Romier C, Reuter K, Suck D, Ficner R
European Molecular Biology Laboratory, Structural Biology Programme, Heidelberg, Germany.
Biochemistry. 1996 Dec 10;35(49):15734-9. doi: 10.1021/bi962003n.
Procaryotic tRNA-guanine transglycosylase (TGT) catalyzes the posttranscriptional base exchange of the queuine precursor 7-aminomethyl-7-deazaguanine (preQ1) with the genetically encoded guanine at the wobble position of tRNAs specific for Asn, Asp, His, and Tyr. The X-ray structures of Zymomonas mobilis TGT and of its complex with preQ1 [Romier, C., Reuter, K., Suck, D., & Ficner, R. (1996) EMBO J. 15, 2850-2857] have revealed a specific preQ1 binding pocket and allowed a proposal for tRNA binding and recognition. We have used band-shift experiments in denaturing conditions to study the enzymatic reaction performed by TGT. The presence of shifted protein bands after incubation with tRNA followed by protein denaturation indicates a reaction mechanism involving a covalent intermediate. Inspection of the X-ray structures and comparison of the different procaryotic TGT sequences highlighted the conserved aspartate 102 as the most likely nucleophile. Mutation of this residue into alanine by site-directed mutagenesis leads to an inactive mutant unable to form a covalent intermediate with tRNA, proving that aspartate 102 is the active site nucleophile in TGT. To investigate the recognition of the wobble guanine in the preQ1 binding pocket, we mutated aspartate 156, the major recognition element for preQ1, into alanine and tyrosine. Both mutants are inactive in producing the final product, but the mutant D156A is able to form the covalent intermediate with tRNA in the first step of the reaction mechanism in comparable amounts to wild-type protein. Therefore, the binding of the wobble guanine in the preQ1 binding pocket is required for the cleavage of the glycosidic bond. The three mutants were crystallized and their X-ray structures determined. The mutants display only subtle changes to the wild-type protein, confirming that the observed biochemical results are due to the chemical substitutions rather than structural rearrangements.
原核生物的tRNA-鸟嘌呤转糖基酶（TGT）催化转录后碱基交换，即queuine前体7-氨甲基-7-脱氮鸟嘌呤（preQ1）与在天冬酰胺、天冬氨酸、组氨酸和酪氨酸特异性tRNA的摆动位置上由基因编码的鸟嘌呤进行碱基交换。运动发酵单胞菌TGT及其与preQ1复合物的X射线结构[Romier, C., Reuter, K., Suck, D., & Ficner, R. (1996) EMBO J. 15, 2850 - 2857]揭示了一个特定的preQ1结合口袋，并提出了tRNA结合和识别的模型。我们在变性条件下进行了凝胶迁移实验，以研究TGT催化的酶促反应。在与tRNA孵育后进行蛋白质变性处理，出现迁移的蛋白质条带，这表明反应机制涉及共价中间体。对X射线结构的检查以及对不同原核生物TGT序列的比较突出显示，保守的天冬氨酸102是最可能的亲核试剂。通过定点诱变将该残基突变为丙氨酸，导致产生一个无活性的突变体，该突变体无法与tRNA形成共价中间体，证明天冬氨酸102是TGT中的活性位点亲核试剂。为了研究preQ1结合口袋中摆动鸟嘌呤的识别情况，我们将preQ1的主要识别元件天冬氨酸156突变为丙氨酸和酪氨酸。这两个突变体在产生最终产物方面均无活性，但突变体D156A在反应机制的第一步能够与tRNA形成共价中间体，其数量与野生型蛋白相当。因此，preQ1结合口袋中摆动鸟嘌呤的结合对于糖苷键的断裂是必需的。这三个突变体均已结晶，并测定了它们的X射线结构。这些突变体与野生型蛋白相比仅显示出细微变化，证实了观察到的生化结果是由于化学取代而非结构重排所致。
