Funatsuki K, Tanaka R, Inagaki S, Konno H, Katoh K, Nakamura H
Aburahi Laboratories, Shionogi and Company, Ltd., Koka, Shiga 520-34, Japan.
J Biol Chem. 1997 May 16;272(20):13302-8. doi: 10.1074/jbc.272.20.13302.
Mutations that exhibit susceptibility to acriflavine have been isolated and classified as acr mutations in Escherichia coli. We cloned the acrB gene, which has been identified as a mutation of the gyrB gene, and found a double point mutation altering two consecutive amino acids (S759R/R760C) in the COOH-terminal region of the gyrase B subunit. The mutant B subunit was found to associate with the A subunit to make the quaternary structure, and the reconstituted gyrase showed an 80-fold reduction of specific activity in DNA supercoiling assay; the sensitivity to acriflavine was not different in the same unit of wild-type and mutant gyrases. The mutant enzyme retained intrinsic ATPase activity, but DNA-dependent stimulation was observed infrequently. A gel shift assay showed that acriflavine inhibited the DNA binding of gyrase. The acrB mutation also reduced significantly the DNA binding of gyrase but did not change the sensitivity to acriflavine. These results revealed that the acrB mutation is related to the inhibitory mechanism of acriflavine; and the acriflavine sensitivity of the mutant, at least in vitro, is caused mainly by reduction of the enzyme activity. Further, our findings suggest that the COOH-terminal region of the B subunit is essential for the initial binding of gyrase to the substrate DNA.
在大肠杆菌中，已分离出对吖啶黄敏感的突变，并将其归类为acr突变。我们克隆了已被鉴定为gyrB基因突变的acrB基因，发现在促旋酶B亚基的COOH末端区域有一个双点突变，改变了两个连续的氨基酸（S759R/R760C）。发现突变的B亚基与A亚基结合形成四级结构，并且重组促旋酶在DNA超螺旋测定中显示比活性降低了80倍；在相同单位的野生型和突变型促旋酶中，对吖啶黄的敏感性没有差异。突变酶保留了内在的ATP酶活性，但很少观察到DNA依赖性刺激。凝胶迁移试验表明，吖啶黄抑制促旋酶与DNA的结合。acrB突变也显著降低了促旋酶与DNA的结合，但没有改变对吖啶黄的敏感性。这些结果表明，acrB突变与吖啶黄的抑制机制有关；突变体对吖啶黄的敏感性，至少在体外，主要是由酶活性降低引起的。此外，我们的研究结果表明，B亚基的COOH末端区域对于促旋酶与底物DNA的初始结合至关重要。
