Molecular Characterization of Two Lysophospholipid:acyl-CoA Acyltransferases Belonging to the MBOAT Family in Nicotiana benthamiana

• Published in: PloS one 2015-12, Vol.10 (12), p.e0144653-e0144653
• Main Authors: Zhang, Donghui, Jasieniecka-Gazarkiewicz, Katarzyna, Wan, Xia, Luo, Ling, Zhang, Yinbo, Banas, Antoni, Jiang, Mulan, Gong, Yangmin
• Format: Article
• Language: English
• Subjects: 1-Acylglycerophosphocholine O-Acyltransferase - genetics; 1-Acylglycerophosphocholine O-Acyltransferase - metabolism; Acetyl-CoA C-Acyltransferase - genetics; Acetyl-CoA C-Acyltransferase - metabolism; Acyltransferase; Agriculture; Amino acid sequence; Amino acids; Animals; Biology; Biosynthesis; Chemical synthesis; Cloning; Cloning, Molecular; Enzymatic activity; Enzyme activity; Enzymes; Fatty acids; Flowers; Gene sequencing; Genomes; Laboratories; Leaves; Lecithin; Linoleic acid; Linolenic acid; Lipids; Lysophosphatidic acid; Lysophosphatidylcholine; Lysophospholipids - metabolism; Membranes; Metabolism; Multigene Family; Nicotiana - enzymology; Nicotiana - genetics; Open Reading Frames; Phosphatidylcholine; Phosphatidylcholines; Phospholipids; Phylogeny; Plant Proteins - genetics; Plant Proteins - metabolism; Platelet activating factor; Polyunsaturated fatty acids; Preferences; Seeds; Sequence Homology, Amino Acid; Substrate Specificity; Tobacco; Yeast; Yeasts
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0144653

In the remodeling pathway for the synthesis of phosphatidylcholine (PC), acyl-CoA-dependent lysophosphatidylcholine (lysoPC) acyltransferase (LPCAT) catalyzes the reacylation of lysoPC. A number of genes encoding LPCATs have been cloned and characterized from several plants in recent years. Using Arabidopsis and other plant LPCAT sequences to screen the genome database of Nicotiana benthamiana, we identified two cDNAs encoding the putative tobacco LPCATs (NbLPCAT1 and NbLPCAT2). Both of them were predicted to encode a protein of 463 amino acids with high similarity to LPCATs from other plants. Protein sequence features such as the presence of at least eight putative transmembrane regions, four highly conserved signature motifs and several invariant residues indicate that NbLPCATs belong to the membrane bound O-acyltransferase family. Lysophospholipid acyltransferase activity of NbLPCATs was confirmed by testing lyso-platelet-activating factor (lysoPAF) sensitivity through heterologous expression of each full-length cDNA in a yeast mutant Y02431 (lca1△) disrupted in endogenous LPCAT enzyme activity. Analysis of fatty acid profiles of phospholipids from the NbLPCAT-expressing yeast mutant Y02431 cultures supplemented with polyunsaturated fatty acids suggested more incorporation of linoleic acid (18:2n6, LA) and α-linolenic acid (18:3n3, ALA) into PC compared to yeast mutant harbouring empty vector. In vitro enzymatic assay demonstrated that NbLPCAT1had high lysoPC acyltransferase activity with a clear preference for α-linolenoyl-CoA (18:3), while NbLPCAT2 showed a high lysophosphatidic acid (lysoPA) acyltransferase activity towards α-linolenoyl-CoA and a weak lysoPC acyltransferase activity. Tissue-specific expression analysis showed a ubiquitous expression of NbLPCAT1 and NbLPCAT2 in roots, stems, leaves, flowers and seeds, and a strong expression in developing flowers. This is the first report on the cloning and characterization of lysophospholipid acyltransferases from N. benthamiana.