Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
Nucleic Acids Res. 2011 Mar;39(4):1576-85. doi: 10.1093/nar/gkq919. Epub 2010 Oct 23.
Wybutosine (yW) is a hypermodified nucleoside found in position 37 of tRNA(Phe), and is essential for correct phenylalanine codon translation. yW derivatives widely exist in eukaryotes and archaea, and their chemical structures have many species-specific variations. Among them, its hydroxylated derivative, hydroxywybutosine (OHyW), is found in eukaryotes including human, but the modification mechanism remains unknown. Recently, we identified a novel Jumonji C (JmjC)-domain-containing protein, TYW5 (tRNA yW-synthesizing enzyme 5), which forms the OHyW nucleoside by carbon hydroxylation, using Fe(II) ion and 2-oxoglutarate (2-OG) as cofactors. In this work, we present the crystal structures of human TYW5 (hTYW5) in the free and complex forms with 2-OG and Ni(II) ion at 2.5 and 2.8 Å resolutions, respectively. The structure revealed that the catalytic domain consists of a β-jellyroll fold, a hallmark of the JmjC domains and other Fe(II)/2-OG oxygenases. hTYW5 forms a homodimer through C-terminal helix bundle formation, thereby presenting a large, positively charged patch involved in tRNA binding. A comparison with the structures of other JmjC-domain-containing proteins suggested a mechanism for substrate nucleotide recognition. Functional analyses of structure-based mutants revealed the essential Arg residues participating in tRNA recognition by TYW5. These findings extend the repertoire of the tRNA modification enzyme into the Fe(II)/2-OG oxygenase superfamily.
假尿嘧啶核苷（yW）是一种在 tRNA（Phe）的 37 位发现的高度修饰核苷，对于正确的苯丙氨酸密码子翻译至关重要。yW 衍生物广泛存在于真核生物和古菌中，其化学结构具有许多种属特异性变化。其中，其羟化衍生物羟假尿嘧啶核苷（OHyW）存在于包括人类在内的真核生物中，但修饰机制尚不清楚。最近，我们鉴定了一种新型的组蛋白去甲基化酶 JmjC 结构域蛋白 TYW5（tRNA yW 合成酶 5），它通过碳羟化作用，使用 Fe(II)离子和 2-氧戊二酸（2-OG）作为辅助因子，形成 OHyW 核苷。在这项工作中，我们分别以 2.5 和 2.8Å 的分辨率展示了人源 TYW5（hTYW5）在游离和与 2-OG 和 Ni(II)离子复合物两种形式的晶体结构。结构揭示了催化结构域由 β-发夹折叠组成，这是 JmjC 结构域和其他 Fe(II)/2-OG 加氧酶的特征。hTYW5 通过 C 端螺旋束形成同源二聚体，从而呈现出一个大的、带正电荷的斑块，参与 tRNA 的结合。与其他含有 JmjC 结构域的蛋白质的结构比较表明了底物核苷酸识别的机制。基于结构的突变体的功能分析揭示了 TYW5 识别 tRNA 所必需的 Arg 残基。这些发现将 tRNA 修饰酶的功能扩展到了 Fe(II)/2-OG 加氧酶超家族。
