Phosphocholine synthesis in spinach:: Characterization of phosphoethanolamine N-methyltransferase

Phosphocholine is a precursor for phosphatidylcholine or it may be hydrolysed to choline, Choline can be oxidized to form the compatible osmolyte glycine betaine which is accumulated by many plants under conditions of osmotic stress. In Spinacia oleracea phosphocholine is synthesized by 3 sequential N-methylations of phosphoethanolamine with the first step catalysed by the enzyme phosphoethanolamine N-methyltransferase (EC 2.1.1.103). This enzyme has been partially purified 5400-fold from spinach leaves using a combination of ammonium sulphate fractionation, followed by chromatographic separations on DEAE-Sepharose, phenyl-Sepharose, omega-amino-hexyl-agarose, Mono Q and adenosine-agarose, Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) separation and silver-staining of the final preparation revealed several polypeptides present, only one of which with an estimated molecular mass of 54 kDa could be photoaffinity cross-linked to the substrate [H-3] S-adenosyl-L-methionine. HPLC gel permeation chromatography was used to obtain an estimate for the native molecular mass of 77 kDa, Enzyme activity was optimal at pH 7.8 in HEPES-KOH buffer, it was inhibited by S-adenosyl-L-homocysteine, phosphocholine, phosphate, Mn2+ and Co2+ but not by ethanolamine, methylethanolamine, dimethylethanolamine, choline, glycine betaine or Mg2+. Using phosphoethanolamine as substrate, the final preparation had a specific activity of 189 nmol mg(-1) protein min(-1). The reaction products were identified and their relative abundance estimated following separation hy TLC as phosphomethylethanolamine (87%), phosphodimethylethanolamine (10%,) and phosphocholine (2%). Thus, a highly purified preparation of phosphoethanolamine N-methyltransferase was shown to catalyse 3 successive N-methylations of phosphoethanolamine. Photoaffinity cross-linking of proteins extracted from leaves of spinach followed by SDS-PAGE and autoradiography shows that a 54-kDa radiolabelled polypeptide was more prominent in extracts from salinized plants and barely visible in extracts from plants exposed to prolonged dark periods, a pattern which corresponds to the salt and light-responsive changes in phosphoethanolamine N-methylating activity. Thus, the production of phosphocholine for glycine betaine accumulation in spinach can be mediated by a single phosphobase N-methyltransferase which is more abundant in salt-stressed plants.