Conserved Cysteine Residues of GidA Are Essential for Biogenesis of 5-Carboxymethylaminomethyluridine at tRNA Anticodon
Conserved Cysteine Residues of GidA Are Essential for Biogenesis of 5-Carboxymethylaminomethyluridine at tRNA Anticodon
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DOI：
10.1016/j.str.2009.03.013
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发表时间：
2009-05-13
期刊：
影响因子：
5.7
通讯作者：
Numata, Tomoyuki
中科院分区：
文献类型：
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作者：
Osawa, Takuo;Ito, Koichi;Numata, Tomoyuki
尿苷(cmnm(5)U)在反密码子第一位的5-羧甲基氨甲基修饰发生在阅读以嘌呤结尾的分裂密码子框的tRNA中。通过限制密码子-反密码子的摆动，这种修饰对于正确的翻译至关重要。两个保守的酶GIDA和MnmE参与了cmnm(5)U的修饰过程。在此，我们测定了Aquifex aeolicus gida在2.3埃分辨率下的晶体结构。该结构揭示了GIDA与FAD的紧密相互作用。基于结构的突变分析使我们能够确定FAD结合位点附近的两个保守的半胱氨酸残基，这两个残基是体内cmnm(5)U修饰所必需的。结合MnmE的突变分析，我们提出了一种cmnm(5)U修饰过程的机制，其中gida而不是MnmE以类似于胸苷合成酶的机制攻击尿苷的C6原子。我们还提出了一个tRNA对接模型，该模型为有效的修饰提供了对tRNA识别机制的结构性见解。
The 5-carboxymethylaminomethyl modification of uridine (cmnm(5)U) at the anticodon first position occurs in tRNAs that read split codon boxes ending with purine. This modification is crucial for correct translation, by restricting codon-anticodon wobbling. Two conserved enzymes, GidA and MnmE, participate in the cmnm(5)U modification process. Here we determined the crystal structure of Aquifex aeolicus GidA at 2.3 angstrom resolution. The structure revealed the tight interaction of GidA with FAD. Structure-based mutation analyses allowed us to identify two conserved Cys residues in the vicinity of the FAD-binding site that are essential for the cmnm(5)U modification in vivo. Together with mutational analysis of MnmE, we propose a mechanism for the cmnm(5)U modification process where GidA, but not MnmE, attacks the C6 atom of uridine by a mechanism analogous to that of thymidylate synthase. We also present a tRNA-docking model that provides structural insights into the tRNA recognition mechanism for efficient modification.