-MS) analysis yielded [M ؊ H]؊ ions, corresponding by exact mass to acyl-PG rather than N-acyl-PE. Collision-induced dissociation mass spectrometry (MS/MS) yielded product ions consistent with acyl-PG. In addition, in vitro enzyme assays using both 32 P-and 14 C-radiolabeled substrates showed that AT1G78690 acylates 1-acyllysophosphatidylethanolamine (1-acyllyso-PE) and 1-acyllysophosphatidylglycerol (1-acyllyso-PG), but not PE or phosphatidylglycerol (PG), to form a diacylated product that co-migrates with PE and PG, respectively. We analyzed the diacylated product formed by AT1G78690 using a combination of base hydrolysis, phospholipase D treatment, ESI-MS, and MS/MS to show that AT1G78690 acylates the sn-2-position of 1-acyllyso-PE and 1-acyllyso-PG. N-Acylphosphatidylethanolamine (N-acyl-PE)2 is an anionic minor membrane glycerophospholipid (GPL) that is abundant in animals, higher plants, and certain microorganisms, such as yeast and Escherichia coli (1-7). This GPL is of particular interest because of its well documented role as the precursor to N-acylethanolamines (NAEs), a class of bioactive lipids that are involved in a variety of physiological processes, such as responses to pathogens, plant development, and germination in plants (8 -10) as well as control of appetite, inflammation, and apoptosis (11-13) in animals.In animals, N-acyl-PE synthesis is catalyzed by two distinct biochemical activities. A Ca 2ϩ -dependent membrane-associated protein has been characterized in brain, testis, and heart (14). This enzyme has been shown to catalyze the transfer of an acyl chain from the sn-1-position of a diacylated GPL, functions most efficiently at alkaline pH, and requires divalent cations (13,15,16). The gene encoding this enzyme has yet to be identified.A lecithin retinol acyltransferase-like protein from rats, RLP-1, has been shown to encode a Ca 2ϩ -independent N-acyltransferase (NAT) (17). It is predominantly expressed in testis and is, therefore, thought to be distinct from the Ca 2ϩ -dependent NAT. Ca 2ϩ -independent NAT activity is detected in both the soluble and membrane fractions when overexpressed in COS-7 cells and does not show preference for transfer of the sn-1 or sn-2 acyl chain to the amine of PE (17). Human and mouse homologs have been cloned and shown to possess similar NAT activity (18).In plants, N-acyl-PE is proposed to be synthesized from PE and unesterified fatty acid...

Altered Lipid Synthesis by Lack of Yeast Pah1 Phosphatidate Phosphatase Reduces Chronological Life Span

“…The fatty acid composition of the mitochondrial lipids after deacylation was determined by gas-liquid chromatography (35) using heptadecanoic acid as an internal standard. The mitochondrial phospholipid molecular species were analyzed by liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (62,63). For radiolabeling of cellular neutral lipids and total phospholipids, cells were grown in SC medium to the late exponential phase (A 600 nm ϳ1).…”

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

“…1; (Faure et al 2009; Coulon et al 2012)). A more recent study reported that this NAPE synthase enzyme may also have lysoglycerophospholipid O -acyltransferase activity (Bulat and Garrett 2011); however, only NAPE formation was demonstrated for this enzyme in planta (Faure et al 2009), so the physiological significance of this O -acyltransferase activity reported in vitro remains unclear.…”

Incorporation of therapeutically modified bacteria into gut microbiota inhibits obesity

“…Food intake, body weight, and fat mass during the saturated and monounsaturated NAPEs expected to exert anorexigenic effects (Supplemental Figure 5). In addition to NAPEs, several species with m/z ions consistent with O-acyl-phosphatidylglycerols (acyl-PG) species (34) were also enriched in pNAPE-EcN (Supplemental Table 2). …”

“…NAPE concentration was quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) after methylamine hydrolysis as we have previously described (33). Because Bulat and Garrett (34) reported that they failed to find evidence for significant synthesis of NAPEs in E. coli expressing At1g76890p, we performed additional experiments to confirm the synthesis of NAPEs category 4: meals >103.3 mg and ≤166.8 mg (mean + 2 SD); category 5: meal events >166.8 mg and ≤230.4 mg (mean + 3 SD). Category X represents meal events >230.4 mg.…”