Matsuda Teppei, Yamagami Ryota, Ihara Aoi, Suzuki Takeo, Hirata Akira, Hori Hiroyuki
Department of Materials Science and Biotechnology, Graduate School of Science and Engineering, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan.
Department of Nature Science, Faculty of Science and Technology, Tokushima University, 2-1 Minamijosanjima-cho, Tokushima 770-8506, Japan.
Nucleic Acids Res. 2025 Jul 8;53(13). doi: 10.1093/nar/gkaf579.
Thermococcus kodakarensis tRNATrp contains 2'-O-methylcytidine at position 6 (Cm6). However, the tRNA methyltransferase responsible for the modification has not been identified. Using comparative genomics, we predicted TK1257 as a candidate gene for the modification enzyme. Biochemical and mass spectrometry studies of purified recombinant TK1257 gene product demonstrated that it possesses a tRNA methyltransferase activity for Cm6 formation. This protein has a highly unusual composition of domains, containing N-terminal ferredoxin-like, SPOUT catalytic, and THUMP domains. Previous to this study, all known THUMP-related tRNA methyltransferases were shown to contain a Rossmann fold catalytic domain and the nucleosides they produced were N2-methylguanosine and/or N2, N2-dimethylguanosine. Therefore, our findings extend the knowledge of architecture of tRNA methyltransferases. We named the TK1257 gene product TrmTS and showed that it can synthesize Am6 and Um6 as well as Cm6. A trmTS gene deletion strain showed slight growth retardation at high temperatures. Site-directed mutagenesis studies revealed catalytically and structurally important amino acid residues in TrmTS and identified a TrmTS-specific linker that is structurally essential. We revealed that TrmTS recognizes the 3'-CCA terminus of tRNA but does not require the three-dimensional structure of tRNA for its activity. Finally, we constructed a model of the binding between TrmTS and tRNA.
嗜热栖热菌的tRNATrp在第6位含有2'-O-甲基胞苷（Cm6）。然而，负责这种修饰的tRNA甲基转移酶尚未被鉴定出来。通过比较基因组学，我们预测TK1257是这种修饰酶的候选基因。对纯化的重组TK1257基因产物进行的生化和质谱研究表明，它具有形成Cm6的tRNA甲基转移酶活性。这种蛋白质具有非常不寻常的结构域组成，包含N端铁氧化还原蛋白样结构域、SPOUT催化结构域和THUMP结构域。在本研究之前，所有已知的与THUMP相关的tRNA甲基转移酶都被证明含有罗斯曼折叠催化结构域，并且它们产生的核苷是N2-甲基鸟苷和/或N2,N2-二甲基鸟苷。因此，我们的发现扩展了对tRNA甲基转移酶结构的认识。我们将TK1257基因产物命名为TrmTS，并表明它可以合成Am6、Um6以及Cm6。一个trmTS基因缺失菌株在高温下表现出轻微的生长迟缓。定点诱变研究揭示了TrmTS中具有催化和结构重要性的氨基酸残基，并确定了一个在结构上必不可少的TrmTS特异性连接子。我们发现TrmTS识别tRNA的3'-CCA末端，但不依赖于tRNA的三维结构来发挥其活性。最后，我们构建了TrmTS与tRNA结合的模型。
