Thermodynamic evaluation of ligand binding in the plant-like phosphoethanolamine methyltransferases of the parasitic nematode Haemonchus contortus.

Nematodes are a major cause of disease and the discovery of new pathways not found in hosts is critical for development of therapeutic targets. Previous studies suggest that Caenorhabditis elegans synthesizes phosphocholine via two S-adenosylmethionine (AdoMet)-dependent phosphoethanolamine methyltransferases (PMT). Here we examine two PMT from the parasitic nematode Haemonchus contortus. Sequence analysis suggests that HcPMT1 contains a methyltransferase domain in the N-terminal half of the protein and that HcPMT2 encodes a C-terminal methyltransferase domain, as in the C. elegans proteins. Kinetic analysis demonstrates that HcPMT1 catalyzes the conversion of phosphoethanolamine to phosphomonomethylethanolamine (pMME) and that HcPMT2 methylates pMME to phosphodimethylethanolamine (pDME) and pDME to phosphocholine. The IC(50) values for miltefosine, sinefungin, amodiaquine, diphenhydramine, and tacrine suggest differences in the active sites of these two enzymes. To examine the interaction of AdoMet and S-adenosylhomocysteine (AdoCys), isothermal titration calorimetry confirmed the presence of a single binding site in each enzyme. Binding of AdoMet and AdoCys is tight (K(d) ∼2-25 µm) over a range of temperatures (5-25 °C) and NaCl concentrations (0.05-0.5 m). Heat capacity changes for AdoMet and AdoCys binding suggests that each HcPMT differs in interaction surface area. Nonlinear van't Hoff plots also indicate a possible conformational change upon AdoMet/AdoCys binding. Functional analysis of the PMT from a parasitic nematode provides new insights on inhibitor and AdoMet/AdoCys binding to these enzymes.

Phosphoethanolamine N-methyltransferase (PMT-1) catalyses the first reaction of a new pathway for phosphocholine biosynthesis in Caenorhabditis elegans.

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.

Defining the role of phosphomethylethanolamine N-methyltransferase from Caenorhabditis elegans in phosphocholine biosynthesis by biochemical and kinetic analysis.

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.

Dynamics of the IRE RNA hairpin loop probed by 2-aminopurine fluorescence and stochastic dynamics simulations.

The iron responsive element (IRE) RNA hairpin loop contains six phylogenetically conserved nucleotides, which constitute part of the sequence-specific binding site of the IRE-binding protein. The NMR structure of the loop has been solved, showing that 3 of the 6 nt are poorly constrained. Here, two purine nucleotides in the IRE loop are individually replaced with the fluorescent purine analog 2-aminopurine (2AP). Steady-state and time-resolved fluorescence methods are used to describe the structure and dynamics of 2AP in the IRE loop. The data indicate that 2AP at the position of the adenosine in the loop moves between stacked and unstacked positions, whereas 2AP at the adjacent guanosine is predominantly solvent exposed. Stochastic dynamics simulations are used to provide a physical description of how those nucleotides might move.