The Glu-84 of the ParC subunit plays critical roles in both topoisomerase IV-quinolone and topoisomerase IV-DNA interactions
The Glu-84 of the ParC subunit plays critical roles in both topoisomerase IV-quinolone and topoisomerase IV-DNA interactions
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DOI：
10.1021/bi026352v
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发表时间：
2002-10-01
期刊：
影响因子：
2.9
通讯作者：
Hiasa, H
中科院分区：
文献类型：
--
作者：
Hiasa, H
DNA旋转酶和拓扑异构酶IV（Topo IV）是喹诺酮类抗菌药物的细胞靶点。ParC亚基的Ser-80和Glu-84已被鉴定为喹诺酮耐药的突变热点。已经构建了含有喹诺酮耐药突变的突变体Topo IV蛋白，并评估了这些突变对Topo IV的影响。S80 L和E84 K突变都消除了喹诺酮类捕获共价Topo IV-DNA复合物的能力，表明ParC的Ser-80和Glu-84对于Topo IV-喹诺酮相互作用都是必需的。此外，E84 K突变大大降低了Topo IV的催化活性。与含有E84 K突变的Topo IV形成的共价Topo IV-DNA复合物比与野生型蛋白形成的那些更稳定。有趣的是，E84 P突变赋予拓扑异构体IV喹诺酮耐药性而不影响其催化活性。E84 P突变抑制共价拓扑IV-DNA复合物的形成时，Mg 2+，而不是Ca 2+，被用作辅因子。这些结果表明Glu-84在Topo IV与DNA的相互作用中起重要作用。因此，ParC的Glu-84对于拓扑异构酶-喹诺酮-DNA三元复合物中Topo IV与喹诺酮药物和DNA的相互作用至关重要。
DNA gyrase and topoisomerase IV (Topo IV) are cellular targets of quinolone antibacterial drugs. The Ser-80 and the Glu-84 of the ParC subunit have been identified as mutational hotspots for quinolone resistance. Mutant Topo IV proteins containing a quinolone resistance-conferring mutation have been constructed, and the effects of these mutations on Topo IV are assessed. Both S80L and E84K mutations abolish the ability of quinolones to trap covalent Topo IV-DNA complexes, demonstrating that both the Ser-80 and the Glu-84 of ParC are essential for Topo IV-quinolone interaction. In addition, the E84K mutation greatly reduces the catalytic activity of Topo IV. Covalent Topo IV-DNA complexes formed with Topo IV containing the E84K mutation are more stable than those formed with the wildtype protein. Interestingly, the E84P mutation confers quinolone resistance to Topo IV without affecting its catalytic activity. The E84P mutation inhibits the formation of covalent Topo IV-DNA complexes when Mg2+, but not Ca2+, is used as a cofactor. These results show that the Glu-84 plays an important role in Topo IV-DNA interaction. Thus, the Glu-84 of ParC is critical for the interactions of Topo IV with both the quinolone drug and the DNA in topoisomerase-quinolone-DNA ternary complexes.