Blaise Mickael, Becker Hubert D, Lapointe Jacques, Cambillau Christian, Giegé Richard, Kern Daniel
Département Machineries Traductionnelles, UPR 9002 du CNRS, Institut de Biologie Moléculaire et Cellulaire, 15, rue René Descartes, 67084 Strasbourg cedex, France.
Biochimie. 2005 Sep-Oct;87(9-10):847-61. doi: 10.1016/j.biochi.2005.03.007. Epub 2005 Apr 8.
Analysis of the completed genome sequences revealed presence in various bacteria of an open reading frame (ORF) encoding a polypeptide chain presenting important similarities with the catalytic domain of glutamyl-tRNA synthetases but deprived of the C-terminal anticodon-binding domain. This paralog of glutamyl-tRNA synthetases, the YadB protein, activates glutamate in the absence of tRNA and transfers the activated glutamate not on tRNA(Glu) but instead on tRNA(Asp). It has been shown that tRNA(Asp) is able to accept two amino acids: aspartate charged by aspartyl-tRNA synthetase and glutamate charged by YadB. The functional properties of YadB contrast with those of the canonical glutamyl-tRNA synthetases, which activate Glu only in presence of the cognate tRNA before aminoacylation of the 3'-end of tRNA. Biochemical approaches and mass spectrometry investigations revealed that YadB transfers the activated glutamate on the cyclopenthene-diol ring of the modified nucleoside queuosine posttranscriptionally inserted at the wobble position of the anticodon-loop to form glutamyl-queuosine. Unstability of the ester bond between the glutamate residue and the cyclopenthene-diol (half-life 7.5 min) explains why until now this modification escaped detection. Among Escherichia coli tRNAs containing queuosine in the wobble position, only tRNA(Asp) is substrate of YadB. Sequence comparison reveals a structural mimicry between the anticodon-stem and loop of tRNA(Asp) and the amino acid acceptor-stem of tRNA(Glu). YadB, renamed glutamyl-Q-tRNA(Asp) synthetase, constitutes the first enzyme structurally related to aminoacyl-tRNA synthetases which catalyzes a hypermodification in tRNA, and whose function seems to be conserved among prokaryotes. The discovery of glutamyl-Q-tRNA(Asp) synthetase breaks down the current paradigm according to which the catalytic domain of aminoacyl-tRNA synthetases recognizes the amino acid acceptor-stem of tRNA and aminoacylates the 3'-terminal ribose. The evolutionary significance of the existence of an aminoacyl-tRNA synthetase paralog dedicated to the hypermodification of a tRNA anticodon will be discussed.
对已完成的基因组序列分析显示，多种细菌中存在一个开放阅读框（ORF），其编码的多肽链与谷氨酰胺 - tRNA合成酶的催化结构域具有重要相似性，但缺少C端反密码子结合结构域。谷氨酰胺 - tRNA合成酶的这种旁系同源物，即YadB蛋白，在没有tRNA的情况下激活谷氨酸，并将活化的谷氨酸转移到tRNA（Asp）上，而不是tRNA（Glu）上。研究表明，tRNA（Asp）能够接受两种氨基酸：由天冬酰胺 - tRNA合成酶负载的天冬氨酸和由YadB负载的谷氨酸。YadB的功能特性与典型的谷氨酰胺 - tRNA合成酶相反，后者仅在同源tRNA存在的情况下激活Glu，然后对tRNA的3'端进行氨酰化。生化方法和质谱研究表明，YadB在转录后将活化的谷氨酸转移到反密码子环环摆动置插入的修饰核苷queuosine的环戊二醇环上，形成谷氨酰 - queuosine。谷氨酸残基与环戊二醇之间酯键的不稳定性（半衰期7.5分钟）解释了为什么到目前为止这种修饰一直未被检测到。在摆动位置含有queuosine的大肠杆菌tRNA中，只有tRNA（Asp）是YadB的底物。序列比较揭示了tRNA（Asp）的反密码子茎环与tRNA（Glu）的氨基酸接受茎之间的结构模拟。YadB，重新命名为谷氨酰 - Q - tRNA（Asp）合成酶，是第一种在结构上与氨酰 - tRNA合成酶相关的酶，它催化tRNA中的超修饰，其功能似乎在原核生物中保守。谷氨酰 - Q - tRNA（Asp）合成酶的发现打破了目前的范式，即氨酰 - tRNA合成酶的催化结构域识别tRNA的氨基酸接受茎并对3'末端核糖进行氨酰化。将讨论存在一种专门用于tRNA反密码子超修饰的氨酰 - tRNA合成酶旁系同源物的进化意义。
