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:phosphoethanolamine N-methyltransferase (PEAMT, EC ). A spinach PEAMT cDNA was isolated by functional complementation of a Schizosaccharomyces pombe cho2(-) mutant and was shown to encode a protein with PEAMT activity and without 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 duplication and fusion. Data base searches suggested that PEAMTs with the same tandem structure are widespread among flowering plants. Size exclusion chromatography of the recombinant enzyme indicates that it exists as a monomer. PEAMT 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 of spinach plants increases PEAMT mRNA abundance and enzyme activity in leaves by about 10-fold, consistent with the high demand in stressed plants for choline to support glycine betaine synthesis.
Nuccio
M L
ML
Horticultural Sciences Department, University of Florida, Gainesville, Florida 32611, USA.
Ziemak
M J
MJ
Henry
S A
SA
Weretilnyk
E A
EA
Hanson
A D
AD
D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005816:Genetic Complementation Test; D008780:Methyltransferases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012492:Salts; D012568:Schizosaccharomyces / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D018724:Spinacia oleracea / Q000201:enzymology / Q000235:genetics*
D018076:DNA, Complementary; D011994:Recombinant Proteins; D012492:Salts; D008780:Methyltransferases; C102844:phosphoethanolamine methyltransferase