Reynolds Jennifer M, Takebe Sachiko, Choi Jae-Yeon, El Bissati Kamal, Witola William H, Bobenchik April M, Hoch Jeffrey C, Voelker Dennis R, Mamoun Choukri Ben
Department of Genetics and Developmental Biology, University of Connecticut Health Center, Framington, CT 06030, USA.
J Biol Chem. 2008 Mar 21;283(12):7894-900. doi: 10.1074/jbc.M709869200. Epub 2008 Jan 4.
The PfPMT enzyme of Plasmodium falciparum, the agent of severe human malaria, is a member of a large family of known and predicted phosphoethanolamine methyltransferases (PMTs) recently identified in plants, worms, and protozoa. Functional studies in P. falciparum revealed that PfPMT plays a critical role in the synthesis of phosphatidylcholine via a plant-like pathway involving serine decarboxylation and phosphoethanolamine methylation. Despite their important biological functions, PMT structures have not yet been solved, and nothing is known about which amino acids in these enzymes are critical for catalysis and binding to S-adenosyl-methionine and phosphoethanolamine substrates. Here we have performed a mutational analysis of PfPMT focused on 24 residues within and outside the predicted catalytic motif. The ability of PfPMT to complement the choline auxotrophy of a yeast mutant defective in phospholipid methylation enabled us to characterize the activity of the PfPMT mutants. Mutations in residues Asp-61, Gly-83 and Asp-128 dramatically altered PfPMT activity and its complementation of the yeast mutant. Our analyses identify the importance of these residues in PfPMT activity and set the stage for advanced structural understanding of this class of enzymes.
恶性疟原虫是导致严重人类疟疾的病原体，其PfPMT酶是最近在植物、蠕虫和原生动物中发现的已知和预测的磷酸乙醇胺甲基转移酶（PMT）大家族的成员。对恶性疟原虫的功能研究表明，PfPMT通过涉及丝氨酸脱羧和磷酸乙醇胺甲基化的类似植物的途径在磷脂酰胆碱的合成中起关键作用。尽管它们具有重要的生物学功能，但PMT的结构尚未得到解析，对于这些酶中的哪些氨基酸对催化以及与S-腺苷甲硫氨酸和磷酸乙醇胺底物的结合至关重要也一无所知。在此，我们对PfPMT进行了突变分析，重点关注预测催化基序内外的24个残基。PfPMT补充磷脂甲基化缺陷酵母突变体胆碱营养缺陷的能力使我们能够表征PfPMT突变体的活性。Asp-61、Gly-83和Asp-128残基的突变显著改变了PfPMT活性及其对酵母突变体的互补作用。我们的分析确定了这些残基在PfPMT活性中的重要性，并为深入了解这类酶的结构奠定了基础。
