In Vivo Evidence for the Specificity of Plasmodium falciparum Phosphoethanolamine Methyltransferase and Its Coupling to the Kennedy Pathway

Abstract

Unlike humans and yeast, Plasmodium falciparum, the agent of the most severe form of human malaria, utilizes host serine as a precursor for the synthesis of phosphatidylcholine via a plant-like pathway involving phosphoethanolamine methylation. The monopartite phosphoethanolamine methyltransferase, Pfpmt, plays an important role in the biosynthetic pathway of this major phospholipid by providing the precursor phosphocholine via a three-step S-adenosyl-L-methionine-dependent methylation of phosphoethanolamine. In vitro studies showed that Pfpmt has strong specificity for phosphoethanolamine. However, the in vivo substrate (phosphoethanolamine or phosphatidylethanolamine)is not yet known. We used yeast as a surrogate system to express Pfpmt and provide genetic and biochemical evidence demonstrating the specificity of Pfpmt for phosphoethanolamine in vivo. Wild-type yeast cells, which inherently lack phosphoethanolamine methylation, acquire this activity as a result of expression of Pfpmt. The Pfpmt restores the ability of a yeast mutant pem1⌬pem2⌬ lacking the phosphatidylethanolamine methyltransferase genes to grow in the absence of choline. Lipid analysis of the Pfpmt-complemented pem1⌬pem2⌬ strain demonstrates the synthesis of phosphatidylcholine but not the intermediates of phosphatidylethanolamine transmethylation. Complementation of the pem1⌬pem2⌬ mutant relies on specific methylation of phosphoethanolamine but not phosphatidylethanolamine. Interestingly, a mutation in the yeast choline-phosphate cytidylyltransferase gene abrogates the complementation by Pfpmt thus demonstrating that Pfpmt activity is directly coupled to the Kennedy pathway for the de novo synthesis of phosphatidylcholine.

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

“…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.…”

“…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).…”

Identification of Gene Encoding Plasmodium knowlesi Phosphatidylserine Decarboxylase by Genetic Complementation in Yeast and Characterization of in Vitro Maturation of Encoded Enzyme

“…Unlike Plasmodium species, S. cerevisiae does not decarboxylate serine to form Etn. Previously, we have successfully used S. cerevisiae as a surrogate system to characterize P. falciparum genes and cDNAs involved in the synthesis and transport of lipid precursors (3,16,17). However, these studies required optimization of cDNAs because of the high A ϩ T content of P. falciparum genes (ϳ81%) (18).…”

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

“…In each case, the committing step appears to be the N-methylation of phosphoethanolamine (PEA) to produce phosphomethylethanolamine (PMEA) (10 -12). The production of PMEA is also a key step in choline biosynthesis in P. falciparum and C. elegans (7,13).…”