The substrate specificity of tRNA (m1G37) methyltransferase (TrmD) from Aquifex aeolicus
The substrate specificity of tRNA (m1G37) methyltransferase (TrmD) from Aquifex aeolicus
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DOI：
10.1111/j.1365-2443.2006.01022.x
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发表时间：
2006-12-01
期刊：
影响因子：
2.1
通讯作者：
Hori, Hiroyuki
中科院分区：
文献类型：
--
作者：
Takeda, Hiroshi;Toyooka, Takashi;Hori, Hiroyuki
转移RNA （m(1)G37）甲基转移酶（TrmD）催化s -腺苷- l-蛋氨酸甲基转移到tRNA中G37的N-1原子。在大肠杆菌细胞中，TrmD甲基化具有G36G37序列的tRNA物种。以前认为G36是TrmD识别的正决定因素。在目前的研究中，我们证明了来自Aquifex aeolicus的TrmD甲基化了具有A36G37序列的tRNA转录本以及具有G36G37序列的tRNA转录本。相比之下，含有嘧啶36g37的tRNA转录本根本没有甲基化。这些底物特异性通过使用16个tRNA转录物的体外动力学分析得到证实。通过液相色谱/质谱法确定了修饰的核苷及其在酵母tRNA（Phe）转录本中的位置。此外，对9个截断的tRNA分子进行了测试，以澄清额外的识别位点。出乎意料的是，风茄TrmD蛋白有效地甲基化了与反密码子臂对应的微螺旋。由于反密码子干的破坏导致甲基接受活性完全丧失，因此反密码子干对于识别是必不可少的。此外，d臂结构的存在抑制了活性。最近，有报道称大肠杆菌TrmD甲基化了含有A36G37序列的酵母tRNA（Phe）。因此，对purine36G37序列的识别可能是真细菌TrmD蛋白的共同特征。
Transfer RNA (m(1)G37) methyltransferase (TrmD) catalyzes methyl-transfer from S-adenosyl-L-methionine to the N-1 atom of G37 in tRNA. In Escherichia coli cells, TrmD methylates tRNA species possessing a G36G37 sequence. It was previously believed that G36 was the positive determinant of TrmD recognition. In the current study, we demonstrate that TrmD from Aquifex aeolicus methylates tRNA transcripts possessing an A36G37 sequence as well as tRNA transcripts possessing a G36G37 sequence. In contrast, tRNA transcripts possessing pyrimidine36G37 were not methylated at all. These substrate specificities were confirmed by an in vitro kinetic assay using 16 tRNA transcripts. The modified nucleoside and the position in yeast tRNA(Phe) transcript were confirmed by LC/MS. Furthermore, nine truncated tRNA molecules were tested to clarify the additional recognition site. Unexpectedly, A. aeolicus TrmD protein efficiently methylated the micro helix corresponding to the anti-codon arm. Because the disruption of the anti-codon stem caused the complete loss of the methyl group acceptance activity, the anti-codon stem is essential for the recognition. Moreover, the existence of the D-arm structure inhibited the activity. Recently, it was reported that E. coli TrmD methylates yeast tRNA(Phe) harboring a sequence A36G37. Thus, recognition of the purine36G37 sequence is probably common to eubacteria TrmD proteins.