Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India.
Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India.
Mol Microbiol. 2023 Jun;119(6):728-738. doi: 10.1111/mmi.15068. Epub 2023 May 15.
DNA gyrase, the sole negative supercoiling type II topoisomerase, is composed of two subunits, GyrA and GyrB, encoded by the gyrA and gyrB genes, respectively, that form a quaternary complex of A B . In this study, we have investigated the assembly of mycobacterial DNA gyrase from its individual subunits, a step prerequisite for its activity. Using analytical size-exclusion chromatography, we show that GyrA from Mycobacterium tuberculosis and Mycobacterium smegmatis forms tetramers (A ) in solution unlike in Escherichia coli and other bacteria where GyrA exists as a dimer. GyrB, however, persists as a monomer, resembling the pattern found in E. coli. GyrB in both mycobacterial species interacts with GyrA and triggers the dissociation of the GyrA tetramer to facilitate the formation of catalytically active A B . Despite oligomerisation, the GyrA tetramer retained its DNA binding ability, and DNA binding had no effect on GyrA's oligomeric state in both species. Moreover, the presence of DNA facilitated the assembly of holoenzyme in the case of M. smegmatis by stabilising the GyrA B tetramer but with little effect in M. tuberculosis. Thus, in addition to the distinct organisation and regulation of the gyr locus in mycobacteria, the enzyme assembly also follows a different pattern.
DNA 回旋酶是唯一的负超螺旋 II 型拓扑异构酶，由两个亚基 GyrA 和 GyrB 组成，分别由 gyrA 和 gyrB 基因编码，形成 A B 的四聚体。在这项研究中，我们研究了分枝杆菌 DNA 回旋酶从其单个亚基组装，这是其活性的前提步骤。使用分析大小排阻色谱法，我们表明结核分枝杆菌和耻垢分枝杆菌的 GyrA 在溶液中形成四聚体（A ），而不是像大肠埃希菌和其他细菌那样，GyrA 作为二聚体存在。然而，GyrB 仍然是单体，类似于在大肠埃希菌中发现的模式。两种分枝杆菌物种的 GyrB 与 GyrA 相互作用，并触发 GyrA 四聚体的解离，以促进催化活性 A B 的形成。尽管发生了寡聚化，但 GyrA 四聚体保留了其 DNA 结合能力，并且 DNA 结合对两种物种中 GyrA 的寡聚状态没有影响。此外，DNA 的存在通过稳定 GyrA B 四聚体促进了 holoenzyme 在耻垢分枝杆菌中的组装，但在结核分枝杆菌中几乎没有影响。因此，除了分枝杆菌中 gyr 基因座的独特组织和调节外，酶的组装也遵循不同的模式。
