Quinolone-Binding Pocket of DNA Gyrase: Role of GyrB

Abstract

DNA gyrase is a prokaryotic type II topoisomerase and a major target of quinolone antibacterials. The majority of mutations conferring resistance to quinolones arise within the quinolone resistance-determining region of GyrA close to the active site (Tyr 122 ) where DNA is bound and cleaved. However, some quinolone resistance mutations are known to exist in GyrB. Present structural data suggest that these residues lie a considerable distance from the quinolone resistance-determining region, and it is not obvious how they affect quinolone action. We have made and purified two such mutant proteins, GyrB(Asp 426 3Asn) and GyrB(Lys 447 3Glu), and characterized them in vitro. We found that the two proteins behave similarly to GyrA quinolone-resistant proteins. We showed that the mutations exert their effect by decreasing the amount of quinolone bound to a gyrase-DNA complex. We suggest that the GyrB residues form part of a quinolonebinding pocket that includes DNA and the quinolone resistance-determining region in GyrA and that large conformational changes during the catalytic cycle of the enzyme allow these regions to come into close proximity.Quinolone drugs are a large and widely used class of synthetic antibacterial compounds (10,11,19). First-generation (acidic) quinolones include nalidixic acid and oxolinic acid (OXO). Subsequent generations have been modified to increase spectrum and potency. The most significant modification has been the addition of a fluorine atom at position C-6 in drugs such as ciprofloxacin (CFX), which results in a considerable increase in activity (30). The newer drugs also commonly contain a secondary amine in addition to the carboxylic acid group common to most quinolones, making them amphoteric rather than acidic (Fig. 1A). More recent modifications that increase drug potency include the presence of a methoxy group at C-8 (48).In Escherichia coli the major target for the quinolones is DNA gyrase (14,35). DNA gyrase is a type II topoisomerase that is able to alter the topological state of DNA by cleaving both strands, passing a double strand of DNA through the gap and resealing the ends (6). In the presence of ATP, this results in negative supercoiling, a reaction unique to gyrase. The crystal structures of the 43-kDa N-terminal domain of GyrB (responsible for ATP hydrolysis and capturing a DNA strand) and a 59-kDa fragment of the 64-kDa N-terminal domain of GyrA (containing the residues for DNA binding and cleavage and the quinolone resistance-determining region [QRDR; residues 67 to 106]) have been determined (24, 39). The structure of the 47-kDa C-terminal domain of GyrB is not known but can be inferred from the analogous enzyme from Saccharomyces cerevisiae, topoisomerase II, for which the crystal structures of a 92-kDa region have been solved (5, 12).Resistance to quinolones commonly arises in DNA gyrase via mutations in the QRDR (44). Such mutations include GyrA(Ser 83 3Trp), which gives Ϸ20-fold resistance to a wide range of quinolones (9, 46). This region of GyrA is close to t...

Synthesis and potential antitumor activity of 7-(4-substituted piperazin-1-yl)-4-oxoquinolines based on ciprofloxacin and norfloxacin scaffolds: in silico studies

“…The antibacterial fluoroquinolones have been found to be one of the fastest growing groups of drugs in recent years [5][6][7][8] . Quinolones are known for their antibacterial and antitumor activities through alteration of the normal functions of bacterial gyrase, and are found to be a topoisomerase II inhibitor in humans [9][10][11][12][13][14][15][16][17][18] . Ciprofloxacin (Figure 1), a commonly used broadspectrum fluoroquinolone antibiotic, has shown anticancer activity in several cancer cell lines 19,20 .…”

Mutation at the “Exit Gate” of the Salmonella Gyrase A Subunit Suppresses a Defect in the Gyrase B Subunit

“…In addition to their effect on quinolone sensitivity, mutations at sites D426 and K447 also inhibit the gyrase catalytic activity (13). It has been proposed that the quinolone-binding site is a pocket surrounded by surfaces involving the quinolone resistance-determining regions of both the GyrA and GyrB proteins (13,27).Isolation of the gyrB651 allele and its phenotypic suppressor, gyrA659. Mutants of Salmonella enterica serovar Typhimurium resistant to low levels of nalidixic acid were isolated by plating 0.2 ml of an overnight liquid culture from strain MA251 (Table 1) on nutrient broth (Difco) agar containing 3 g/ml Nal.…”

Studying the interaction between gyrase and DNA using magnetic tweezers

“…According with our results, biochemistry research [32] indicated that, norfloxacin almost did not bind with gyrase or DNA when only one of them existed; but a large number of norfloxacin was bound if both existed. Moreover, biochemistry research [33] and crystal structure [34] showed that norfloxacin together with DNA and gyrase formed a stable complex where norfloxacin bound near active site of GyrA Tyr122 and interact with GyrB and DNA. Besides, gyrase could change the geometric scale of DNA supercoils, yielding chiral differences, clearly indicating that it has different effects on positive and negative supercoils.…”