cDNA cloning of phosphoethanolamine N-methyltransferase from spinach by complementation in Schizosaccharomyces pombe and characterization of the recombinant enzyme

The N-methylation of phosphoethanolamine is the committing step in choline biogenesis in plants and is catalyzed by S-adenosyl-L-methionine:phosphoeth anolamine N-methyltransferase (PEAMT, EC 2.1.1.103). A spinach PEAMT cDNA w as isolated by functional complementation of a Schizosaccharomyces pombe ch o2(-) mutant and was shown to encode a protein with PEAMT activity and with out ethanolamine- or phosphatidylethanolamine N-methyltransferase activity. The PEAMT cDNA specifies a 494-residue polypeptide comprising two similar, tandem methyltransferase domains, implying that PEAMT arose by gene duplic ation and fusion. Data base searches suggested that PEAMTs with the same ta ndem structure are widespread among flowering plants. Size exclusion chroma tography of the recombinant enzyme indicates that it exists as a monomer, P EAMT catalyzes not only the first N-methylation of phosphoethanolamine but also the two subsequent N-methylations, yielding phosphocholine. Monomethyl - and dimethylphosphoethanolamine are detected as reaction intermediates. A truncated PEAMT lacking the C-terminal methyltransferase domain catalyzes only the first methylation. Phosphocholine inhibits both the wild type and the truncated enzyme, although the latter is less sensitive. Salinization o f spinach plants increases PEAMT mRNA abundance and enzyme activity in leav es by about 16-fold, consistent with the high demand in stressed plants for choline to support glycine betaine synthesis.