Department of Biology, Washington University in St. Louis, One Brookings Drive, St. Louis, MO, 63130, United States.
Department of Biology, Washington University in St. Louis, One Brookings Drive, St. Louis, MO, 63130, United States.
Mol Biochem Parasitol. 2020 Jul;238:111291. doi: 10.1016/j.molbiopara.2020.111291. Epub 2020 May 30.
In free-living and parasitic nematodes, the methylation of phosphoethanolamine to phosphocholine provides a key metabolite to sustain phospholipid biosynthesis for growth and development. Because the phosphoethanolamine methyltransferases (PMT) of nematodes are essential for normal growth and development, these enzymes are potential targets of inhibitor design. The pine wilt nematode (Bursaphelenchus xylophilus) causes extensive damage to trees used for lumber and paper in Asia. As a first step toward testing BxPMT1 as a potential nematicide target, we determined the 2.05 Å resolution x-ray crystal structure of the enzyme as a dead-end complex with phosphoethanolamine and S-adenosylhomocysteine. The three-dimensional structure of BxPMT1 served as a template for site-directed mutagenesis to probe the contribution of active site residues to catalysis and phosphoethanolamine binding using steady-state kinetic analysis. Biochemical analysis of the mutants identifies key residues on the β1d-α6 loop (W123F, M126I, and Y127F) and β1e-α7 loop (S155A, S160A, H170A, T178V, and Y180F) that form the phosphobase binding site and suggest that Tyr127 facilitates the methylation reaction in BxPMT1.
在自由生活和寄生线虫中，磷酸乙醇胺的甲基化为维持生长和发育所需的磷脂生物合成提供了关键代谢物。由于线虫的磷酸乙醇胺甲基转移酶（PMT）对于正常生长和发育至关重要，因此这些酶是抑制剂设计的潜在靶标。松材线虫（Bursaphelenchus xylophilus）在亚洲造成了用于木材和纸张的树木的广泛破坏。作为测试 BxPMT1 作为潜在杀线虫剂靶标的第一步，我们确定了该酶与磷酸乙醇胺和 S-腺苷同型半胱氨酸形成的死端复合物的 2.05 Å 分辨率 X 射线晶体结构。BxPMT1 的三维结构被用作定点诱变的模板，通过稳态动力学分析探测催化和磷酸乙醇胺结合的活性位点残基的贡献。对突变体的生化分析确定了形成磷酸碱基结合位点的β1d-α6 环（W123F、M126I 和 Y127F）和β1e-α7 环（S155A、S160A、H170A、T178V 和 Y180F）上的关键残基，并表明 Tyr127 有助于 BxPMT1 中的甲基化反应。
