Topoisomerase IV-quinolone interactions are mediated through a water-metal ion bridge: mechanistic basis of quinolone resistance

Abstract

Although quinolones are the most commonly prescribed antibacterials, their use is threatened by an increasing prevalence of resistance. The most common causes of quinolone resistance are mutations of a specific serine or acidic residue in the A subunit of gyrase or topoisomerase IV. These amino acids are proposed to serve as a critical enzyme-quinolone interaction site by anchoring a water-metal ion bridge that coordinates drug binding. To probe the role of the proposed water-metal ion bridge, we characterized wild-type, GrlAE85K, GrlAS81F/E85K, GrlAE85A, GrlAS81F/E85A and GrlAS81F Bacillus anthracis topoisomerase IV, their sensitivity to quinolones and related drugs and their use of metal ions. Mutations increased the Mg2+ concentration required to produce maximal quinolone-induced DNA cleavage and restricted the divalent metal ions that could support quinolone activity. Individual mutation of Ser81 or Glu85 partially disrupted bridge function, whereas simultaneous mutation of both residues abrogated protein–quinolone interactions. Results provide functional evidence for the existence of the water-metal ion bridge, confirm that the serine and glutamic acid residues anchor the bridge, demonstrate that the bridge is the primary conduit for interactions between clinically relevant quinolones and topoisomerase IV and provide a likely mechanism for the most common causes of quinolone resistance.

Fluoroquinolone-Gyrase-DNA Complexes

“…In the binding mode revealed by crystal structures (Fig. 1B), the carboxyl end of fluoroquinolones forms a stabilizing magnesium-water bridge with GyrA residues 83 and 87 (17,18). This magnesium-mediated binding contributes to drug activity, and the loss of the interaction correlates with GyrA-mediated quinolone resistance (17,18).…”

“…1B). The quinolone 3-carboxyl, along with an aspartic/glutamic acid and a serine in helix-IV of GyrA (ParC), participates in a magnesium-water bridge that stabilizes the drug-enzyme-DNA complex (17,18). Although existing crystal structures explain many aspects of quinolone action, it is not clear why a spontaneous GyrA resis-tance substitution in Mycobacterium smegmatis (GyrA-Cys 89 ) preferentially restricts the bacteriostatic action of fluoroquinolones that have bulky substitutions at the distal end of the fluoroquinolone C-7 piperazinyl ring (19): this region of the drug should be far from the GyrA-Cys 89 residue in the cleaved complex.…”

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

“…Recent structural (19) and functional (20,21) studies with topoisomerase IV indicate that quinolones interact with bacterial type II enzymes primarily through a water-metal ion bridge. This bridge is formed by a divalent metal ion that is chelated by the C3/C4 keto acid of the drug and stabilized by four water molecules (19).…”

“…Amino acid residues A90 and D94 occur at the positions that, by sequence homology, are predicted to anchor the water-metal ion bridge if it is used to mediate quinolone-enzyme interactions in this species (19)(20)(21). The GyrA A90V , GyrA…”

“…In several other species, quinazolinediones containing a 3′-(aminomethyl) pyrrolidinyl [3′-(AM)P] (or a related) group at C7 maintain activity against enzymes containing mutations that disrupt the water-metal ion bridge by forming binding interactions primarily through the C7 substituent (20,21,32,(38)(39)(40)(41)(42). As seen in Fig.…”