The isolation and characterization in yeast of a gene for arabidopsis S-adenosylmethionine:phospho-ethanolamin N-methyltransferase
The isolation and characterization in yeast of a gene for arabidopsis S-adenosylmethionine:phospho-ethanolamin N-methyltransferase
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DOI：
10.1104/pp.124.4.1800
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发表时间：
2000-12-01
期刊：
影响因子：
7.4
通讯作者：
McGraw, P
中科院分区：
文献类型：
--
作者：
Bolognese, CP;McGraw, P
酿酒酵母opi 3突变株不具有催化磷脂酰胆碱（PC）生物合成途径中的两个末端甲基化的磷脂N-甲基转移酶。这导致中间体磷脂酰单甲基乙醇胺的积累，引起温度敏感性生长表型。拟南芥cDNA文库被用来分离三个重叠的质粒，补充了温度敏感的表型。磷脂分析表明，克隆的cDNA的存在下，导致磷脂酰单甲基乙醇胺的水平降低了65倍，并显着，但不相等，PC的生产增加。序列分析表明，该cDNA与OPI 3或CHO 2（唯一的其他酵母磷脂N-甲基转移酶）不同源，但与其他几类甲基转移酶相似。S-腺苷甲硫氨酸：磷酸基N-甲基转移酶测定显示，该cDNA催化磷酸乙醇胺的三个连续甲基化形成磷酸胆碱。磷酸胆碱通过CDP-胆碱途径转化为PC，解释了cDNA赋予酵母突变株的表型。因此，该基因被命名为AtNMT 1。这种酶的鉴定和未能分离出植物磷脂N-甲基转移酶表明，酵母和某些植物用于合成PC的途径之间存在根本差异。
Saccharomyces cerevisiae opi3 mutant strains do not have the phospholipid N-methyltransferase that catalyzes the two terminal methylations in the phosphatidylcholine (PC) biosynthetic pathway. This results in a build up of the intermediate phosphatidylmonomethylethanolamine, causing a temperature-sensitive growth phenotype. An Arabidopsis cDNA library was used to isolate three overlapping plasmids that complemented the temperature-sensitive phenotype. Phospholipid analysis showed that the presence of the cloned cDNA caused a 65-fold reduction in the level of phosphatidylmonomethylethanolamine and a significant, though not equivalent, increase in the production of PC. Sequence analysis established that the cDNA was not homologous to OPI3 or to CHO2, the only other yeast phospholipid N-methyltransferase, but was similar to several other classes of methyltransferases. S-adenosyl-Met:phospho-base N-methyltransferase assays revealed that the cDNA catalyzed the three sequential methylations of phospho-ethanolamine to form phospho-choline. Phospho-choline is converted to PC by the CDP-choline pathway, explaining the phenotype conferred upon the yeast mutant strain by the cDNA. In accordance with this the gene has been named AtNMT1. The identification of this enzyme and the failure to isolate a plant phospholipid N-methyltransferase suggests that there are fundamental differences between the pathways utilized by yeast and by some plants for synthesis of PC.