Stengl Bernhard, Meyer Emmanuel A, Heine Andreas, Brenk Ruth, Diederich François, Klebe Gerhard
Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, Marbacher Weg 6, 35032 Marburg, Germany.
J Mol Biol. 2007 Jul 13;370(3):492-511. doi: 10.1016/j.jmb.2007.04.008. Epub 2007 Apr 12.
The bacterial tRNA-guanine transglycosylase (TGT) is a tRNA modifying enzyme catalyzing the exchange of guanine 34 by the modified base preQ1. The enzyme is involved in the infection pathway of Shigella, causing bacterial dysentery. As no crystal structure of the Shigella enzyme is available the homologous Zymomonas mobilis TGT was used for structure-based drug design resulting in new, potent, lin-benzoguanine-based inhibitors. Thorough kinetic studies show size-dependent inhibition of these compounds resulting in either a competitive or non-competitive blocking of the base exchange reaction in the low micromolar range. Four crystal structures of TGT-inhibitor complexes were determined with a resolution of 1.58-2.1 A. These structures give insight into the structural flexibility of TGT necessary to perform catalysis. In three of the structures molecular rearrangements are observed that match with conformational changes also noticed upon tRNA substrate binding. Several water molecules are involved in these rearrangement processes. Two of them demonstrate the structural and catalytic importance of water molecules during TGT base exchange reaction. In the fourth crystal structure the inhibitor unexpectedly interferes with protein contact formation and crystal packing. In all presently known TGT crystal structures the enzyme forms tightly associated homodimers internally related by crystallographic symmetry. Upon binding of the fourth inhibitor the dimer interface, however, becomes partially disordered. This result prompted further analyses to investigate the relevance of dimer formation for the functional protein. Consultation of the available TGT structures and sequences from different species revealed structural and functional conservation across the contacting residues. This suggests that bacterial and eukaryotic TGT could possibly act as homodimers in catalysis. It is hypothesized that one unit of the dimer performs the catalytic reaction whereas the second is required to recognize and properly orient the bound tRNA for the catalytic reaction.
细菌的tRNA-鸟嘌呤转糖基酶（TGT）是一种tRNA修饰酶，可催化鸟嘌呤34与修饰碱基preQ1的交换。该酶参与志贺氏菌的感染途径，可引发细菌性痢疾。由于尚无志贺氏菌该酶的晶体结构，因此利用同源的运动发酵单胞菌TGT进行基于结构的药物设计，从而得到了新型、强效的基于连苯并鸟嘌呤的抑制剂。深入的动力学研究表明，这些化合物具有大小依赖性抑制作用，在低微摩尔范围内导致碱基交换反应的竞争性或非竞争性阻断。测定了TGT-抑制剂复合物的四个晶体结构，分辨率为1.58-2.1埃。这些结构揭示了TGT进行催化所必需的结构灵活性。在其中三个结构中观察到分子重排，这与tRNA底物结合时也注意到的构象变化相匹配。几个水分子参与了这些重排过程。其中两个水分子证明了水分子在TGT碱基交换反应中的结构和催化重要性。在第四个晶体结构中，抑制剂意外地干扰了蛋白质接触的形成和晶体堆积。在目前所有已知的TGT晶体结构中，该酶通过晶体学对称性在内部形成紧密结合的同型二聚体。然而，在结合第四种抑制剂后，二聚体界面变得部分无序。这一结果促使进一步分析，以研究二聚体形成对功能蛋白的相关性。查阅不同物种的可用TGT结构和序列，发现接触残基在结构和功能上具有保守性。这表明细菌和真核TGT在催化过程中可能作为同型二聚体起作用。据推测，二聚体的一个单元进行催化反应，而另一个单元则需要识别并正确定向结合的tRNA以进行催化反应。
