In plants and Plasmodium falciparum, the synthesis of phosphatidylcholine requires the conversion of phosphoethanolamine to phosphocholine by phosphoethanolamine methyltransferase (PEAMT). This pathway differs from the metabolic route of phosphatidylcholine synthesis used in mammals and, on the basis of bioinformatics, was postulated to function in the nematode Caenorhabditis elegans. Here we describe the cloning and biochemical characterization of a PEAMT from C. elegans (gene, pmt-2; protein, PMT-2). Although similar in size to the PEAMT from plants, which contain two tandem methyltransferase domains, PMT-2 retains only the C-terminal methyltransferase domain. RNA-mediated interference experiments in C. elegans show that PMT-2 is essential for worm viability and that choline supplementation rescues the RNAi-generated phenotype. Unlike the plant and Plasmodium PEAMT, which catalyze all three methylations in the pathway, PMT-2 catalyzes only the last two steps in the pathway, i.e., the methylation of phosphomonomethylethanolamine (P-MME) to phosphodimethylethanolamine (P-DME) and of P-DME to phosphocholine. Analysis of initial velocity patterns suggests a random sequential kinetic mechanism for PMT-2. Product inhibition by S-adenosylhomocysteine was competitive versus S-adenosylmethionine and noncompetitive versus P-DME, consistent with formation of a dead-end complex. Inhibition by phosphocholine was competitive versus each substrate. Fluorescence titrations show that all substrates and products bind to the free enzyme. The biochemical data are consistent with a random sequential kinetic mechanism for PMT-2. This work provides a kinetic basis for additional studies on the reaction mechanism of PEAMT. Our results indicate that nematodes also use the PEAMT pathway for phosphatidylcholine biosynthesis. If the essential role of PMT-2 in C. elegans is conserved in parasitic nematodes of mammals and plants, then inhibition of the PEAMT pathway may be a viable approach for targeting these parasites with compounds of medicinal or agronomic value.

The development of nematicides targeting parasitic nematodes of animals and plants requires the identification of biochemical targets not found in host organisms. Recent studies suggest that Caenorhabditis elegans synthesizes phosphocholine through the action of PEAMT (S-adenosyl-L-methionine:phosphoethanolamine N-methyltransferases) that convert phosphoethanolamine into phosphocholine. Here, we examine the function of a PEAMT from C. elegans (gene: pmt-1; protein: PMT-1). Our analysis shows that PMT-1 only catalyses the conversion of phosphoethanolamine into phospho-monomethylethanolamine, which is the first step in the PEAMT pathway. This is in contrast with the multifunctional PEAMT from plants and Plasmodium that perform multiple methylations in the pathway using a single enzyme. Initial velocity and product inhibition studies indicate that PMT-1 uses a random sequential kinetic mechanism and is feedback inhibited by phosphocholine. To examine the effect of abrogating PMT-1 activity in C. elegans, RNAi (RNA interference) experiments demonstrate that pmt-1 is required for worm growth and development and validate PMT-1 as a potential target for inhibition. Moreover, providing pathway metabolites downstream of PMT-1 reverses the RNAi phenotype of pmt-1. Because PMT-1 is not found in mammals, is only distantly related to the plant PEAMT and is conserved in multiple parasitic nematodes of humans, animals and crop plants, inhibitors targeting it may prove valuable in human and veterinary medicine and agriculture.

Three unrestrained stochastic dynamics simulations have been carried out on the RNA hairpin GGAC[UUCG] GUCC, using the AMBER94 force field (Cornell et al., 1995. J. Am. Chem. Soc. 117:5179-5197) in MacroModel 5.5 (Mohamadi et al., 1990. J. Comp. Chem. 11:440-467) and either the GB/SA continuum solvation model (Still et al., 1990. J. Am. Chem. Soc. 112:6127-6129) or a linear distance-dependent dielectric (1/R) treatment. The linear distance-dependent treatment results in severe distortion of the nucleic acid structure, restriction of all hydroxyl dihedrals, and collapse of the counterion atmosphere over the course of a 5-ns simulation. An additional vacuum simulation without counterions shows somewhat improved behavior. In contrast, the two GB/SA simulations (1.149 and 3.060 ns in length) give average structures within 1.2 A of the initial NMR structure and in excellent agreement with results of an earlier explicit solvent simulation (Miller and Kollman, 1997. J. Mol. Biol. 270:436-450). In a 3-ns GB/SA simulation starting with the incorrect UUCG tetraloop structure (Cheong et al., 1990. Nature. 346:680-682), this loop conformation converts to the correct loop geometry (Allain and Varani, 1995. J. Mol. Biol. 250:333-353), suggesting enhanced sampling relative to the previous explicit solvent simulation. Thermodynamic effects of 2'-deoxyribose substitutions of loop nucleotides were experimentally determined and are found to correlate with the fraction of time the ribose 2'-OH is hydrogen bonded and the distribution of the hydroxyl dihedral is observed in the GB/SA simulations. The GB/SA simulations thus appear to faithfully represent structural features of the RNA without the computational expense of explicit solvent.

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Background: Phosphoethanolamine methyltransferases (PMT) are required for growth and development of nematodes. Result: Biochemical studies of two PMT from a parasitic nematode reveal key similarities and differences. Conclusion: Although the nematode PMT are conserved, the domains that catalyze specific reactions may undergo different conformational changes upon ligand binding. Significance: Because the PMT are not found in mammals, these proteins are potential antiparasitic targets for human and veterinary medicine.