Defining the Role of Phosphomethylethanolamine N-Methyltransferase from Caenorhabditis elegans in Phosphocholine Biosynthesis by Biochemical and Kinetic Analysis

Abstract

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.

Choline Transport and de novo Choline Synthesis Support Acetylcholine Biosynthesis in Caenorhabditis elegans Cholinergic Neurons

“…Recently, Palavalli et al (2006) demonstrated that a major pathway for phosphocholine biosynthesis in C. elegans is through the methylation of phosphoethanolamine. Conversion of phosphocholine to choline by dephosphorylation could potentially provide a source of choline for ACh synthesis.…”

“…Conversion of phosphocholine to choline by dephosphorylation could potentially provide a source of choline for ACh synthesis. Through RNA interference, Palavalli et al (2006) demonstrated that the C. elegans phosphoethanolamine methyltransferase gene (pmt-2) is essential for growth and development and that supplementation with choline can bypass the requirement for this enzyme.…”

“…We generated an RNAi construct as described by Palavalli et al (2006) to knock down synthesis of choline through the pmt-2-dependent pathway. L4 hermaphrodites were exposed to RNAi in the presence or absence of exogenous choline (30 mm).…”

“…We constructed a pmt-2 RNAi plasmid (RM#895p) in the pPD129.36 plasmid vector (Timmons and Fire 1998) using the primers described by Palavalli et al (2006). Bacteria containing this construct or the control plasmid (pPD129.36) were spread on NGM-L plates (without streptomycin) supplemented with isopropyl 1-thio-b-dgalactopyranoside (IPTG) and ampicillin.…”

“…L4 hermaphrodites were washed briefly in M9 buffer and then transferred to RNAi plates. Plates were scored 4 days later as described by Palavalli et al (2006), and behavioral analyses were conducted at this stage. Initially, all RNAi experiments were performed on worms carrying the rrf-3(pk1426) mutation, which results in enhanced sensitivity to RNAi in the nervous system (Simmer et al 2002).…”