Hyperthermophilic topoisomerase I from Thermotoga maritima.: A very efficient enzyme that functions independently of zinc binding.
Hyperthermophilic topoisomerase I from Thermotoga maritima.: A very efficient enzyme that functions independently of zinc binding.
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DOI：
10.1074/jbc.m107714200
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发表时间：
2001-12-07
影响因子：
4.8
通讯作者：
de la Tour, CB
中科院分区：
文献类型：
--
作者：
Viard, T;Lamour, V;de la Tour, CB
拓扑异构酶是嗜热生物适应高温的关键酶，通过控制DNA超螺旋状态而发挥作用。我们在这里集中在拓扑异构酶I从极端嗜热菌海栖热袍菌（最佳生长温度，80 degreesC）。为了确定该酶与其嗜温同系物的性质相比，我们过表达T。海洋拓扑异构酶I的大肠杆菌，并纯化它接近同质。我们证明了T. maritima拓扑异构酶I表现出非常高的DNA松弛活性。对各种单链寡核苷酸上的切割位点的作图表明，在切割的位置-4处强烈偏好胞嘧啶，这是E.大肠杆菌拓扑异构酶I和古细菌反向旋转酶。正如预期的那样，推定的活性位点Tyr 288突变为Phe导致完全失活的蛋白质。目的探讨T.在maritima拓扑异构酶I中，已经用在四半胱氨酸基序上突变的蛋白质进行了实验。引人注目的是，结果表明锌结合不是DNA松弛活性所必需的，与E.大肠杆菌酶。此外，在该突变体中，热稳定性和切割特异性均未改变。这一发现揭示了锌结合基序在T。maritima拓扑异构酶I，并建议这种超嗜热拓扑异构酶具有不同的机制，从它的嗜温同系物。
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 degreesC). 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.