Molecular and Biochemical Characterization of a Cold-Regulated PhosphoethanolamineN-Methyltransferase from Wheat

Abstract

A cDNA that encodes a methyltransferase (MT) was cloned from a cold-acclimated wheat (Triticum aestivum) cDNA library. Molecular analysis indicated that the enzyme WPEAMT (wheat phosphoethanolamine [P-EA] MT) is a bipartite protein with two separate sets of S-adenosyl-l-Met-binding domains, one close to the N-terminal end and the second close to the C-terminal end. The recombinant protein was found to catalyze the three sequential methylations of P-EA to form phosphocholine, a key precursor for the synthesis of phosphatidylcholine and glycine betaine in plants. Deletion and mutation analyses of the two S-adenosyl-l-Met-binding domains indicated that the N-terminal domain could perform the three N-methylation steps transforming P-EA to phosphocholine. This is in contrast to the MT from spinach (Spinacia oleracea), suggesting a different functional evolution for the monocot enzyme. The truncated C-terminal and the N-terminal mutated enzyme were only able to methylate phosphomonomethylethanolamine and phosphodimethylethanolamine, but not P-EA. This may suggest that the C-terminal part is involved in regulating the rate and the equilibrium of the three methylation steps. Northern and western analyses demonstrated that both Wpeamt transcript and the corresponding protein are up-regulated during cold acclimation. This accumulation was associated with an increase in enzyme activity, suggesting that the higher activity is due to de novo protein synthesis. The role of this enzyme during cold acclimation and the development of freezing tolerance are discussed.Winter survival and crop productivity are influenced by many different winter stresses, such as freezing temperature, length of freezing period, ice encasement, flooding, and oxidative stress caused by low temperature (LT)-induced photoinhibition (Fowler et al., 1999). Exposure of plants to LT produces morphological, physiological, and biochemical changes that are often highly correlated with plant freezing tolerance (FT) and winter survival. These changes are regulated by LT at the gene expression level. Cold-regulated genes and their products have been identified and characterized in many species (Thomashow, 1999). The complexity of the LT response has made it difficult to separate genes responsible for LT acclimation and cold hardiness from those associated with metabolic adjustments to LT. To characterize the genetic components associated with LT response, a better understanding of the LTresponsive genes responsible for adaptation to winter stresses and their interaction with the environment is needed (Fowler et al., 1999).

Characterization of Three O-Methyltransferases Involved in Noscapine Biosynthesis in Opium Poppy

“…Similarly, orcinol OMT was reported to carry out two sequential O-methylations in rose (Rosa 3 hybrida) scent biosynthesis (Lavid et al, 2002;Scalliet et al, 2002). Sequential methylation has also been reported for certain N-methyltransferases (Nuccio et al, 2000;Charron et al, 2002), but these proteins possess a different structural domain. In comparison, SOMT1 accepted both benzylisoquinoline and protoberberine substrates, showed no discrimination toward N-desmethyl or N-methylated BIAs, and was able to catalyze sequential O-methylations on both the isoquinoline and benzyl moieties.…”

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.…”

“…1B). The plant PMT contain tandem methyltransferase domains in a single polypeptide, in which the N-terminal domain catalyzes the initial methylation of pEA to phosphomonomethylethanolamine (pMME) and the C-terminal domain catalyzes the methylations of pMME to phosphodimethylethanolamine (pDME) and pDME to pCho (7,8). The PMT from P. falciparum is half the length of the plant enzyme and consists of a single methyltransferase domain that methylates all three phosphobases (13)(14).…”

PG12, a Phospholipid Analog with Potent Antimalarial Activity, Inhibits Plasmodium falciparum CTP:Phosphocholine Cytidylyltransferase Activity

“…PfPMT catalyzes the three-step S-adenosylmethionine methylation of phosphoethanolamine to form phosphocholine, which is used as a substrate by PfCCT and subsequently by PfCEPT to form PtdCho (2,12). The transmethylation of phosphoethanolamine to form phosphocholine also exists in plants, worms, and other protozoa but not in mammals (11,(13)(14)(15)(16). Recent genetic studies have shown that parasites lacking a functional SDPM pathway are severely altered in their development, produce half the number of nuclei and daughter parasites compared with wild-type P. falciparum, and show increased cell death (17).…”