大肠杆菌拓扑异构酶I结合重金属的特性及功能影响

Effect of heavy metal binding on the function of Escherichia coli topoisomerase I

摘要:

【背景】大肠杆菌拓扑异构酶I (Escherichia coli topoisomerase I，E. coli TopA)在DNA复制、转录、重组和基因表达调控等过程发挥关键作用。研究表明E. coli TopA只有结合锌离子才具有活性，然而E. coli TopA能否结合其他金属离子尤其是重金属离子，以及结合其他金属后是否具有活性，目前仍不清楚。【目的】探究大肠杆菌拓扑异构酶I是否结合环境中常见重金属离子，研究重金属离子结合E. coli TopA蛋白后对其活性的影响。【方法】在分别添加有锌、钴、镍、镉、铁、汞、砷、铬、铅、铜离子的M9基础培养中表达、纯化出E. coli TopA蛋白，并对纯化得到的蛋白用电感耦合等离子体质谱仪进行相应金属离子含量的测定；利用表达E. coli TopA锌指结构的突变体蛋白鉴定重金属离子的结合位点；通过体外超螺旋DNA松弛实验测定不同金属结合E. coli TopA的拓扑异构酶活性；通过测定蛋白内源性荧光推测不同金属结合E. coli TopA的空间构象差异。【结果】E. coli TopA在体内除了能结合锌和铁之外，还能够结合钴、镍、镉3种离子，但是不能结合汞、砷、铬、铅、铜离子。钴、镍、镉结合形式的E. coli TopA，每个蛋白分子最多可以结合3个相应的金属离子，他们与TopA蛋白的结合位点也是位于3个锌指结构域，而且每个锌指结构域结合1个金属离子。此外，E. coli TopA结合钴、镍、镉离子后，其DNA拓扑异构酶活性并未受到影响，可能是由于钴、镍、镉离子结合形式的E. coli TopA蛋白，其空间构象与锌结合形式相比并未发生显著变化。【结论】由于DNA拓扑异构酶在维持细胞正常生理功能中发挥关键作用，研究表明E. coli TopA的功能不会受到常见重金属的干扰(不结合或者结合后活性无影响)，这也有可能是大肠杆菌在进化过程中产生的对抗环境中重金属离子毒害作用的一种自我保护和耐受机制，具有重要的生理意义。

Abstract:

[Background] Escherichia coli topoisomerase I (E. coli TopA) plays essential role in DNA replication, transcription, recombination and regulation of gene expression. It is active only if it is bound with zinc. However, it remains unknown if it could bind other metals, especially heavy metals. [Objective] This study is aimed to characterize the binding activity of E. coli TopA to some toxic heavy metals in the environment, and to verify the potential effect of heavy metals on the topoisomerase activity of E. coli TopA. [Methods] We expressed and purified E. coli TopA from E. coli cells grown in M9 minimal media supplemented with exogenous zinc, cobalt, nickel, cadmium, iron, mercury, arsenic, chromium, lead or copper separately. The metal contents of purified TopA proteins were determined by ICP-MS (Inductively coupled plasma mass spectrometry). By using E. coli TopA mutants in which the zinc-finger motifs were disrupted, the binding sites of these metals in TopA were identified. The in vitro DNA relaxation assay was conducted to study the topoisomerase activity of E. coli TopA bound with different metals. Finally, intrinsic fluorescences of E. coli TopA bound with different metals were also measured to discover potential conformation diversity among them. [Results] We found that E. coli TopA could maximally bind three atoms of cobalt, nickel or cadmium per protein monomer respectively, while could not bind mercury, arsenic, chromium, lead or copper. The binding sites of these three metals in TopA were just located in the zinc-finger motifs, in which one atom of metal iron was coordinated with each zinc-finger motif respectively. It also showed that the DNA topoisomerase activity of TopA remained active when bound with cobalt, nickel or cadmium. Moreover, intrinsic fluorescence measurement showed that the protein conformation of cobalt, nickel or cadmium bound TopA would be similar as that of zinc bound. [Conclusion] In light of the crucial role of DNA topoisomerase in maintenance of cell viability, these results indicate that the topoisomerase activity of TopA would not be disrupted by common heavy metals (no binding or binding without inhibition of enzyme activity), which could be considered as an evolutionary mechanism for E. coli in resistance to toxicity of heavy metals in environment.