Role of the Water–Metal Ion Bridge in Mediating Interactions between Quinolones and Escherichia coli Topoisomerase IV

Abstract

Although quinolones have been in clinical use for decades, the mechanism underlying drug activity and resistance has remained elusive. However, recent studies indicate that clinically relevant quinolones interact with Bacillus anthracis (Gram-positive) topoisomerase IV through a critical water–metal ion bridge and that the most common quinolone resistance mutations decrease drug activity by disrupting this bridge. As a first step toward determining whether the water–metal ion bridge is a general mechanism of quinolone–topoisomerase interaction, we characterized drug interactions with wild-type Escherichia coli (Gram-negative) topoisomerase IV and a series of ParC enzymes with mutations (S80L, S80I, S80F, and E84K) in the predicted bridge-anchoring residues. Results strongly suggest that the water–metal ion bridge is essential for quinolone activity against E. coli topoisomerase IV. Although the bridge represents a common and critical mechanism that underlies broad-spectrum quinolone function, it appears to play different roles in B. anthracis and E. coli topoisomerase IV. The water–metal ion bridge is the most important binding contact of clinically relevant quinolones with the Gram-positive enzyme. However, it primarily acts to properly align clinically relevant quinolones with E. coli topoisomerase IV. Finally, even though ciprofloxacin is unable to increase levels of DNA cleavage mediated by several of the Ser80 and Glu84 mutant E. coli enzymes, the drug still retains the ability to inhibit the overall catalytic activity of these topoisomerase IV proteins. Inhibition parallels drug binding, suggesting that the presence of the drug in the active site is sufficient to diminish DNA relaxation rates.

Fluoroquinolone interactions with Mycobacterium tuberculosis gyrase: Enhancing drug activity against wild-type and resistant gyrase

“…Results indicate that the water-metal ion bridge is partially functional in WT gyrase and that the most common resistance mutations cause a decrease in bridge-mediated drug affinity for the enzyme. In contrast to other species (32,33), quinolone interactions within the gyrase-cleaved DNA complex depend more heavily on substituents at C7 and C8. Based on an analysis of structure-activity relationships at these two positions, we identified fluoroquinolones that display significantly improved activity against WT and resistant M. tuberculosis gyrase compared with moxifloxacin.…”

“…3, Left). The amino acid at this position plays a critical role in anchoring the water-metal ion bridge in other bacterial type II enzymes (20,21,33). Results with WT gyrase were compared with those of mutant enzymes containing a serine or a valine in place of the alanine at position 90.…”

“…It is likely that the alanine at position 90 in WT gyrase, although it does not serve as a bridge anchor, still allows D94 to anchor the bridge. However, because M. tuberculosis gyrase has only one available amino acid to anchor the bridge, fluoroquinolone interactions are weaker than seen with species that encode two amino acid bridge anchors (20,21,33). Substitution of A90 with the larger valine may cause quinolone resistance by interfering with the ability of water molecules to coordinate properly with the Mg 2+ or by impeding D94 interactions with the bridge.…”

“…In other bacterial type II topoisomerases, mutations in bridgeanchoring residues that partially disrupt the water-metal ion bridge often restrict the variety of metal ions that can be used to form the bridge (21,33). Therefore, we examined the ability of a second divalent metal ion, Mn 2+ , to support ciprofloxacin activity against WT gyrase and enzymes with mutations at position 90 (Fig.…”

“…With Bacillus anthracis topoisomerase IV, it primarily acts as a binding contact (20,21). However, with Escherichia coli topoisomerase IV, it has little effect on drug affinity and is believed to properly position the drug in a manner that promotes DNA cleavage (33). Therefore, to determine whether the water-metal ion bridge plays a major role in drug-enzyme binding in M. tuberculosis gyrase, we used a competition approach and examined the effects of ciprofloxacin on DNA cleavage induced by 10 μM 8-methyl-3′-(AM)Pdione in two enzymes with impaired bridge function (GyrA A90V and GyrA…”