Type II topoisomerases alter DNA topology by forming a covalent DNA-cleavage complex that allows DNA transport through a double-stranded DNA break. We present the structures of cleavage complexes formed by the Streptococcus pneumoniae ParC breakage-reunion and ParE TOPRIM domains of topoisomerase IV stabilized by moxifloxacin and clinafloxacin, two antipneumococcal fluoroquinolones. These structures reveal two drug molecules intercalated at the highly bent DNA gate and help explain antibacterial quinolone action and resistance.
Type II DNA topoisomerases are ubiquitous enzymes with essential functions in DNA replication, recombination and transcription. They change DNA topology by forming a transient covalent cleavage complex with a gate-DNA duplex that allows transport of a second duplex though the gate. Despite its biological importance and targeting by anticancer and antibacterial drugs, cleavage complex formation and reversal is not understood for any type II enzyme. To address the mechanism, we have used X-ray crystallography to study sequential states in the formation and reversal of a DNA cleavage complex by topoisomerase IV from Streptococcus pneumoniae, the bacterial type II enzyme involved in chromosome segregation. A high resolution structure of the complex captured by a novel antibacterial dione reveals two drug molecules intercalated at a cleaved B-form DNA gate and anchored by drug-specific protein contacts. Dione release generated drug-free cleaved and resealed DNA complexes in which the DNA gate instead adopts an unusual A/B-form helical conformation with a Mg2+ ion repositioned to coordinate each scissile phosphodiester group and promote reversible cleavage by active-site tyrosines. These structures, the first for putative reaction intermediates of a type II topoisomerase, suggest how a type II enzyme reseals DNA during its normal reaction cycle and illuminate aspects of drug arrest important for the development of new topoisomerase-targeting therapeutics.
Ciprofloxacin-resistant mutants of Streptococcus pneumoniae 7785 were generated by stepwise selection at increasing drug concentrations. Sequence analysis of PCR products from the strains was used to examine the quinolone resistance-determining regions of the GyrA and GyrB proteins of DNA gyrase and the analogous regions of the ParC and ParE subunits of DNA topoisomerase IV. First-step mutants exhibiting low-level resistance had no detectable changes in their topoisomerase quinolone resistance-determining regions, suggesting altered permeation or another novel resistance mechanism. Nine of 10 second-step mutants exhibited an alteration in ParC at Ser-79 to Tyr or Phe or at Ala-84 to Thr. Third- and fourth-step mutants displaying high-level ciprofloxacin resistance were found to have, in addition to the ParC alteration, a change in GyrA at residues equivalent to Escherichia coli GyrA resistance hot spots Ser-83 and Asp-87 or in GyrB at Asp-435 to Asn, equivalent to E. coli Asp-426, part of a highly conserved EGDSA motif in GyrB. No ParE changes were observed. Complementary analysis of two S. pneumoniae clinical isolates displaying low-level resistance to ciprofloxacin revealed a ParC change at Ser-79 to Phe or Arg-95 to Cys but no changes in GyrA, GyrB, or ParE. A highly resistant isolate, in addition to a ParC mutation, had a GyrA alteration at the residue equivalent to E. coli Asp-87. Thus, in both laboratory strains and clinical isolates, ParC mutations preceded those in GyrA, suggesting that topoisomerase IV is a primary topoisomerase target and gyrase is a secondary target for ciprofloxacin in S. pneumoniae.
Genome studies suggest that DNA gyrase is the sole type II topoisomerase and likely the unique target of quinolones in Mycobacterium tuberculosis. Despite the emerging importance of quinolones in the treatment of mycobacterial disease, the slow growth and high pathogenicity of M. tuberculosis have precluded direct purification of its gyrase and detailed analysis of quinolone action. To address these issues, we separately overexpressed the M. tuberculosis DNA gyrase GyrA and GyrB subunits as His-tagged proteins in Escherichia coli from pET plasmids carrying gyrA and gyrB genes. The soluble 97-kDa GyrA and 72-kDa GyrB subunits were purified by nickel chelate chromatography and shown to reconstitute an ATP-dependent DNA supercoiling activity. The drug concentration that inhibited DNA supercoiling by 50% (IC 50 ) was measured for 22 different quinolones, and values ranged from 2 to 3 g/ml (sparfloxacin, sitafloxacin, clinafloxacin, and gatifloxacin) to >1,000 g/ml (pipemidic acid and nalidixic acid). By comparison, MICs measured against M. tuberculosis ranged from 0.12 g/ml (for gatifloxacin) to 128 g/ml (both pipemidic acid and nalidixic acid) and correlated well with the gyrase IC 50 s (R 2 ؍ 0.9). Quinolones promoted gyrase-mediated cleavage of plasmid pBR322 DNA due to stabilization of the cleavage complex, which is thought to be the lethal lesion. Surprisingly, the measured concentrations of drug inducing 50% plasmid linearization correlated less well with the MICs (R 2 ؍ 0.7). These findings suggest that the DNA supercoiling inhibition assay may be a useful screening test in identifying quinolones with promising activity against M. tuberculosis. The quinolone structure-activity relationship demonstrated here shows that C-8, the C-7 ring, the C-6 fluorine, and the N-1 cyclopropyl substituents are desirable structural features in targeting M. tuberculosis gyrase.Fluoroquinolones are active against Mycobacterium tuberculosis and are the first new antimycobacterial drugs to be available since the discovery of rifampin (7,13,40). Fluoroquinolones are part of the drug regimens now recommended for treating rifampin-resistant tuberculosis (6, 10). Ofloxacin and ciprofloxacin have a bacteriostatic antimycobacterial activity (13,20,40), but several new fluoroquinolones, such as sparfloxacin and moxifloxacin, show high bactericidal activity against M. tuberculosis that compares with that of major antituberculous drugs in animal models (20). By contrast, other new fluoroquinolones, such as gemifloxacin and trovafloxacin, are less active than ofloxacin against M. tuberculosis.Studies of other bacterial species suggest that the differences in intrinsic activity observed between quinolones are mainly related to differences in quinolone inhibition of the targets. This has been demonstrated for the differences in activities of several compounds against a given bacterial species, e.g., nalidixic acid and ciprofloxacin against Escherichia coli (17), and of a given compound against several species, e.g., ciprofloxacin against...
DNA topoisomerase IV mediates chromosome segregation and is a potential target for antibacterial agents including new antipneumococcal fluoroquinolones. We have used hybridization to a Staphylococcus aureus gyrB probe in concert with chromosome walking to isolate the Streptococcus pneumoniae parE-parC locus, lying downstream of a putative new insertion sequence and encoding 647-residue ParE and 823-residue ParC subunits of DNA topoisomerase IV. These proteins exhibited greatest homology respectively to the GrlB (ParE) and GrlA (ParC) subunits of S. aureus DNA topoisomerase IV. When combined, whole-cell extracts of Escherichia coli strains expressing S. pneumoniae ParC or ParE proteins reconstituted a salt-insensitive ATP-dependent decatenase activity characteristic of DNA topoisomerase IV. A second gyrB homolog isolated from S. pneumoniae encoded a 648-residue protein which we identified as GyrB through its close homology both to counterparts in S. aureus and Bacillus subtilis and to the product of the S. pneumoniae nov-1 gene that confers novobiocin resistance. gyrB was not closely linked to gyrA. To examine the role of DNA topoisomerase IV in fluoroquinolone action and resistance in S. pneumoniae, we isolated mutant strains stepwise selected for resistance to increasing concentrations of ciprofloxacin. We analysed four low-level resistant mutants and showed that Ser-79 of ParC, equivalent to resistance hotspots Ser-80 of GrlA and Ser-84 of GyrA in S. aureus, was in each case substituted with Tyr. These results suggest that DNA topoisomerase IV is an important target for fluoroquinolones in S. pneumoniae and establish this organism as a useful gram-positive system for resistance studies.
gyrA and parC mutations have been identified inn Streptococcus pneumoniae mutants stepwise selected for resistance to sparfloxacin, an antipneumococcal fluoroquinolone. GyrA mutations (at the position equivalent to resistance hot spot Ser-83 in Escherichia coli GyrA) were found in all 17 first-step mutants examined and preceded DNA topoisomerase IV parC mutations (at Ser-79 or Glu-83), which appeared only in second-step mutants. The targeting of gyrase by sparfloxacin in S. pneumoniae but of topoisomerase IV by ciprofloxacin indicates that target preference can be altered by changes in quinolone structure.
Quinazoline-2,4-diones, such as PD 0305970, are new DNA gyrase and topoisomerase IV (topo IV) inhibitors with potent activity against gram-positive pathogens, including quinolone-resistant isolates. The mechanistic basis of dione activity vis-à-vis quinolones is not understood. We present evidence for Streptococcus pneumoniae gyrase and topo IV that PD 0305970 and quinolones interact differently with the enzyme breakage-reunion and Toprim domains, DNA, and Mg 2؉ -four components that are juxtaposed in the topoisomerase cleavage complex to effect DNA scission. First, PD 0305970 targets primarily gyrase in Streptococcus pneumoniae. However, unlike quinolones, which select predominantly for gyrA (or topo IV parC) mutations in the breakage-reunion domain, unusually the dione selected for novel mutants with alterations that map to a region of the Toprim domain of GyrB (R456H and E474A or E474D) or ParE (D435H and E475A). This "dione resistance-determining region" overlaps the GyrB quinolone resistance-determining region and the region that binds essential Mg 2؉ ions, each function involving conserved EGDSA and PLRGK motifs. Second, dione-resistant gyrase and topo IV were inhibited by ciprofloxacin, whereas quinolone-resistant enzymes (GyrA S81F and ParC S79F) remained susceptible to PD 0305970. Third, dione-promoted DNA cleavage by gyrase occurred at a distinct repertoire of sites, implying that structural differences with quinolones are sensed at the DNA level. Fourth, unlike the situation with quinolones, the Mg 2؉ chelator EDTA did not reverse dione-induced gyrase cleavage nor did the dione promote Mg 2؉ -dependent DNA unwinding. It appears that PD 0305970 interacts uniquely to stabilize the cleavage complex of gyrase/topo IV perhaps via an altered orientation directed by the bidentate 3-amino-2,4-dione moiety.The established and emerging resistance of gram-positive pathogens to fluoroquinolones, macrolides, and beta-lactams has underlined the need for new antimicrobial agents (2,6,28,32,35,46,52). Recent work has shown that quinazolinediones constitute a promising new class of antibacterial inhibitors of DNA gyrase and topoisomerase IV (topo IV) that share structural similarity with fluoroquinolones (14,50,51). Among this group of inhibitors, the 3-aminoquinazolinedione PD 0305790 (3-amino-7-[(R)-3-[(S)-1-aminoethyl]-pyrrolidin-1-yl]-1-cyclopropyl-6-fluoro-8-methyl-1H-quinazoline-2,4-dione) (Fig. 1) has particularly potent activity against susceptible and drugresistant gram-positive species, especially Streptococcus pneumoniae (23). The drug differs most notably from quinolones, e.g., ciprofloxacin, in having the 3-aminoquinazoline 2,4-dione system in place of the 3-carboxy-substituted 4-quinolone ring (Fig. 1). Surprisingly, these subtle differences in drug structure confer activity against quinolone-resistant strains (23).Initial work with Escherichia coli gyrase has shown that, like quinolones, 3-aminodiones, such as PD 0305790, stabilize an enzyme "cleavage complex" on DNA (50). This complex is thought to ref...
Topoisomerase (topo) IV and gyrase are bacterial type IIA DNA topoisomerases essential for DNA replication and chromosome segregation that act via a transient double-stranded DNA break involving a covalent enzyme-DNA "cleavage complex." Despite their mechanistic importance, the DNA breakage determinants are not understood for any bacterial type II enzyme. We investigated DNA cleavage by Streptococcus pneumoniae topo IV and gyrase stabilized by gemifloxacin and other antipneumococcal fluoroquinolones. Topo IV and gyrase induce distinct but overlapping repertoires of double-strand DNA breakage sites that were essentially identical for seven different quinolones and were augmented (in intensity) by positive or negative supercoiling. Sequence analysis of 180 topo IV and 126 gyrase sites promoted by gemifloxacin on pneumococcal DNA revealed the respective consensus sequences: G(G/C)(A/T)A*GNNCT(T/A)N(C/A) and GN 4 G(G/C)(A/C)G*GNNCTTN(C/A) (preferred bases are underlined; disfavored bases are in small capitals; N indicates no preference; and asterisk indicates DNA scission between ؊1 and ؉1 positions). Both enzymes show strong preferences for bases clustered symmetrically around the DNA scission site, i.e. ؉1G/؉4C, ؊4G/؉8C, and particularly the novel ؊2A/؉6T, but with no preference at ؉2/؉3 within the staggered 4-bp overhang. Asymmetric elements include ؊3G and several unfavored bases. These cleavage preferences, the first for Gram-positive type IIA topoisomerases, differ markedly from those reported for Escherichia coli topo IV (consensus (A/G)*T/A) and gyrase, which are based on fewer sites. However, both pneumococcal enzymes cleaved an E. coli gyrase site suggesting overlap in gyrase determinants. We propose a model for the cleavage complex of topo IV/gyrase that accommodates the unique ؊2A/ ؉6T and other preferences.Type IIA DNA topoisomerases mediate the ATP-dependent transport of one DNA helix through another DNA segment via a transient enzyme-bridged double-strand break (1-4). This maneuver allows the control of DNA topology essential for a variety of DNA transactions, including DNA replication, transcription, and recombination. Bacteria usually express two essential type IIA enzymes, DNA topoisomerase (topo) 1 IV and DNA gyrase (5, 6), which were first identified in Escherichia coli (7,8). Gyrase is unique in catalyzing the negative supercoiling of closed circular DNA. It is believed to act ahead of replication forks promoting fork progression by removing the positive supercoils that arise during DNA unwinding (9). By contrast, topo IV relaxes positive and negative supercoils and has evolved as an efficient DNA decatenase with a specialized role in separating daughter chromosomes at cell division (9). In addition to their biological functions, topo IV and gyrase are also targets for the quinolone class of antibacterial drugs (5, 10).Topo IV and gyrase are tetrameric complexes comprising two subunits each of ParC and ParE, and GyrA and GyrB, respectively (5). The ParC and GyrA subunits are homologous in their ...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.