The DNA Gyrase-Quinolone Complex

Abstract

Quinolone binding to the gyrase-DNA complex induces a conformational change that results in the blocking of supercoiling. Under these conditions gyrase is still capable of ATP hydrolysis which now proceeds through an alternative pathway involving two different conformations of the enzyme (Kampranis, S. C., and Maxwell, A. (1998) J. Biol. Chem. 269, 22606 -22614). The kinetics of ATP hydrolysis via this pathway have been studied and found to differ from those of the reaction of the drug-free enzyme. The quinolone-characteristic ATPase rate is DNA-dependent and can be induced in the presence of DNA fragments as small as 20 base pairs. By observing the conversion of the ATPase rate to the quinolone characteristic rate, the formation and dissociation of the gyrase-DNA-quinolone complex can be monitored. Comparison of the time dependence of the conversion of the gyrase ATPase with that of DNA cleavage reveals that formation of the gyrase-DNA-quinolone complex does not correspond to the formation of cleaved DNA. Quinolone-induced DNA cleavage proceeds via a mechanism consisting of two cleavage events that is modulated in the presence of a nucleotide cofactor. We demonstrate that quinolone binding and drug-induced DNA cleavage are separate processes constituting two sequential steps in the mechanism of action of quinolones on DNA gyrase.DNA gyrase is the intracellular target of the quinolone group of antibacterial agents. The addition of quinolone drugs to an in vitro reaction containing DNA gyrase, relaxed closed-circular DNA, and ATP leads to the inhibition of supercoiling (2). If this reaction is terminated by the addition of a protein denaturant, like SDS, the DNA is found to be cleaved in both strands with a gyrase A subunit (GyrA) 1 covalently attached to each 5Ј-phosphoryl terminus (3). Quinolones also inhibit the relaxation, catenation, and decatenation reactions of gyrase (2, 4).The molecular details of the interaction of the drugs with the gyrase-DNA complex are unknown. When the binding of the drugs to the enzyme was investigated, insignificant binding of quinolones to the A or B subunit (GyrB) or to the gyrase holoenzyme (A 2 B 2 ) was detected (5-7). Binding of the drugs to the DNA is weak, and more efficient in the case of singlecompared with double-stranded DNA (8,9). Quinolones were found to bind strongly to the complex formed by gyrase and DNA, and ATP appeared to assist this interaction (5). These results led to the proposal that the drugs bind to the singlestranded DNA region that is revealed when gyrase cleaves DNA during turnover (10). This proposal was addressed by site-directed mutagenesis of the gyrase active site. Mutation of tyrosine 122 to serine or phenylalanine abolished supercoiling and quinolone-induced DNA cleavage but the mutants could still bind the drugs equally well as wild-type (11). Clearly DNA cleavage is not required for drug binding. Similar results have also been obtained with topoisomerase IV (12).Using limited proteolysis we found that quinolones stabilize a conformational ...

Conformational Changes in DNA Gyrase Revealed by Limited Proteolysis

“…4). The concentration of quinolones that is required to reveal the complex IIIcharacteristic signature is significantly higher than that required to inhibit the enzyme (40). This is probably a manifestation of the degradation of the 33-kDa domains resulting in the destabilization of the enzyme-DNA-quinolone complex.…”

“…We suggest that inhibition of supercoiling by quinolones is the consequence of a drug-induced conformational change involving the 47-kDa domains of GyrB and that DNA cleavage is a subsequent slower step. Further evidence for this proposal is presented in the accompanying paper (40).…”

“…This is probably a manifestation of the degradation of the 33-kDa domains resulting in the destabilization of the enzyme-DNA-quinolone complex. Indeed, the A64 2 B 2 -DNA-quinolone complex was found to be comparatively unstable, requiring higher concentrations of quinolone for its formation (40).…”

In vitro characterization of DNA gyrase inhibition by microcin B17 analogs with altered bisheterocyclic sites

“…These results may suggest that MccB17 can interact with gyrase at two different stages in the reaction pathway. Quinolones bind to the gyrase-DNA complex in the absence of DNA cleavage (26), and the effects of ciprofloxacin can be separated into two components, suggesting that there are two sequential steps in the mechanism of action of quinolones, binding followed by trapping of the covalent complex (27). Further experiments will be required to characterize the mechanism of action of MccB17 and the nature of the complexes formed.…”

The Action of the Bacterial Toxin Microcin B17

“…To explain the origin of the biphasic kinetics observed in the religation reactions, we propose a mechanistic scheme for the cleavage-religation reactions of gyrase. Earlier reports on gyrase and topo II suggested that both the cleavage and religation reactions occurred via a two-step process involving a nicked DNA intermediate (29,30,36). The first analysis of the kinetic curves of the cleavage and the religation reactions used a simple model of two irreversible consecutive reactions (29,36).…”