The Putative N-acylphosphatidylethanolamine Synthase from Arabidopsis Thaliana Is a Lysoglycerophospholipid Acyltransferase

At1g78690p, a gene found in Arabidopsis thaliana, has been reported to encode a Nacyltransferase (NAT) that transfers an acyl chain from acyl-CoA to the head-group of phosphatidylethanolamine (PE) to form N-acyl phosphatidylethanolamine (N-acyl PE). Our investigation suggests that At1g78690p is not a PE-dependent NAT, but is instead a lysoglycerophospholipid O-acyltransferase. We over-expressed At1g78690p in Escherichia coli, extracted the cellular lipids, and identified the accumulating glycerophospholipid as acylphosphatidylglycerol (acyl PG). Electrospray ionization quadrupole time-offlight 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 P and C radiolabeled substrates showed that At1g78690p acylates 1-acyl lysophosphatidylethanolamine (1-acyl lyso PE) and 1-acyl lysophosphatidylglycerol (1-acyl lyso PG), but not PE or phosphatidylglycerol (PG), to form a di-acylated product that co-migrates with PE and PG respectively. We analyzed the di-acylated product formed by At1g78690p using a combination of base hydrolysis, phospholipase D treatment, ESI-MS and MS/MS to show that At1g78690p acylates the sn-2 position of 1-acyl lyso PE and 1-acyl lyso PG.