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

作者：Lavanya H. Palavalli, Katherine M. Brendza, William Haakenson, Rebecca E. Cahoon, Merry McLaird, Leslie M. Hicks, James P. McCarter, D. Jeremy Williams, Michelle Coutu Hresko, Joseph M. Jez · 发表于：Biochemistry · 年份：2006 · DOI：10.1021/bi060199d · 被引用次数：72 · 研究领域：Cassava research and cyanide、Genetics, Aging, and Longevity in Model Organisms、Child Nutrition and Water Access

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 su...