Nakada N, Gmünder H, Hirata T, Arisawa M
Nippon Roche Research Center, Kamakura, Japan.
J Biol Chem. 1995 Jun 16;270(24):14286-91. doi: 10.1074/jbc.270.24.14286.
The mechanism of inhibition of DNA gyrase by cyclothialidine, a novel gyrase inhibitor isolated from Streptomyces filipinensis NR0484, has been studied further by using [14C]benzoylcyclothialidine and a reconstituted Escherichia coli gyrase system consisting of the A subunit, the B subunit and relaxed ColE1 DNA. The mechanism of inhibition was also studied with the 43-kDa N-terminal fragment of the B subunit. The [14C]benzoylcyclothialidine could bind to the B subunit alone but not to the A subunit nor to the plasmid DNA alone. Furthermore, the compound also bound to the 43-kDa N-terminal fragment of the B subunit. Scatchard analysis of [14C]benzoylcyclothialidine binding to DNA gyrase showed that the binding affinity of the compound increased, depending on the assembly of the gyrase (A2B2). DNA complex. This suggests that the binding site of cyclothialidine on the B subunit or its vicinity causes a conformational change during the assembly of the gyrase.DNA complex (increase in affinity: B-->A2B2-->A2B2.DNA). Furthermore, displacement curves of [14C]benzoylcyclothialidine binding by nonlabeled cyclothialidine, ATP analogues, and coumarin antibiotics indicated that cyclothialidine, coumarins, and ATP share a common (or overlapping) site of action on the B subunit of DNA gyrase; however, the microenvironment of the binding sites may differ.
从菲律宾链霉菌NR0484中分离得到的新型DNA促旋酶抑制剂环硫噻啶对DNA促旋酶的抑制机制，已通过使用[14C]苯甲酰环硫噻啶以及由A亚基、B亚基和松弛型ColE1 DNA组成的重组大肠杆菌促旋酶系统进行了进一步研究。还使用B亚基的43 kDa N端片段研究了抑制机制。[14C]苯甲酰环硫噻啶可单独与B亚基结合，但不与A亚基或单独的质粒DNA结合。此外，该化合物还与B亚基的43 kDa N端片段结合。对[14C]苯甲酰环硫噻啶与DNA促旋酶结合的Scatchard分析表明，该化合物的结合亲和力增加，这取决于促旋酶（A2B2）.DNA复合物的组装情况。这表明环硫噻啶在B亚基上或其附近的结合位点在促旋酶.DNA复合物组装过程中引起了构象变化（亲和力增加：B→A2B2→A2B2.DNA）。此外，未标记的环硫噻啶、ATP类似物和香豆素类抗生素对[14C]苯甲酰环硫噻啶结合的置换曲线表明，环硫噻啶、香豆素和ATP在DNA促旋酶B亚基上有共同（或重叠）的作用位点；然而，结合位点的微环境可能不同。
