Viard T, Lamour V, Duguet M, Bouthier de la Tour C
Laboratoire d'Enzymologie des Acides Nucléiques, Institut de Génétique et Microbiologie, UMR 8621 CNRS, Bâtiment 400, Université de Paris Sud, Centre d'Orsay, 91405 Orsay Cedex, France.
J Biol Chem. 2001 Dec 7;276(49):46495-503. doi: 10.1074/jbc.M107714200. Epub 2001 Sep 27.
Topoisomerases, by controlling DNA supercoiling state, are key enzymes for adaptation to high temperatures in thermophilic organisms. We focus here on the topoisomerase I from the hyperthermophilic bacterium Thermotoga maritima (optimal growth temperature, 80 degrees C). To determine the properties of the enzyme compared with those of its mesophilic homologs, we overexpressed T. maritima topoisomerase I in Escherichia coli and purified it to near homogeneity. We show that T. maritima topoisomerase I exhibits a very high DNA relaxing activity. Mapping of the cleavage sites on a variety of single-stranded oligonucleotides indicates a strong preference for a cytosine at position -4 of the cleavage, a property shared by E. coli topoisomerase I and archaeal reverse gyrases. As expected, the mutation of the putative active site Tyr 288 to Phe led to a totally inactive protein. To investigate the role of the unique zinc motif (Cys-X-Cys-X(16)-Cys-X-Cys) present in T. maritima topoisomerase I, experiments have been performed with the protein mutated on the tetracysteine motif. Strikingly, the results show that zinc binding is not required for DNA relaxation activity, contrary to the E. coli enzyme. Furthermore, neither thermostability nor cleavage specificity is altered in this mutant. This finding opens the question of the role of the zinc-binding motif in T. maritima topoisomerase I and suggests that this hyperthermophilic topoisomerase possesses a different mechanism from its mesophilic homolog.
拓扑异构酶通过控制DNA超螺旋状态，是嗜热生物适应高温的关键酶。我们在此聚焦于嗜热细菌海栖热袍菌（最适生长温度80摄氏度）的拓扑异构酶I。为了确定该酶与其嗜温同源物相比的特性，我们在大肠杆菌中过表达了海栖热袍菌拓扑异构酶I，并将其纯化至近乎同质。我们表明，海栖热袍菌拓扑异构酶I表现出非常高的DNA松弛活性。在多种单链寡核苷酸上对切割位点进行定位表明，在切割位点的-4位强烈偏好胞嘧啶，这是大肠杆菌拓扑异构酶I和古菌反向回旋酶共有的特性。正如预期的那样，将推定的活性位点酪氨酸288突变为苯丙氨酸导致蛋白质完全无活性。为了研究海栖热袍菌拓扑异构酶I中独特的锌基序（半胱氨酸- X -半胱氨酸- X(16)-半胱氨酸- X -半胱氨酸）的作用，我们对该蛋白质的四半胱氨酸基序进行了突变实验。令人惊讶的是，结果表明与大肠杆菌酶相反，DNA松弛活性不需要锌结合。此外，该突变体的热稳定性和切割特异性均未改变。这一发现引发了关于海栖热袍菌拓扑异构酶I中锌结合基序作用的问题，并表明这种嗜热拓扑异构酶具有与其嗜温同源物不同的机制。
