Watanabe Yasunori, Kumeta Hiroyuki, Watanabe Seiya
Faculty of Science, Yamagata University, Yamagata, Japan.
Graduate School of Life Science, Hokkaido University, Sapporo, Hokkaido, Japan.
J Biol Chem. 2025 May;301(5):108507. doi: 10.1016/j.jbc.2025.108507. Epub 2025 Apr 11.
In phosphatidylcholine (PC)-containing bacteria, PC is synthesized by phospholipid N-methyltransferases (Pmts) and plays an important role in the interactions between symbiotic and pathogenic bacteria and their eukaryotic host cells. Pmts catalyze the SAM-dependent three methylation reactions of the head group of phosphatidylethanolamine (PE) to form PC through monomethyl PE and dimethyl PE. However, the precise molecular mechanisms underlying PC biosynthesis by PmtA remain largely unclear, owing to the lack of structural information. Here, we determined the crystal structures of Agrobacterium tumefaciens Pmt (AtPmtA) in complex with SAH or 5'-methylthioadenosine. Crystal structures and NMR analysis revealed the binding mode of AtPmtA to SAH in solution. Structure-based mutational analyses showed that a conserved tyrosine residue in the substrate-binding groove is involved in methylation. Furthermore, we showed that differences in substrate specificity among Pmt homologs were determined by whether the amino acid residues comprising the substrate-binding groove were isoleucine or phenylalanine. These findings provide a structural basis for understanding the mechanisms underlying Pmts-mediated PC biosynthesis.
在含有磷脂酰胆碱（PC）的细菌中，PC由磷脂N-甲基转移酶（Pmts）合成，并且在共生细菌和致病细菌与其真核宿主细胞之间的相互作用中发挥重要作用。Pmts催化磷脂酰乙醇胺（PE）头部基团的依赖于S-腺苷甲硫氨酸（SAM）的三步甲基化反应，通过单甲基PE和二甲基PE形成PC。然而，由于缺乏结构信息，PmtA介导PC生物合成的确切分子机制在很大程度上仍不清楚。在这里，我们确定了与SAH或5'-甲硫基腺苷结合的根癌土壤杆菌Pmt（AtPmtA）的晶体结构。晶体结构和核磁共振分析揭示了AtPmtA在溶液中与SAH的结合模式。基于结构的突变分析表明，底物结合凹槽中的一个保守酪氨酸残基参与甲基化。此外，我们表明Pmt同源物之间底物特异性的差异取决于构成底物结合凹槽的氨基酸残基是异亮氨酸还是苯丙氨酸。这些发现为理解Pmts介导PC生物合成的机制提供了结构基础。
