Structural basis of nonnatural amino acid recognition by an engineered aminoacyl-tRNA synthetase for genetic code expansion
Structural basis of nonnatural amino acid recognition by an engineered aminoacyl-tRNA synthetase for genetic code expansion
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DOI：
10.1073/pnas.0407039102
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发表时间：
2005-02-01
影响因子：
11.1
通讯作者：
Yokoyama, S
中科院分区：
文献类型：
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作者：
Kobayashi, T;Sakamoto, K;Yokoyama, S
通过对含有Y37V和Q195C突变(37V195C)的大肠杆菌酪氨酰-tRNA合成酶(TyrRS)的改造，扩展了真核系统中的遗传密码，该酶特异性地识别3-碘-L-酪氨酸而不是L-酪氨酸。在本研究中，我们在2.0埃分辨率下测定了大肠杆菌酪氨酸催化结构域37V195C突变体的3-碘-L-酪氨酸和L-酪氨酸结合结构。Val-37的γ-甲基和Cys-195的硫原子使van der Waals与3-碘-L-酪氨酸的碘原子接触。Val-37和Cys-195侧链被相邻残基刚性固定，形成TyrRS的疏水核心。这两个突变在氨酰化反应的第一步(氨基酸活化步骤)中对3-碘-L酪氨酸选择性识别的主要作用是不同的：Y37V突变消除了与碘原子的致命空间斥力，Q195C突变减少了L酪氨酸的错误识别。37V195C突变体TyrRS与L-酪氨酸二烯酸酯类似物的结构也得到了解析，表明在第二步(氨基酰转移步骤)中，3-碘-L-酪氨酸和L-酪氨酸侧链被类似地区分。这些结果表明，37V195C突变体上的氨基酸结合口袋对3-碘-L-酪氨酸的识别是优化的。
The genetic code in a eukaryotic system has been expanded by the engineering of Escherichia coli tyrosyl-tRNA synthetase (TyrRS) with the Y37V and Q195C mutations (37V195C), which specifically recognize 3-iodo-L-tyrosine rather than L-tyrosine. In the present study, we determined the 3-iodo-L-tyrosine- and L-tyrosine-bound structures of the 37V195C mutant of the E. coli TyrRS catalytic domain at 2.0-Angstrom resolution. The gamma-methyl group of Val-37 and the sulfur atom of Cys-195 make van der Waals contacts with the iodine atom of 3-iodo-L-tyrosine. The Val-37 and Cys-195 side chains are rigidly fixed by the neighboring residues forming the hydrophobic core of the TyrRS. The major roles of the two mutations are different for the 3-iodo-L-tyrosine-selective recognition in the first step of the aminoacylation reaction (the amino acid activation step): the Y37V mutation eliminates the fatal steric repulsion with the iodine atom, and the Q195C mutation reduces the L-tyrosine misrecognition. The structure of the 37V195C mutant TyrRS complexed with an L-tyrosyladenylate analogue was also solved, indicating that the 3-iodo-L-tyrosine and L-tyrosine side chains are similarly discriminated in the second step (the aminoacyl transfer step). These results demonstrate that the amino acid-binding pocket on the 37V195C mutant is optimized for specific 3-iodo-L-tyrosine recognition.