Nicholls Robert A, Morgan Harry, Warren Anna J, Ward Simon E, Long Fei, Murshudov Garib N, Sutormin Dmitry, Bax Benjamin D
Scientific Computing Department, UKRI Science and Technology Facilities Council, Harwell Campus, Didcot, Oxfordshire OX11 0DE, UK.
Medicines Discovery Institute, Cardiff University, Cardiff CF10 3AT, UK.
Int J Mol Sci. 2024 Dec 24;26(1):33. doi: 10.3390/ijms26010033.
DNA gyrase is a bacterial type IIA topoisomerase that can create temporary double-stranded DNA breaks to regulate DNA topology and an archetypical target of antibiotics. The widely used quinolone class of drugs use a water-metal ion bridge in interacting with the GyrA subunit of DNA gyrase. Zoliflodacin sits in the same pocket as quinolones but interacts with the GyrB subunit and also stabilizes lethal double-stranded DNA breaks. Gepotidacin has been observed to sit on the twofold axis of the complex, midway between the two four-base-pair separated DNA-cleavage sites and has been observed to stabilize singe-stranded DNA breaks. Here, we use information from three crystal structures of complexes of DNA gyrase (one with a precursor of gepotidacin and one with the progenitor of zoliflodacin) to propose a simple single moving metal-ion-catalyzed DNA-cleavage mechanism. Our model explains why the catalytic tyrosine is in the tyrosinate (negatively charged) form for DNA cleavage. Movement of a single catalytic metal-ion (Mg or Mn) guides water-mediated protonation and cleavage of the scissile phosphate, which is then accepted by the catalytic tyrosinate. Type IIA topoisomerases need to be able to rapidly cut the DNA when it becomes positively supercoiled (in front of replication forks and transcription bubbles) and we propose that the original purpose of the small Greek Key domain, common to all type IIA topoisomerases, was to allow access of the catalytic metal to the DNA-cleavage site. Although the proposed mechanism is consistent with published data, it is not proven and other mechanisms have been proposed. Finally, how such mechanisms can be experimentally distinguished is considered.
DNA促旋酶是一种细菌IIA型拓扑异构酶，可产生临时双链DNA断裂以调节DNA拓扑结构，也是抗生素的典型作用靶点。广泛使用的喹诺酮类药物通过水-金属离子桥与DNA促旋酶的GyrA亚基相互作用。佐利氟达辛与喹诺酮类药物位于同一口袋中，但与GyrB亚基相互作用，还能稳定致死性双链DNA断裂。已观察到吉波达辛位于复合物的二重轴上，在两个相隔四个碱基对的DNA切割位点之间的中间位置，并且已观察到它能稳定单链DNA断裂。在此，我们利用来自DNA促旋酶复合物的三种晶体结构的信息（一种与吉波达辛的前体形成复合物，一种与佐利氟达辛的祖代形成复合物）提出一种简单的单移动金属离子催化的DNA切割机制。我们的模型解释了为什么催化酪氨酸在DNA切割时处于酪氨酸盐（带负电荷）形式。单个催化金属离子（镁或锰）的移动引导水介导的质子化和可切割磷酸酯的切割，然后被催化酪氨酸盐接受。IIA型拓扑异构酶需要能够在DNA变为正超螺旋时（在复制叉和转录泡前方）快速切割DNA，我们提出所有IIA型拓扑异构酶共有的小希腊钥匙结构域的最初目的是允许催化金属进入DNA切割位点。尽管所提出的机制与已发表的数据一致，但尚未得到证实，也有人提出了其他机制。最后，考虑了如何通过实验区分这些机制。
