Effects of mutagenesis in the switch I region and conserved arginines of Escherichia coli MnmE protein, a GTPase involved in tRNA modification
Effects of mutagenesis in the switch I region and conserved arginines of Escherichia coli MnmE protein, a GTPase involved in tRNA modification
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DOI：
10.1074/jbc.m503223200
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发表时间：
2005-09-02
影响因子：
4.8
通讯作者：
Armengod, ME
中科院分区：
文献类型：
--
作者：
Martínez-Vicente, M;Yim, L;Armengod, ME
MnmE 是一种进化保守的三结构域 GTP 酶，参与 tRNA 修饰。与 Ras 蛋白相比，MnmE 表现出较高的内在 GTP 酶活性，并且需要 GTP 水解才能发挥功能活性。它的 G 结构域保留了完整蛋白的 GTP 酶活性，因此，它应该包含负责此活性的催化残基。在这项工作中，对 MnmE G 结构域所有保守精氨酸残基的突变分析表明，与其他 GTP 酶不同，MnmE 不使用精氨酸指来驱动催化。此外，我们发现位于开关 I 区域的 G2 基序 ( (249)GTTRD(253)) 中的残基对于 GTP 结合并不重要，但在稳定过渡态方面发挥一定作用，特别是 Gly(249) 和 Thr(251)。另一方面，导致 GTP 酶活性轻微丧失的 G2 突变导致 MnmE 蛋白无功能。这表明 GTP 水解是 MnmE 介导 tRNA 修饰的必要但非充分条件。与 GTP 水解相关的开关 I 区域的构象变化似乎对于 MnmE 的功能至关重要，而 G2 基序的不变苏氨酸 (Thr251) 对于这种变化至关重要，因为它不能被丝氨酸取代。 MnmE 缺陷会导致生长受损，当参与解码过程的其他基因缺陷同时存在时，这种情况会加剧。这种行为让人想起在酵母中发现的行为，并强调了 tRNA 修饰对基因表达的重要性。
MnmE is an evolutionarily conserved, three domain GTPase involved in tRNA modification. In contrast to Ras proteins, MnmE exhibits a high intrinsic GTPase activity and requires GTP hydrolysis to be functionally active. Its G domain conserves the GTPase activity of the full protein, and thus, it should contain the catalytic residues responsible for this activity. In this work, mutational analysis of all conserved arginine residues of the MnmE G- domain indicates that MnmE, unlike other GTPases, does not use an arginine finger to drive catalysis. In addition, we show that residues in the G2 motif ( (249)GTTRD(253)), which resides in the switch I region, are not important for GTP binding but play some role in stabilizing the transition state, specially Gly(249) and Thr(251). On the other hand, G2 mutations leading to a minor loss of the GTPase activity result in a non- functional MnmE protein. This indicates that GTP hydrolysis is a required but non- sufficient condition so that MnmE can mediate modification of tRNA. The conformational change of the switch I region associated with GTP hydrolysis seems to be crucial for the function of MnmE, and the invariant threonine ( Thr251) of the G2 motif would be essential for such a change, because it cannot be substituted by serine. MnmE defects result in impaired growth, a condition that is exacerbated when defects in other genes involved in the decoding process are simultaneously present. This behavior is reminiscent to that found in yeast and stresses the importance of tRNA modification for gene expression.