Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Nature. 2010 Sep 30;467(7315):612-6. doi: 10.1038/nature09411.
In most bacteria and all archaea, glutamyl-tRNA synthetase (GluRS) glutamylates both tRNA(Glu) and tRNA(Gln), and then Glu-tRNA(Gln) is selectively converted to Gln-tRNA(Gln) by a tRNA-dependent amidotransferase. The mechanisms by which the two enzymes recognize their substrate tRNA(s), and how they cooperate with each other in Gln-tRNA(Gln) synthesis, remain to be determined. Here we report the formation of the 'glutamine transamidosome' from the bacterium Thermotoga maritima, consisting of tRNA(Gln), GluRS and the heterotrimeric amidotransferase GatCAB, and its crystal structure at 3.35 A resolution. The anticodon-binding body of GluRS recognizes the common features of tRNA(Gln) and tRNA(Glu), whereas the tail body of GatCAB recognizes the outer corner of the L-shaped tRNA(Gln) in a tRNA(Gln)-specific manner. GluRS is in the productive form, as its catalytic body binds to the amino-acid-acceptor arm of tRNA(Gln). In contrast, GatCAB is in the non-productive form: the catalytic body of GatCAB contacts that of GluRS and is located near the acceptor stem of tRNA(Gln), in an appropriate site to wait for the completion of Glu-tRNA(Gln) formation by GluRS. We identified the hinges between the catalytic and anticodon-binding bodies of GluRS and between the catalytic and tail bodies of GatCAB, which allow both GluRS and GatCAB to adopt the productive and non-productive forms. The catalytic bodies of the two enzymes compete for the acceptor arm of tRNA(Gln) and therefore cannot assume their productive forms simultaneously. The transition from the present glutamylation state, with the productive GluRS and the non-productive GatCAB, to the putative amidation state, with the non-productive GluRS and the productive GatCAB, requires an intermediate state with the two enzymes in their non-productive forms, for steric reasons. The proposed mechanism explains how the transamidosome efficiently performs the two consecutive steps of Gln-tRNA(Gln) formation, with a low risk of releasing the unstable intermediate Glu-tRNA(Gln).
在大多数细菌和所有古菌中，谷氨酰-tRNA 合成酶（GluRS）将 tRNA（Glu）和 tRNA（Gln）都氨酰化，然后通过依赖于 tRNA 的酰胺转移酶将 Glu-tRNA（Gln）选择性地转化为 Gln-tRNA（Gln）。两种酶识别其底物 tRNA（s）的机制，以及它们在 Gln-tRNA（Gln）合成中相互合作的方式，仍有待确定。在这里，我们报道了来自嗜热栖热菌（Thermotoga maritima）的“谷氨酰胺转酰胺酶体”的形成，它由 tRNA（Gln）、GluRS 和异源三聚体酰胺转移酶 GatCAB 组成，并以 3.35 Å 的分辨率报告了其晶体结构。GluRS 的反密码子结合体识别 tRNA（Gln）和 tRNA（Glu）的共同特征，而 GatCAB 的尾部结合体以 tRNA（Gln）特异性的方式识别 L 形 tRNA（Gln）的外角。GluRS 处于有活性形式，因为其催化体结合在 tRNA（Gln）的氨基酸接受臂上。相比之下，GatCAB 处于无活性形式：GatCAB 的催化体与 GluRS 的催化体接触，并位于 tRNA（Gln）的接受茎附近，位于适当的位置，等待 GluRS 完成 Glu-tRNA（Gln）的形成。我们确定了 GluRS 的催化体和反密码子结合体之间以及 GatCAB 的催化体和尾部结合体之间的铰链，这允许 GluRS 和 GatCAB 采用有活性和无活性的形式。两种酶的催化体争夺 tRNA（Gln）的接受臂，因此不能同时处于有活性形式。从当前的氨酰化状态（有活性的 GluRS 和无活性的 GatCAB）过渡到假定的酰胺化状态（无活性的 GluRS 和有活性的 GatCAB），需要两种酶处于无活性形式的中间状态，这是出于空间位阻的原因。所提出的机制解释了转酰胺酶体如何高效地完成 Gln-tRNA（Gln）形成的两个连续步骤，同时降低了释放不稳定中间产物 Glu-tRNA（Gln）的风险。
