Crystal structure of QueC from Bacillus subtilis:: An enzyme involved in preQ1 biosynthesis
Crystal structure of QueC from Bacillus subtilis:: An enzyme involved in preQ1 biosynthesis
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DOI：
10.1002/prot.22098
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发表时间：
2008-08-15
影响因子：
2.9
通讯作者：
Huang, Raven H.
中科院分区：
文献类型：
--
作者：
Cicmil, Nenad;Huang, Raven H.
迄今为止，已在各种 RNA 分子中鉴定出 107 个修饰核苷酸。 1 这些修饰的核苷酸大部分出现在转移 RNA (tRNA) 中，由几种不同的 RNA 修饰酶形成，这些酶在转录后水平催化它们的反应。从化学角度来看，RNA 修饰的程度差异很大，从甲基化等简单修饰到将 queuosine (Q) 掺入 tRNA 等复杂修饰。复杂的 RNA 修饰，也称为超修饰，通常发生在 tRNA 反密码子附近。这些修饰改善了核糖体翻译蛋白质的解码步骤中 mRNA 和 tRNA 之间的密码子：反密码子相互作用，从而提高了蛋白质合成的效率和保真度。在细菌中，导致 Q 掺入四种 tRNA（tRNAHis、tRNAAsp、tRNAAsn 和 tRNATyr）摆动位置（位置 34）的酶促反应可分为三个阶段（图 S1，补充在线材料）。第一阶段是以GTP为起始原料的preQ1的生物合成。第二阶段是 tRNA 鸟嘌呤转糖基酶 (TGT) 将 tRNA 摆动位置处的碱基鸟嘌呤 (G) 替换为 preQ1。第三阶段是tRNA中preQ1进一步转化为Q，涉及两个酶促反应：QueA催化的S-AdoMet依赖性烷基化和未知酶催化的B12依赖性还原（图S1）。对TGT的广泛生化和结构研究使我们能够更好地了解TGT催化反应的分子识别和反应机制。 2, 3 根据最近的生化研究，我们还获得了有关 QueA 催化反应的大量生化知识。 4, 5 另一方面，由于缺乏遗传和生化数据，对 preQ1 的生物合成了解甚少。最近，de Crecy-Lagard 及其同事进行了生物信息学搜索，揭示了 queC、queD、queE 和 queF 4 个基因的产物参与 preQ1 生物合成。 6 迄今为止，仅对一种基因产物 QueF 进行了生化表征，7, 8，并且上述四种基因产物中的任何一种都没有可用的结构信息。我们的实验室对 tRNA 中的 Q 修饰有着长期的兴趣。此前，我们报道了 TGT 与茎环 RNA 复合物的结构研究。 3 为了更好地了解 preQ1 的生物合成，我们从枯草芽孢杆菌中克隆、过表达和纯化了 queC、queD、queE 和 queF 的基因产物，用于生化和结构研究。本文描述的是枯草芽孢杆菌QueC的晶体结构。
To date, 107 modified nucleotides have been identified in various RNA molecules. 1 These modified nucleotides, most of which occur in transfer RNA (tRNA), are formed by several different RNA modifying enzymes that catalyze their reaction at the posttranscriptional level. From a chemical point of view, the degree of an RNA modification varies significantly, ranging from a simple modification such as methylation to a complicated one such as incorporation of queuosine (Q) into tRNA. Complex RNA modifications, also called hypermodifications, usually occur near the anticodon of tRNAs. These modifications improve codon: anticodon interaction between mRNA and tRNA during the decoding step of protein translation by the ribosome, and as a result, enhance the efficiency and fidelity of protein synthesis. In bacteria, enzymatic reactions leading to incorporation of Q into the wobble position (position 34) of four tRNAs (tRNAHis, tRNAAsp, tRNAAsn, and tRNATyr) can be divided into three stages (Fig. S1, Supplemental Online Material). The first stage is the biosynthesis of preQ1 with GTP as the starting material. The second stage is the replacement of the base guanine (G) with preQ1 at the wobble position of tRNA, carried out by tRNA guanine transglycosylase (TGT). The third stage is further transformation of preQ1 to Q in tRNA involving two enzymatic reactions: a S-AdoMet-dependent alkylation catalyzed by QueA and a B12-dependent reduction catalyzed by an unknown enzyme (Fig. S1). Extensive biochemical and structural studies on TGT have allowed us to better understand the molecular recognition and reaction mechanism of the TGT-catalyzed reaction. 2, 3 We have also gained substantial biochemical knowledge regarding the QueA-catalyzed reaction based on recent biochemical studies. 4, 5 On the other hand, the biosynthesis of preQ1 is much less understood due to the lack of genetic and biochemical data. Recently, de Crecy-Lagard and coworkers carried out a bioinformatics search, revealing that the products of four genes, queC, queD, queE, and queF, are involved in preQ1 biosynthesis. 6 To date, only one gene product QueF has been biochemically characterized, 7, 8 and no structural information is available for any of the four gene products mentioned above.Our laboratory has a long-standing interest in Q modification in tRNA. Previously, we have reported structural studies of TGT in complex with a stem-loop RNA. 3 To better understand the biosynthesis of preQ1, we have cloned, overexpressed, and purified gene products of queC, queD, queE, and queF from B. subtilis for biochemical and structural studies. Described herein is the crystal structure of B. subtilis QueC.