title: The Glu-84 of the ParC subunit plays critical roles in both topoisomerase IV-quinolone and topoisomerase IV-DNA interactions.
abstract: 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 wild-type 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 Mg(2+), but not Ca(2+), 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.
authors.affiliation: Department of Pharmacology, University of Minnesota Medical School, Minneapolis, MN 55455, USA. hiasa001@tc.umn.edu
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Hiasa
mesh_terms: D042462:4-Quinolones; D000890:Anti-Infective Agents / Q000737:chemistry*; D002118:Calcium / Q000737:chemistry; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D004249:DNA Damage / Q000235:genetics; D027101:DNA Topoisomerase IV / Q000737:chemistry* / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry*; D024881:Drug Resistance, Bacterial; D004795:Enzyme Stability / Q000235:genetics; D018698:Glutamic Acid / Q000737:chemistry* / Q000235:genetics; D007930:Leucine / Q000235:genetics; D008239:Lysine / Q000235:genetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D021122:Protein Subunits; D012694:Serine / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D042462:4-Quinolones; D000890:Anti-Infective Agents; D004269:DNA, Bacterial; D046911:Macromolecular Substances; D021122:Protein Subunits; D018698:Glutamic Acid; D012694:Serine; D027101:DNA Topoisomerase IV; D007930:Leucine; D008274:Magnesium; D008239:Lysine; D002118:Calcium