Faculty Scholarship

 DNA gyrase and topoisomerase IV control bacterial DNA topology by breaking DNA, passing duplex DNA through the break, and then resealing the break. This process is subject to reversible corruption by fluoroquinolones, antibacterials that form drug-enzyme-DNA complexes in which the DNA is broken. The complexes, called cleaved complexes because of the presence of DNA breaks, have been crystallized and found to have thefluoroquinolone C-7ring systemfacing the GyrB/ParE subunits. As expected from x-ray crystallography, a thiol-reactive, C-7-modifiedchloroacetylderivativeofciprofloxacin(CipAcCl) formed cross-linked cleaved complexes with mutant GyrB-Cys466 gyrase as evidenced by resistance to reversal by both EDTAandthermaltreatments.Surprisingly,cross-linking was also readily seen with complexes formed by mutant GyrAG81Cgyrase, thereby revealing a novel drug-gyrase interaction not observed in crystal structures. The cross-link between fluoroquinolone and GyrA-G81C gyrase correlated with exceptional bacteriostatic activity for Cip-AcCl with a quinoloneresistant GyrA-G81C variant of Escherichia coli and its Mycobacteriumsmegmatisequivalent(GyrA-G89C).Cip-AcClmediated, irreversible inhibition of DNA replication provided further evidence for a GyrA-drug cross-link. Collectively these data establish the existence of interactions between the fluoroquinoloneC-7ringandbothGyrAandGyrB.BecausetheGyrAGly81andGyrB-Glu466residuesarefarapart(17Å)inthecrystal structureofcleavedcomplexes,twomodesofquinolonebinding mustexist. The presence of two binding modesraises the possibility that multiple quinolone-enzyme-DNA complexes can form, a discovery that opens new avenues for exploring and exploiting relationships between drug structure and activity with type II DNAtopoisomerases.