Topoisomerase IV Catalysis and the Mechanism of Quinolone Action

Abstract

Topoisomerase IV is a bacterial type II topoisomerase that is essential for proper chromosome segregation and is a target for quinolone-based antimicrobial agents. Despite the importance of this enzyme to the survival of prokaryotic cells and to the treatment of bacterial infections, relatively little is known about the details of its catalytic mechanism or the basis by which quinolones alter its enzymatic functions. Therefore, a series of experiments that analyzed individual steps of the topoisomerase IV catalytic cycle were undertaken to address these critical mechanistic issues. The following conclusions were drawn. First, equilibrium levels of DNA cleavage mediated by the bacterial enzyme were considerably (>10-fold) higher than those observed with its eukaryotic counterparts. To a large extent, this reflected decreased rates of DNA religation. Second, the preference of topoisomerase IV for catalyzing DNA decatenation over relaxation reflects increased rates of strand passage and enzyme recycling rather than a heightened recognition of intermolecular DNA helices. Third, quinolones stimulate topoisomerase IV-mediated DNA cleavage both by increasing rates of DNA scission and by inhibiting religation of cleaved DNA. Finally, quinolones inhibit the overall catalytic activity of topoisomerase IV primarily by interfering with enzyme-ATP interactions.Type II topoisomerases alter the topological state of the genetic material by passing an intact double helix through a transient double-stranded break that they generate in a separate DNA segment (1-6). Eubacteria contain two distinct members of this enzyme class, DNA gyrase and topoisomerase IV (6). The most well characterized of the two, DNA gyrase, was discovered over 2 decades ago (7). It is unique among type II enzymes in that it is the only known topoisomerase (prokaryotic or eukaryotic) that is capable of introducing negative superhelical twists into relaxed DNA molecules (7). DNA gyrase plays critical roles in DNA replication, recombination, and transcription, as well as in the maintenance of genomic superhelical density (1,2,5,6,8,9).The second prokaryotic type II enzyme, topoisomerase IV, is comprised of the products of the parC and parE genes (10). Although it was long known that ParC and ParE both were required for proper chromosome segregation in Escherichia coli (11, 12), it was not realized until 1990 that these two polypeptides together constitute a novel type II topoisomerase (10). Consistent with its critical role in chromosome segregation, topoisomerase IV displays a prejudice for catalyzing intermolecular DNA strand passage events (i.e. catenation/decatenation reactions) as opposed to intramolecular events (i.e. DNA relaxation reactions) (13-16). This prejudice notwithstanding, topoisomerase IV appears to relax DNA in vivo, and recent evidence suggests that this relaxation activity plays an important role in maintaining levels of DNA supercoiling in E. coli (17).Beyond their essential physiological functions, prokaryotic type II topoisomerases are t...

Topoisomerase IV, alone, unknots DNA in E. coli

“…A type-2 topoisomerase will remove a catenane before it removes a supercoil node, even when supercoil nodes are in vast excess (Roca and Wang 1994;Ullsperger and Cozzarelli 1996). In addition, these enzymes will hydrolyze ATP more efficiently in the presence of a catenane node than a supercoil node (Anderson et al 1998). The type-2 topoisomerases that have been tested preferentially unknot rather than knot DNA, which also indicates that these enzymes can recognize the global topology of DNA (Rybenkov et al 1997a).…”

Quinolone Resistance Due to Reduced Target Enzyme Expression

“…The lethal activity of quinolones is thought to result from the release of lethal, double-stranded breaks in DNA, an event that appears to require an additional poorly defined cellular function after formation of the ternary complex (9,16,34). Thus, quinolones convert type II topo-isomerases to potent poisons which form DNA lesions that create double-stranded breaks in the genome (2,29,31).…”

The Action of the Bacterial Toxin Microcin B17

“…As regards religation, CFX has an effect comparable with that in the cleavage reaction, although in this case largely KЈ 2 is affected. The action of CFX on the gyrase cleavage-religation equilibrium resembles that observed on topo IV, where the rate of DNA cleavage is increased and the religation of cleaved DNA inhibited (40), but differs from the eukaryotic topo II, where quinolones enhance the forward rate of cleavage with little effect on religation (41,42). The lack of effect of OXO on religation could be attributable to the reaction conditions, e.g.…”