The Isolation and Characterization in Yeast of a Gene for ArabidopsisS-Adenosylmethionine:Phospho-EthanolamineN-Methyltransferase

Abstract

Saccharomyces cerevisiae opi3 mutant strains do not have the phospholipid N-methyltransferase that catalyzes the two terminal methylations in the phosphatidylcholine (PC) biosynthetic pathway. This results in a build up of the intermediate phosphatidylmonomethylethanolamine, causing a temperature-sensitive growth phenotype. An Arabidopsis cDNA library was used to isolate three overlapping plasmids that complemented the temperature-sensitive phenotype. Phospholipid analysis showed that the presence of the cloned cDNA caused a 65-fold reduction in the level of phosphatidylmonomethylethanolamine and a significant, though not equivalent, increase in the production of PC. Sequence analysis established that the cDNA was not homologous to OPI3 or to CHO2, the only other yeast phospholipid N-methyltransferase, but was similar to several other classes of methyltransferases. S-adenosyl-Met:phospho-base N-methyltransferase assays revealed that the cDNA catalyzed the three sequential methylations of phospho-ethanolamine to form phospho-choline. Phospho-choline is converted to PC by the CDP-choline pathway, explaining the phenotype conferred upon the yeast mutant strain by the cDNA. In accordance with this the gene has been named AtNMT1. The identification of this enzyme and the failure to isolate a plant phospholipid N-methyltransferase suggests that there are fundamental differences between the pathways utilized by yeast and by some plants for synthesis of PC.

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

“…motifs (8,13,14). Isothermal titration calorimetry (ITC) was used to evaluate the stoichiometry and energetics of ligand binding to AtPMT2.…”

“…by functional complementation of a yeast mutant (13). 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).…”

Thermodynamic Evaluation of Ligand Binding in the Plant-like Phosphoethanolamine Methyltransferases of the Parasitic Nematode Haemonchus contortus

“…These pathways allow for the use of exogenous choline and other phospholipids to be used for the synthesis of the major membrane phospholipid. In contrast, plants endogenously synthesize phosphocholine (pCho) 2 via the sequential methylation of phosphoethanolamine (pEA) in the phosphobase methylation pathway (3)(4)(5)(6)(7)(8). Interestingly, the plant-like phosphobase methylation pathway is also found in the free-living nematode Caenorhabditis elegans and the protozoan parasite Plasmodium falciparum, which causes malaria (9 -14).…”

“…1A) (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). Although the PMT found in plants, nematodes, and protozoans all catalyze the S-adenosylmethionine (AdoMet)-dependent methylation of pEA to pCho, the physical organization of the methyltransferase domains in these enzymes from plants (type 1), protozoa (type 2), and nematodes (type 3) differs dramatically (Fig.…”

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).…”