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

被引:86
作者
Nuccio, ML
Ziemak, MJ
Henry, SA
Weretilnyk, EA
Hanson, AD
机构
[1] Univ Florida, Dept Hort Sci, Gainesville, FL 32611 USA
[2] Carnegie Mellon Univ, Dept Biol Sci, Pittsburgh, PA 15213 USA
[3] McMaster Univ, Dept Biol, Hamilton, ON L8S 4K1, Canada
关键词
D O I
10.1074/jbc.275.19.14095
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 2.1.1.103). 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 16-fold, consistent with the high demand in stressed plants for choline to support glycine betaine synthesis.
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页码:14095 / 14101
页数:7
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