Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.
Nucleic Acids Res. 2012 Oct;40(19):9774-87. doi: 10.1093/nar/gks704. Epub 2012 Jul 27.
DNA gyrase is the only type II topoisomerase in Mycobacterium tuberculosis and needs to catalyse DNA supercoiling, relaxation and decatenation reactions in order to fulfil the functions normally carried out by gyrase and DNA topoisomerase IV in other bacteria. We have obtained evidence for the existence of a Ca(2+)-binding site in the GyrA subunit of M. tuberculosis gyrase. Ca(2+) cannot support topoisomerase reactions in the absence of Mg(2+), but partial removal of Ca(2+) from GyrA by dialysis against EGTA leads to a modest loss in relaxation activity that can be restored by adding back Ca(2+). More extensive removal of Ca(2+) by denaturation of GyrA and dialysis against EGTA results in an enzyme with greatly reduced enzyme activities. Mutation of the proposed Ca(2+)-binding residues also leads to loss of activity. We propose that Ca(2+) has a regulatory role in M. tuberculosis gyrase and suggest a model for the modulation of gyrase activity by Ca(2+) binding.
DNA 回旋酶是结核分枝杆菌中唯一的 II 型拓扑异构酶，需要催化 DNA 的超螺旋化、松弛和解链反应，以完成通常由其他细菌中的回旋酶和 DNA 拓扑异构酶 IV 执行的功能。我们已经获得了结核分枝杆菌回旋酶 GyrA 亚基中存在 Ca(2+)结合位点的证据。在没有 Mg(2+)的情况下，Ca(2+)不能支持拓扑异构酶反应，但通过用 EGTA 透析部分去除 GyrA 中的 Ca(2+)会导致松弛活性适度丧失，可通过添加 Ca(2+)来恢复。通过变性 GyrA 和用 EGTA 透析更彻底地去除 Ca(2+)会导致酶活性大大降低的酶。突变拟议的 Ca(2+)结合残基也会导致活性丧失。我们提出 Ca(2+)在结核分枝杆菌回旋酶中具有调节作用，并提出了 Ca(2+)结合调节回旋酶活性的模型。
