Identification of Phosphomethylethanolamine N-Methyltransferase from Arabidopsis and Its Role in Choline and Phospholipid Metabolism

Abstract

Three sequential methylations of phosphoethanolamine (PEA) are required for the synthesis of phosphocholine (PCho) in plants. A cDNA encoding an N-methyltransferase that catalyzes the last two methylation steps was cloned from Arabidopsis by heterologous complementation of a Saccharomyces cerevisiae cho2, opi3 mutant. The cDNA encodes phosphomethylethanolamine N-methyltransferase (PMEAMT), a polypeptide of 475 amino acids that is organized as two tandem methyltransferase domains. PMEAMT shows 87% amino acid identity to a related enzyme, phosphoethanolamine N-methyltransferase, an enzyme in plants that catalyzes all three methylations of PEA to PCho. PMEAMT cannot use PEA as a substrate, but assays using phosphomethylethanolamine as a substrate result in both phosphodimethylethanolamine and PCho as products. PMEAMT is inhibited by the reaction products PCho and S-adenosyl-L-homocysteine, a property reported for phosphoethanolamine N-methyltransferase from various plants. An Arabidopsis mutant with a T-DNA insertion associated with locus At1g48600 showed no transcripts encoding PMEAMT. Shotgun lipidomic analyses of leaves of atpmeamt and wild-type plants generated phospholipid profiles showing the content of phosphatidylmethylethanolamine to be altered relative to wild type with the content of a 34:3 lipid molecular species 2-fold higher in mutant plants. In S. cerevisiae, an increase in PtdMEA in membranes is associated with reduced viability. This raises a question regarding the role of PMEAMT in plants and whether it serves to prevent the accumulation of PtdMEA to potentially deleterious levels.Choline occurs in plants as free choline, phosphocholine (PCho) 2

Conformational changes in the di-domain structure of Arabidopsis phosphoethanolamine methyltransferase leads to active-site formation

“…One report examining AtPMT2 suggests that this enzyme lacks the ability to convert pEA to pMME (i.e. the first step in the phosphobase methylation pathway) and that its gene knock-out alters levels of one phospholipid form (26). No information is available on AtPMT3, and the substrate preferences of the AtPMT have not been systematically examined.…”

“…2E). At the metabolic level, previous studies describe a modest 10% decrease in PtdCho in roots and leaves of the atpmt1/xipotl1 mutant (25) and only a 2-fold decrease for one polar lipid (34:3) species in the atpmt2 knock-outs (26). We compared the polar lipid profiles of wild-type and atpmt3 knock-out plants, which indicate no significant differences (supplemental Fig.…”

“…Examination of the role of each AtPMT isoform in Arabidopsis has been limited to the xipotl1 mutant phenotype of atpmt1, which suggests a key role for this enzyme in root development (24,25), and a report of a change in one phospholipid form in atpmt2 knock-out lines (26). For a comparison of the role of each isoform in planta, T-DNA insertion mutants for each atpmt were obtained with the knock-out confirmed by reverse transcript PCR (Fig.…”

“…Subsequent work demonstrated that silencing of atpmt1 resulted in temperature-sensitive male sterility and salt hypersensitivity and that the Arabidopsis xipotl1 mutant, which disrupts atpmt1, is characterized by a short primary root, increased lateral root formation, and short epidermal cells (24,25). Limited analysis of AtPMT2 suggests that this protein may not catalyze the methylation of pEA, but only the conversion of pMME to pCho (26). No phenotypic or functional information is available on AtPMT3, and no detailed biochemical examination of either AtPMT1 or AtPMT3 has been described.…”

Lipid Biosynthesis and Protein Concentration Respond Uniquely to Phosphate Supply during Leaf Development in Highly Phosphorus-Efficient Hakea prostrata

“…Consistent with higher phospholipid levels, HpNMT2 transcripts encoding a putative phosphocholine-producing enzyme involved in de novo phospholipid biosynthesis (Bolognese and McGraw, 2000;Jost et al, 2009;BeGora et al, 2010) were more abundant in young than in mature leaves of P-limited H. prostrata. Interestingly, higher Po concentrations and NMT transcript abundance in young leaf tissues are also striking features of a highly P-efficient wheat (Triticum aestivum) cultivar (Aziz et al, 2014).…”