Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
J Mol Biol. 2010 Aug 20;401(3):323-33. doi: 10.1016/j.jmb.2010.06.046. Epub 2010 Jun 30.
tRNA:m(5)C methyltransferase Trm4 generates the modified nucleotide 5-methylcytidine in archaeal and eukaryotic tRNA molecules, using S-adenosyl-l-methionine (AdoMet) as methyl donor. Most archaea and eukaryotes possess several Trm4 homologs, including those related to diseases, while the archaeon Methanocaldococcus jannaschii has only one gene encoding a Trm4 homolog, MJ0026. The recombinant MJ0026 protein catalyzed AdoMet-dependent methyltransferase activity on tRNA in vitro and was shown to be the M. jannaschii Trm4. We determined the crystal structures of the substrate-free M. jannaschii Trm4 and its complex with sinefungin at 1.27 A and 2.3 A resolutions, respectively. This AdoMet analog is bound in a negatively charged pocket near helix alpha8. This helix can adopt two different conformations, thereby controlling the entry of AdoMet into the active site. Adjacent to the sinefungin-bound pocket, highly conserved residues form a large, positively charged surface, which seems to be suitable for tRNA binding. The structure explains the roles of several conserved residues that were reportedly involved in the enzymatic activity or stability of Trm4p from the yeast Saccharomyces cerevisiae. We also discuss previous genetic and biochemical data on human NSUN2/hTrm4/Misu and archaeal PAB1947 methyltransferase, based on the structure of M. jannaschii Trm4.
tRNA：m(5)C 甲基转移酶 Trm4 利用 S-腺苷甲硫氨酸 (AdoMet) 作为甲基供体，在古菌和真核生物 tRNA 分子中生成修饰核苷酸 5-甲基胞苷。大多数古菌和真核生物都拥有几个 Trm4 同源物，包括与疾病相关的同源物，而古菌 Methanocaldococcus jannaschii 只有一个基因编码 Trm4 同源物 MJ0026。重组 MJ0026 蛋白在体外催化 tRNA 上的 AdoMet 依赖性甲基转移酶活性，并被证明是 M. jannaschii Trm4。我们分别以 1.27Å 和 2.3Å 的分辨率测定了无底物的 M. jannaschii Trm4 及其与 sinefungin 的复合物的晶体结构。这种 AdoMet 类似物结合在靠近α8 螺旋的带负电荷的口袋中。该螺旋可以采用两种不同的构象，从而控制 AdoMet 进入活性位点。紧邻 sinefungin 结合口袋的是高度保守的残基形成的大的正电荷表面，这似乎适合 tRNA 结合。该结构解释了几个保守残基的作用，这些残基据报道参与了酿酒酵母 Saccharomyces cerevisiae 的 Trm4p 的酶活性或稳定性。我们还根据 M. jannaschii Trm4 的结构讨论了先前关于人类 NSUN2/hTrm4/Misu 和古菌 PAB1947 甲基转移酶的遗传和生化数据。
