Increased sensitivity to quinolone antibacterials can be engineered in human topoisomerase IIα by selective mutagenesis
Increased sensitivity to quinolone antibacterials can be engineered in human topoisomerase IIα by selective mutagenesis
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DOI：
10.1006/jmbi.2000.3892
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发表时间：
2000-07-14
影响因子：
5.6
通讯作者：
Maxwell, A
中科院分区：
文献类型：
--
作者：
Hammonds, TR;Foster, SR;Maxwell, A
在DNA回转酶的A亚基中，药物-DNA相互作用的潜在区域已经从晶体学研究中确定。局部氨基酸序列已与酵母拓扑异构酶II和人类拓扑异构酶II α的相似区域进行了比较。人类拓扑异构酶II α中位于762、763和766位的三个非保守的、可能被溶剂接近的残基位于保守区域之间。GyrA中相应的残基（83、84和87）在喹诺酮耐药菌中具有较高的突变频率。在试图设计环丙沙星对这种酶的敏感性时，已经产生了人类拓扑异构酶II α的突变：M762S， S763A和M766D（每个突变为存在于gyrase中的相同氨基酸），以及M762S/S763A双突变体和三突变体。这些酶被引入对温度敏感的酵母菌株中，缺乏拓扑异构酶II，用于体内研究，并且在体外研究中过量生产。M766D突变使酶在不允许的温度下不能支持温度敏感菌株。然而，M766D和三突变体酶都可以过量产生，并且在体外具有充分的活性。双突变体切割DNA的能力受损，催化活性降低。三重突变使体外对环丙沙星的敏感性增加三倍，对一系列其他喹诺酮类药物的敏感性相似。喹诺酮类药物CP-115,953是一种细菌和真核生物拓扑异构酶II毒药，其活性不受这些突变的影响。发现该区域的突变增加了酶对插入DNA的抗肿瘤药物m-AMSA和ellipticine的敏感性，但赋予了对非插入药物etopo苷、tenipo苷和merbarone的抗性，这种作用在三突变体中最大。因此，我们已经证明了该区域在确定拓扑异构酶II对药物的敏感性方面的重要性，并设计了对喹诺酮类药物的敏感性增加。(C) 2000年学术出版社。
A potential region of drug-DNA interaction in the A subunit of DNA gyrase has previously been identified from crystallographic studies. The local amino acid sequence has been compared with similar regions in yeast topoisomerase II and human topoisomerase II alpha. Three nonconserved, potentially solvent-accessible residues at positions 762, 763 and 766 in human topoisomerase II alpha lie between well-conserved regions. The corresponding residues in GyrA (83, 84 and 87) have a high frequency of mutation in quinolone-resistant bacteria. Mutations in human topoisomerase II alpha have been generated in an attempt to engineer ciprofloxacin sensitivity into this enzyme: M762S, S763A and M766D (each mutated to the identical amino acid present in gyrase), along with an M762S/S763A double mutant and a triple mutant. These enzymes were introduced into a temperature-sensitive yeast strain, deficient in topoisomerase II, for in vivo studies, and were overproduced for in vitro studies. The M766D mutation renders the enzyme incapable of supporting the temperature-sensitive strain at a non-permissive temperature. However, both M766D and the triple mutant enzymes can be overproduced and are fully active in vitro. The double mutant was impaired in its ability to cleave DNA and had reduced catalytic activity. The triple mutation confers a three-fold increase in sensitivity to ciprofloxacin in vitro and similar sensitivities to a range of other quinolones. The activity of the quinolone CP-115,953, a bacterial and eukaryotic topoisomerase II poison, was unaffected by any of these mutations. Mutations in this region were found to increase the sensitivity of the enzyme to the DNA intercalating anti-tumour agents m-AMSA and ellipticine, but confer resistance to the non-intercalating agents etoposide, teniposide and merbarone, an effect that was maximal in the triple mutant. We have therefore shown the importance of this region in determining the sensitivity of topoisomerase II to drugs and have engineered increased sensitivity to quinolones. (C) 2000 Academic Press.