Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India.
Arch Biochem Biophys. 2012 Dec 15;528(2):197-203. doi: 10.1016/j.abb.2012.10.004. Epub 2012 Oct 17.
Type I DNA topoisomerases from bacteria catalyse relaxation of negatively supercoiled DNA in a Mg(2+) dependent manner. Although topoisomerases of distinct classes have been subjected for anti-cancer and anti-infective drug development, bacterial type I enzymes are way behind in this regard. Our studies with Mycobacterium smegmatis topoisomerase I (MstopoI) revealed several of its distinct properties compared to the well studied Escherichia coli topoisomerase I (EctopoI) suggesting the possibility of targeting the mycobacterial enzyme for inhibitor development. Here, we describe Mycobacterium tuberculosis topoisomerase I (MttopoI) and compare its properties with MstopoI and EctopoI. The enzyme cleaves DNA at preferred sites in a pattern similar to its ortholog from M. smegmatis. Oligonucleotides containing the specific recognition sequence inhibited the activity of the enzyme in a manner similar to that of MstopoI. Substitution of the acidic residues, D111 and E115 which are involved in Mg(2+) co-ordination, to alanines affected the DNA relaxation activity. Unlike the wild type enzyme, D111A was dependent on Mg(2+) for DNA cleavage and both the mutants were compromised in religation. The monoclonal antibody (mAb), 2F3G4, developed against MstopoI inhibited the relaxation activity of MttopoI. These studies affirm the characteristics of MttopoI to be similar to MstopoI and set a stage to target it for the development of specific small molecule inhibitors.
I 型 DNA 拓扑异构酶可依赖于 Mg2+催化负超螺旋 DNA 的松弛。尽管不同类型的拓扑异构酶已被用于抗癌和抗感染药物的开发，但 I 型细菌酶在这方面还远远落后。我们对耻垢分枝杆菌拓扑异构酶 I（MstopoI）的研究揭示了其与广泛研究的大肠杆菌拓扑异构酶 I（EctopoI）相比具有许多独特的特性，这表明靶向结核分枝杆菌酶进行抑制剂开发是有可能的。在这里，我们描述了结核分枝杆菌拓扑异构酶 I（MttopoI），并将其与 MstopoI 和 EctopoI 进行了比较。该酶在与 M. smegmatis 同源物相似的模式下在优选的位点切割 DNA。含有特定识别序列的寡核苷酸以类似于 MstopoI 的方式抑制酶的活性。酸性残基 D111 和 E115 的取代，这些残基参与 Mg2+配位，会影响 DNA 松弛活性。与野生型酶不同，D111A 依赖于 Mg2+进行 DNA 切割，并且两个突变体在重连接中都受到影响。针对 MstopoI 开发的单克隆抗体（mAb）2F3G4 抑制了 MttopoI 的松弛活性。这些研究证实了 MttopoI 的特性与 MstopoI 相似，并为针对其开发特异性小分子抑制剂奠定了基础。
