Phosphocholine synthesis in spinach: Characterization of phosphoethanolamine N-methyltransferase

Phosphocholine is a precursor for phosphatidylcholine or it may be hydrolys ed to choline, Choline can be oxidized to form the compatible osmolyte glyc ine 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 combinat ion of ammonium sulphate fractionation, followed by chromatographic separat ions on DEAE-Sepharose, phenyl-Sepharose, omega-amino-hexyl-agarose, Mono Q and adenosine-agarose, Sodium dodecyl sulphate-polyacrylamide gel electrop horesis (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 substra te [H-3] S-adenosyl-L-methionine. HPLC gel permeation chromatography was us ed to obtain an estimate for the native molecular mass of 77 kDa, Enzyme ac tivity was optimal at pH 7.8 in HEPES-KOH buffer, it was inhibited by S-ade nosyl-L-homocysteine, phosphocholine, phosphate, Mn2+ and Co2+ but not by e thanolamine, methylethanolamine, dimethylethanolamine, choline, glycine bet aine or Mg2+. Using phosphoethanolamine as substrate, the final preparation had a specific activity of 189 nmol mg(-1) protein min(-1). The reaction p roducts were identified and their relative abundance estimated following se paration hy TLC as phosphomethylethanolamine (87%), phosphodimethylethanola mine (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 prot eins extracted from leaves of spinach followed by SDS-PAGE and autoradiogra phy shows that a 54-kDa radiolabelled polypeptide was more prominent in ext racts from salinized plants and barely visible in extracts from plants expo sed to prolonged dark periods, a pattern which corresponds to the salt and light-responsive changes in phosphoethanolamine N-methylating activity. Thu s, the production of phosphocholine for glycine betaine accumulation in spi nach can be mediated by a single phosphobase N-methyltransferase which is m ore abundant in salt-stressed plants.