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首页> 外文期刊>Biochemistry >Defining the Role of Phosphomethylethanolamine N-Methyltransferase from Caenorhabditis elegans in Phosphocholine Biosynthesis by Biochemical and Kinetic Analysis
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Defining the Role of Phosphomethylethanolamine N-Methyltransferase from Caenorhabditis elegans in Phosphocholine Biosynthesis by Biochemical and Kinetic Analysis

机译：通过生化和动力学分析确定秀丽隐杆线虫的磷酸甲基乙醇胺N-甲基转移酶在磷酸胆碱生物合成中的作用

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In plants and Plasmodium falciparum,the synthesis of phosphatidylcholine requires the conversion of phosphoethanolamine to phosphocholine by phosphoemanolamine methyltransferase(PEAMT).This pathway differs from the metabolic route of phosphatidylcholine synthesis used in mammals and,on the basis of bioinformatics,was postulated to function in the nematode Caenorhabditis elegans.Here we describe the cloning and biochemical characterization of a PEAMT from C.elegans(gene,pmt-2;protein,PMT-2).Although similar in size to the PEAMT from plants,which contain two tandem methyltransferase domains,PMT-2 retains only the C-terminal methyltransferase domain.RNA-mediated interference experiments in C.elegans show that PMT-2 is essential for worm viability and that choline supplementation rescues the RNAi-generated phenotype.Unlike the plant and Plasmodium PEAMT,which catalyze all three methylations in the pathway,PMT-2 catalyzes only the last two steps in the pathway,i.e.,the methylation of phosphomonomethylethanolamine(P-MME)to phosphodimethylethanolamine(P-DME)and of P-DME to phosphocholine.Analysis of initial velocity patterns suggests a random sequential kinetic mechanism for PMT-2.Product inhibition by S-adenosylhomocysteine was competitive versus 5-adenosylmethionine and noncompetitive versus P-DME,consistent with formation of a dead-end complex.Inhibition by phosphocholine was competitive versus each substrate.Fluorescence titrations show that all substrates and products bind to the free enzyme.The biochemical data are consistent with a random sequential kinetic mechanism for PMT-2.This work provides a kinetic basis for additional studies on the reaction mechanism of PEAMT.Our results indicate that nematodes also use the PEAMT pathway for phosphatidylcholine biosynthesis.If the essential role of PMT-2 in C.elegans is conserved in parasitic nematodes of mammals and plants,then inhibition of the PEAMT pathway may be a viable approach for targeting these parasites with compounds of medicinal or agronomic value.
机译：在植物和恶性疟原虫中，磷脂酰胆碱的合成需要将磷酸乙醇胺通过磷酸氨醇胺甲基转移酶（PEAMT）转化为磷酸胆碱。该途径不同于哺乳动物所用的磷脂酰胆碱合成的代谢途径，并且根据生物信息学被认为在哺乳动物中起作用这里我们描述了线虫的PEAMT的克隆和生化特性（基因，pmt-2;蛋白质，PMT-2）。尽管大小与植物的PEAMT相似，但包含两个串联的甲基转移酶结构域。 ，PMT-2仅保留C端甲基转移酶结构域。秀丽隐杆线虫中RNA介导的干扰实验表明，PMT-2对蠕虫的生存能力至关重要，胆碱的补充可以挽救RNAi产生的表型。与植物和疟原虫PEAMT不同，它催化该途径中的所有三个甲基化，PMT-2仅催化该途径中的最后两个步骤，即磷酸的甲基化O-单甲基乙醇胺（P-MME）转化为磷酸二甲基乙醇胺（P-DME）以及P-DME转化为磷酸胆碱。分析初始速度模式表明PMT-2具有随机顺序动力学机制。S-腺苷同型半胱氨酸对产物的抑制作用与5-腺苷甲硫氨酸和非竞争性与P-DME竞争，与形成死胡同的复合物一致。磷酸胆碱的抑制作用与每种底物竞争性。荧光滴定表明，所有底物和产物均与游离酶结合。生化数据与随机顺序动力学机制一致这项工作为进一步研究PEAMT的反应机理提供了动力学基础。我们的结果表明，线虫也利用PEAMT途径进行磷脂酰胆碱的生物合成。如果PMT-2在秀丽隐杆线虫中的重要作用得以保留哺乳动物和植物的寄生线虫，然后抑制PEAMT途径可能是一种可行的方法，可以通过复合物靶向这些寄生虫具有药用或农艺价值。

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