Pessi Gabriella, Choi Jae-Yeon, Reynolds Jennifer M, Voelker Dennis R, Mamoun Choukri Ben
Center for Microbial Pathogenesis and Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, Connecticut 06030, USA.
J Biol Chem. 2005 Apr 1;280(13):12461-6. doi: 10.1074/jbc.M414626200. Epub 2005 Jan 21.
Unlike humans and yeast, Plasmodium falciparum, the agent of the most severe form of human malaria, utilizes host serine as a precursor for the synthesis of phosphatidylcholine via a plant-like pathway involving phosphoethanolamine methylation. The monopartite phosphoethanolamine methyltransferase, Pfpmt, plays an important role in the biosynthetic pathway of this major phospholipid by providing the precursor phosphocholine via a three-step S-adenosyl-L-methionine-dependent methylation of phosphoethanolamine. In vitro studies showed that Pfpmt has strong specificity for phosphoethanolamine. However, the in vivo substrate (phosphoethanolamine or phosphatidylethanolamine) is not yet known. We used yeast as a surrogate system to express Pfpmt and provide genetic and biochemical evidence demonstrating the specificity of Pfpmt for phosphoethanolamine in vivo. Wild-type yeast cells, which inherently lack phosphoethanolamine methylation, acquire this activity as a result of expression of Pfpmt. The Pfpmt restores the ability of a yeast mutant pem1Deltapem2Delta lacking the phosphatidylethanolamine methyltransferase genes to grow in the absence of choline. Lipid analysis of the Pfpmt-complemented pem1Deltapem2Delta strain demonstrates the synthesis of phosphatidylcholine but not the intermediates of phosphatidylethanolamine transmethylation. Complementation of the pem1Deltapem2Delta mutant relies on specific methylation of phosphoethanolamine but not phosphatidylethanolamine. Interestingly, a mutation in the yeast choline-phosphate cytidylyltransferase gene abrogates the complementation by Pfpmt thus demonstrating that Pfpmt activity is directly coupled to the Kennedy pathway for the de novo synthesis of phosphatidylcholine.
与人类和酵母不同，导致最严重形式人类疟疾的恶性疟原虫通过涉及磷酸乙醇胺甲基化的类植物途径利用宿主丝氨酸作为合成磷脂酰胆碱的前体。单部分磷酸乙醇胺甲基转移酶Pfpmt通过磷酸乙醇胺的三步S-腺苷-L-甲硫氨酸依赖性甲基化提供前体磷酸胆碱，在这种主要磷脂的生物合成途径中发挥重要作用。体外研究表明Pfpmt对磷酸乙醇胺具有很强的特异性。然而，体内底物（磷酸乙醇胺或磷脂酰乙醇胺）尚不清楚。我们使用酵母作为替代系统来表达Pfpmt，并提供遗传和生化证据证明Pfpmt在体内对磷酸乙醇胺的特异性。天然缺乏磷酸乙醇胺甲基化的野生型酵母细胞由于Pfpmt的表达而获得这种活性。Pfpmt恢复了缺乏磷脂酰乙醇胺甲基转移酶基因的酵母突变体pem1Δpem2Δ在没有胆碱的情况下生长的能力。对Pfpmt互补的pem1Δpem2Δ菌株的脂质分析表明合成了磷脂酰胆碱，但没有磷脂酰乙醇胺转甲基化的中间体。pem1Δpem2Δ突变体的互补依赖于磷酸乙醇胺的特异性甲基化，而不是磷脂酰乙醇胺。有趣的是，酵母胆碱磷酸胞苷转移酶基因的突变消除了Pfpmt的互补作用，从而证明Pfpmt活性直接与磷脂酰胆碱从头合成的肯尼迪途径相关。
