Publications

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

  Palavalli L.H., Brendza K.M., Haakenson W., Cahoon R.E., McLaird M., Hicks L.M., McCarter J.P., Williams D.J., Hresko M.C., Jez J.M.

  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 ba ... >> More

  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. << Less

  Biochemistry 45:6056-6065(2006) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Purification of phosphatidylethanolamine N-methyltransferase from rat liver.

  Ridgway N.D., Vance D.E.

  Phosphatidylethanolamine (PE) N-methyltransferase catalyzes the synthesis of phosphatidylcholine by the stepwise transfer of methyl groups from S-adenosylmethionine to the amino head group of PE. PE N-methyltransferase was solubilized from a microsomal membrane fraction of rat liver using the noni ... >> More

  Phosphatidylethanolamine (PE) N-methyltransferase catalyzes the synthesis of phosphatidylcholine by the stepwise transfer of methyl groups from S-adenosylmethionine to the amino head group of PE. PE N-methyltransferase was solubilized from a microsomal membrane fraction of rat liver using the nonionic detergent Triton X-100 and purified to apparent homogeneity. Specific activities of PE N-methyltransferase with PE, phosphatidyl-N-monomethylethanolamine (PMME), and phosphatidyl-N,N-dimethylethanolamine (PDME) as substrates were 0.63, 8.59, and 3.75 mumol/min/mg protein, respectively. The purified enzyme was composed of a single subunit with a molecular mass of 18.3 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Methylation activities dependent on the presence of PE, PMME, and PDME and the 18.3-kDa protein co-eluted when purified PE N-methyltransferase was subjected to gel filtration on Sephacryl S-300 in the presence of 0.1% Triton X-100. All three methylation activities eluted with a Stokes radius 2.1 A greater than that determined for pure Triton micelles (molecular mass difference of 27.4 kDa). Two-dimensional analysis of PE N-methyltransferase employing nonequilibrium pH gradient gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the enzyme is composed of a single isoform. Analysis of enzyme activity using PE, PMME, and PDME at various Triton X-100 concentrations indicated the enzyme follows the "surface dilution" model proposed for other enzymes that act at the surface of mixed micelle substrates. Initial velocity data for all three lipid substrates (at fixed concentrations of Triton X-100) were highly cooperative in nature. Hill numbers for PMME and PDME ranged from 3 at 0.5 mM Triton to 6 at 2.0 mM Triton. All three methylation activities had a pH optimum of 10. These results provide evidence that a single membrane-bound enzyme catalyzes all three methylation steps for the conversion of PE to phosphatidylcholine. << Less

  J. Biol. Chem. 262:17231-17239(1987) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Yeast phosphatidylethanolamine methylation pathway. Cloning and characterization of two distinct methyltransferase genes.

  Kodaki T., Yamashita S.

  The structural genes (PEM1 and PEM2) encoding the enzymes involved in the yeast phosphatidylethanolamine (PE) methylation pathway were cloned by means of genetic complementation using yeast mutants. The cloned genes were expressed in a yeast mutant that was completely deficient in the PE methylati ... >> More

  The structural genes (PEM1 and PEM2) encoding the enzymes involved in the yeast phosphatidylethanolamine (PE) methylation pathway were cloned by means of genetic complementation using yeast mutants. The cloned genes were expressed in a yeast mutant that was completely deficient in the PE methylation pathway. The membrane fraction obtained from the transformants carrying PEM1 only catalyzed the conversion of PE to phosphatidyl-N-monomethylethanolamine (PMME), the first step of the methylation pathway. Therefore, the enzyme encoded by PEM1 was designated as PE methyltransferase. In contrast, the membrane fraction from the transformants carrying PEM2 catalyzed the synthesis of phosphatidylcholine (PC) from PE, indicating that it contains all of the three methylation activities. PMME and phosphatidyl-N,N-dimethylethanolamine were found to be utilized more preferentially than PE. Because of its rather broad substrate specificity, the enzyme encoded by PEM2 is designated as phospholipid methyltransferase. The results of phospholipid composition analysis showed that the PEM1 transformant accumulated PMME whereas the PEM2 transformant contained a decreased amount of PC. Both genes were required for maintenance of the PC content of the yeast at a normal level. The results of nucleotide sequence analysis demonstrated that the coding frames of the PEM1 and PEM2 genes were capable of encoding 869- and 206-amino acid residues with calculated molecular weights of 101,202 and 23,150, respectively. The sizes of the PEM1 and PEM2 transcripts detected in the exponentially growing wild-type yeast were consistent with those of the deduced translation products. PE methyltransferase exhibits internal sequence homology as well as homology with phospholipid methyltransferase, suggesting that these two methyltransferase genes evolved through gene duplication. Furthermore, there was significant sequence homology between PE methyltransferase and bovine phenylethanolamine N-methyltransferase, and between phospholipid methyltransferase and Escherichia coli S-adenosylmethionine-6-N',N'-adenosyl (rRNA) dimethyltransferase. << Less

  J. Biol. Chem. 262:15428-15435(1987) [PubMed] [EuropePMC]