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.

在自由生活的和寄生的线虫中，磷酸乙醇胺甲基化为磷酸胆碱提供了关键的代谢产物，以维持磷脂的生物合成以促进生长和发育。由于线虫的磷酸乙醇胺甲基转移酶（PMT）对于正常的生长和发育至关重要，因此这些酶是抑制剂设计的潜在目标。松萎线虫（Bursaphelenchus xylophilus）对亚洲用于木材和造纸的树木造成广泛破坏。作为测试BxPMT1作为潜在杀线虫靶标的第一步，我们确定了该酶的2.05Å分辨率X射线晶体结构为与磷酸乙醇胺和S-腺苷同型半胱氨酸的末端复合物。BxPMT1的三维结构用作定点诱变的模板，以使用稳态动力学分析探讨活性位点残基对催化和磷酸乙醇胺结合的贡献。突变体的生化分析确定了β1d-α6环（W123F，M126I和Y127F）和β1e-α7环（S155A，S160A，H170A，T178V和Y180F）上的关键残基，这些残基形成了磷酸化碱基结合位点，提示Tyr127有助于BxPMT1中的甲基化反应。