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Neil Osheroff - One of the best experts on this subject based on the ideXlab platform.
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Bimodal Actions of a Naphthyridone/Aminopiperidine-Based Antibacterial That Targets Gyrase and Topoisomerase IV
Biochemistry, 2019Co-Authors: Elizabeth G. Gibson, Keir C Neuman, Alexandria A. Oviatt, Monica Cacho, Pan F. Chan, Neil OsheroffAbstract:Gyrase and Topoisomerase IV are the targets of fluoroquinolone antibacterials. However, the rise in antimicrobial resistance has undermined the clinical use of this important drug class. Therefore,...
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bimodal actions of a naphthyridone aminopiperidine based antibacterial that targets gyrase and Topoisomerase IV
Biochemistry, 2019Co-Authors: Elizabeth G. Gibson, Keir C Neuman, Alexandria A. Oviatt, Monica Cacho, Pan F. Chan, Neil OsheroffAbstract:Gyrase and Topoisomerase IV are the targets of fluoroquinolone antibacterials. However, the rise in antimicrobial resistance has undermined the clinical use of this important drug class. Therefore,...
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actIVities of gyrase and Topoisomerase IV on positIVely supercoiled dna
Nucleic Acids Research, 2017Co-Authors: Rachel E Ashley, Charles L Turnbough, Keir C Neuman, Sylvia A. Mcpherson, Andrew Dittmore, Neil OsheroffAbstract:: Although bacterial gyrase and Topoisomerase IV have critical interactions with positIVely supercoiled DNA, little is known about the actions of these enzymes on overwound substrates. Therefore, the abilities of Bacillus anthracis and Escherichia coli gyrase and Topoisomerase IV to relax and cleave positIVely supercoiled DNA were analyzed. Gyrase removed positIVe supercoils ∼10-fold more rapidly and more processIVely than it introduced negatIVe supercoils into relaxed DNA. In time-resolved single-molecule measurements, gyrase relaxed overwound DNA with burst rates of ∼100 supercoils per second (average burst size was 6.2 supercoils). Efficient positIVe supercoil removal required the GyrA-box, which is necessary for DNA wrapping. Topoisomerase IV also was able to distinguish DNA geometry during strand passage and relaxed positIVely supercoiled substrates ∼3-fold faster than negatIVely supercoiled molecules. Gyrase maintained lower levels of cleavage complexes with positIVely supercoiled (compared with negatIVely supercoiled) DNA, whereas Topoisomerase IV generated similar levels with both substrates. Results indicate that gyrase is better suited than Topoisomerase IV to safely remove positIVe supercoils that accumulate ahead of replication forks. They also suggest that the wrapping mechanism of gyrase may have evolved to promote rapid removal of positIVe supercoils, rather than induction of negatIVe supercoils.
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Bacillus anthracis GrlAV96A Topoisomerase IV, a Quinolone Resistance Mutation That Does Not Affect the Water-Metal Ion Bridge
Antimicrobial agents and chemotherapy, 2014Co-Authors: Katie J. Aldred, Charles L Turnbough, Sylvia A. Mcpherson, Robert J. Kerns, Erin J. Breland, Neil OsheroffAbstract:ABSTRACT The rise in quinolone resistance is threatening the clinical use of this important class of broad-spectrum antibacterials. Quinolones kill bacteria by increasing the level of DNA strand breaks generated by the type II Topoisomerases gyrase and Topoisomerase IV. Most commonly, resistance is caused by mutations in the serine and acidic amino acid residues that anchor a water-metal ion bridge that facilitates quinolone-enzyme interactions. Although other mutations in gyrase and Topoisomerase IV have been reported in quinolone-resistant strains, little is known regarding their contributions to cellular quinolone resistance. To address this issue, we characterized the effects of the V96A mutation in the A subunit of Bacillus anthracis Topoisomerase IV on quinolone actIVity. The results indicate that this mutation causes an ∼3-fold decrease in quinolone potency and reduces the stability of covalent Topoisomerase IV-cleaved DNA complexes. However, based on metal ion usage, the V96A mutation does not disrupt the function of the water-metal ion bridge. A similar level of resistance to quinazolinediones (which do not use the bridge) was seen. V96A is the first Topoisomerase IV mutation distal to the water-metal ion bridge demonstrated to decrease quinolone actIVity. It also represents the first A subunit mutation reported to cause resistance to quinazolinediones. This cross-resistance suggests that the V96A change has a global effect on the structure of the drug-binding pocket of Topoisomerase IV.
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Role of the water-metal ion bridge in mediating interactions between quinolones and Escherichia coli Topoisomerase IV.
Biochemistry, 2014Co-Authors: Katie J. Aldred, Keir C Neuman, Robert J. Kerns, Erin J. Breland, Vladislava Vlčková, Marie-paule Strub, Neil OsheroffAbstract: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.
Katie J. Aldred - One of the best experts on this subject based on the ideXlab platform.
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Bacillus anthracis GrlAV96A Topoisomerase IV, a Quinolone Resistance Mutation That Does Not Affect the Water-Metal Ion Bridge
Antimicrobial agents and chemotherapy, 2014Co-Authors: Katie J. Aldred, Charles L Turnbough, Sylvia A. Mcpherson, Robert J. Kerns, Erin J. Breland, Neil OsheroffAbstract:ABSTRACT The rise in quinolone resistance is threatening the clinical use of this important class of broad-spectrum antibacterials. Quinolones kill bacteria by increasing the level of DNA strand breaks generated by the type II Topoisomerases gyrase and Topoisomerase IV. Most commonly, resistance is caused by mutations in the serine and acidic amino acid residues that anchor a water-metal ion bridge that facilitates quinolone-enzyme interactions. Although other mutations in gyrase and Topoisomerase IV have been reported in quinolone-resistant strains, little is known regarding their contributions to cellular quinolone resistance. To address this issue, we characterized the effects of the V96A mutation in the A subunit of Bacillus anthracis Topoisomerase IV on quinolone actIVity. The results indicate that this mutation causes an ∼3-fold decrease in quinolone potency and reduces the stability of covalent Topoisomerase IV-cleaved DNA complexes. However, based on metal ion usage, the V96A mutation does not disrupt the function of the water-metal ion bridge. A similar level of resistance to quinazolinediones (which do not use the bridge) was seen. V96A is the first Topoisomerase IV mutation distal to the water-metal ion bridge demonstrated to decrease quinolone actIVity. It also represents the first A subunit mutation reported to cause resistance to quinazolinediones. This cross-resistance suggests that the V96A change has a global effect on the structure of the drug-binding pocket of Topoisomerase IV.
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Role of the water-metal ion bridge in mediating interactions between quinolones and Escherichia coli Topoisomerase IV.
Biochemistry, 2014Co-Authors: Katie J. Aldred, Keir C Neuman, Robert J. Kerns, Erin J. Breland, Vladislava Vlčková, Marie-paule Strub, Neil OsheroffAbstract: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.
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overcoming target mediated quinolone resistance in Topoisomerase IV by introducing metal ion independent drug enzyme interactions
ACS Chemical Biology, 2013Co-Authors: Katie J. Aldred, Charles L Turnbough, Sylvia A. Mcpherson, Robert J. Kerns, Heidi A Schwanz, Gangqin Li, Neil OsheroffAbstract:Quinolones, which target gyrase and Topoisomerase IV, are the most widely prescribed antibacterials worldwide. Unfortunately, their use is threatened by the increasing prevalence of target-mediated drug resistance. Greater than 90% of mutations that confer quinolone resistance act by disrupting enzyme–drug interactions coordinated by a critical water–metal ion bridge. Quinazolinediones are quinolone-like drugs but lack the skeletal features necessary to support the bridge interaction. These compounds are of clinical interest, however, because they retain actIVity against the most common quinolone resistance mutations. We utilized a chemical biology approach to determine how quinazolinediones overcome quinolone resistance in Bacillus anthracis Topoisomerase IV. Quinazolinediones that retain actIVity against quinolone-resistant Topoisomerase IV do so primarily by establishing novel interactions through the C7 substituent, rather than the drug skeleton. Because some quinolones are highly actIVe against human...
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Topoisomerase IV-quinolone interactions are mediated through a water-metal ion bridge: mechanistic basis of quinolone resistance
Nucleic Acids Research, 2013Co-Authors: Katie J. Aldred, Charles L Turnbough, Sylvia A. Mcpherson, Robert J. Kerns, Neil OsheroffAbstract: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.
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Drug Interactions with Bacillus anthracis Topoisomerase IV: Biochemical Basis for Quinolone Action and Resistance
Biochemistry, 2011Co-Authors: Katie J. Aldred, Charles L Turnbough, Sylvia A. Mcpherson, Robert J. Kerns, Pengfei Wang, David E. Graves, Neil OsheroffAbstract:Bacillus anthracis, the causatIVe agent of anthrax, is considered a serious threat as a bioweapon. The drugs most commonly used to treat anthrax are quinolones, which act by increasing the levels of DNA cleavage mediated by Topoisomerase IV and gyrase. Quinolone resistance most often is associated with specific serine mutations in these enzymes. Therefore, to determine the basis for quinolone action and resistance, we characterized wild-type B. anthracis Topoisomerase IV, the GrlAS81F and GrlAS81Y quinolone-resistant mutants, and the effects of quinolones and a related quinazolinedione on these enzymes. Ser81 is believed to anchor a water–Mg2+ bridge that coordinates quinolones to the enzyme through the C3/C4 keto acid. Consistent with this hypothesized bridge, ciprofloxacin required increased Mg2+ concentrations to support DNA cleavage by GrlAS81F Topoisomerase IV. The three enzymes displayed similar catalytic actIVities in the absence of drugs. However, the resistance mutations decreased the affinity of...
David C Hooper - One of the best experts on this subject based on the ideXlab platform.
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interaction of the plasmid encoded quinolone resistance protein qnra with escherichia coli Topoisomerase IV
Antimicrobial Agents and Chemotherapy, 2005Co-Authors: John H Tran, George A Jacoby, David C HooperAbstract:Purified QnrA blocked ciprofloxacin inhibition of Topoisomerase IV, just as QnrA was previously found to prevent quinolone inhibition of DNA gyrase. With a gel displacement assay, tagged QnrA was shown to bind to Topoisomerase IV and its subunits in a reaction that did not depend on the presence of DNA, quinolone, or ATP.
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Dual Targeting of Topoisomerase IV and Gyrase To Reduce Mutant Selection: Direct Testing of the Paradigm by Using WCK-1734, a New Fluoroquinolone, and Ciprofloxacin
Antimicrobial agents and chemotherapy, 2005Co-Authors: Jacob Strahilevitz, David C HooperAbstract:Quinolones that act equally against DNA gyrase and Topoisomerase IV are a desirable modality to decrease the selection of resistant strains. We first determined by genetic and biochemical studies in Staphylococcus aureus that the primary target enzyme of WCK-1734, a new quinolone, was DNA gyrase. A single mutation in gyrase, but not Topoisomerase IV, caused a two- to fourfold increase in the MIC. Studies with purified Topoisomerase IV and gyrase from S. aureus also showed that gyrase was more sensitIVe than Topoisomerase IV to WCK-1734 (50% inhibitory concentration, 1.25 and 2.5 to 5.0 μg/ml, respectIVely; 50% stimulation of cleavage complex formation, 0.62 and 2.5 to 5.0 μg/ml, respectIVely). To test the effect of balanced actIVity of quinolones against the two target enzymes, we measured the frequency of selection of mutants with ciprofloxacin (which targets Topoisomerase IV) and WCK-1734 alone and in combination. With the combination of ciprofloxacin and WCK-1734, each at its MIC, the ratio of frequency of mutants selected was significantly lower than that with each drug alone at two times their respectIVe MICs. We further characterized resistant strains selected with the combination of ciprofloxacin and WCK-1734 and found evidence to suggest the existence of novel mutational mechanisms for low-level quinolone resistance. By use of a combination of differentially targeting quinolones, this study provides novel data in direct support of the paradigm for dual targeting of quinolone action and reduced development of resistance.
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dual targeting of dna gyrase and Topoisomerase IV target interactions of garenoxacin bms 284756 t 3811me a new desfluoroquinolone
Antimicrobial Agents and Chemotherapy, 2002Co-Authors: Dilek Ince, Christine L Silver, Xiamei Zhang, David C HooperAbstract:We determined the target enzyme interactions of garenoxacin (BMS-284756, T-3811ME), a novel desfluoroquinolone, in Staphylococcus aureus by genetic and biochemical studies. We found garenoxacin to be four- to eightfold more actIVe than ciprofloxacin against wild-type S. aureus. A single Topoisomerase IV or gyrase mutation caused only a 2- to 4-fold increase in the MIC of garenoxacin, whereas a combination of mutations in both loci caused a substantial increase (128-fold). Overexpression of the NorA efflux pump had minimal effect on resistance to garenoxacin. With garenoxacin at twice the MIC, selection of resistant mutants (<7.4 × 10−12 to 4.0 × 10−11) was 5 to 6 log units less than that with ciprofloxacin. Mutations inside or outside the quinolone resistance-determining regions (QRDR) of either Topoisomerase IV, or gyrase, or both were selected in single-step mutants, suggesting dual targeting of Topoisomerase IV and gyrase. Three of the novel mutations were shown by genetic experiments to be responsible for resistance. Studies with purified Topoisomerase IV and gyrase from S. aureus also showed that garenoxacin had similar actIVity against Topoisomerase IV and gyrase (50% inhibitory concentration, 1.25 to 2.5 and 1.25 μg/ml, respectIVely), and although its actIVity against Topoisomerase IV was 2-fold greater than that of ciprofloxacin, its actIVity against gyrase was 10-fold greater. This study provides the first genetic and biochemical data supporting the dual targeting of Topoisomerase IV and gyrase in S. aureus by a quinolone as well as providing genetic proof for the expansion of the QRDRs to include the 5′ terminus of grlB and the 3′ terminus of gyrA.
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dual targeting of dna gyrase and Topoisomerase IV target interactions of garenoxacin bms 284756 t 3811me a new desfluoroquinolone
Antimicrobial Agents and Chemotherapy, 2002Co-Authors: Dilek Ince, Christine L Silver, Xiamei Zhang, David C HooperAbstract:We determined the target enzyme interactions of garenoxacin (BMS-284756, T-3811ME), a novel desfluoroquinolone, in Staphylococcus aureus by genetic and biochemical studies. We found garenoxacin to be four- to eightfold more actIVe than ciprofloxacin against wild-type S. aureus. A single Topoisomerase IV or gyrase mutation caused only a 2- to 4-fold increase in the MIC of garenoxacin, whereas a combination of mutations in both loci caused a substantial increase (128-fold). Overexpression of the NorA efflux pump had minimal effect on resistance to garenoxacin. With garenoxacin at twice the MIC, selection of resistant mutants (<7.4 × 10−12 to 4.0 × 10−11) was 5 to 6 log units less than that with ciprofloxacin. Mutations inside or outside the quinolone resistance-determining regions (QRDR) of either Topoisomerase IV, or gyrase, or both were selected in single-step mutants, suggesting dual targeting of Topoisomerase IV and gyrase. Three of the novel mutations were shown by genetic experiments to be responsible for resistance. Studies with purified Topoisomerase IV and gyrase from S. aureus also showed that garenoxacin had similar actIVity against Topoisomerase IV and gyrase (50% inhibitory concentration, 1.25 to 2.5 and 1.25 μg/ml, respectIVely), and although its actIVity against Topoisomerase IV was 2-fold greater than that of ciprofloxacin, its actIVity against gyrase was 10-fold greater. This study provides the first genetic and biochemical data supporting the dual targeting of Topoisomerase IV and gyrase in S. aureus by a quinolone as well as providing genetic proof for the expansion of the QRDRs to include the 5′ terminus of grlB and the 3′ terminus of gyrA.
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selectIVe targeting of Topoisomerase IV and dna gyrase in staphylococcus aureus different patterns of quinolone induced inhibition of dna synthesis
Antimicrobial Agents and Chemotherapy, 2000Co-Authors: Benedicte Fournier, Xilin Zhao, Karl Drlica, David C HooperAbstract:The effect of quinolones on the inhibition of DNA synthesis in Staphylococcus aureus was examined by using single resistance mutations in parC or gyrA to distinguish action against gyrase or Topoisomerase IV, respectIVely. Norfloxacin preferentially attacked Topoisomerase IV and blocked DNA synthesis slowly, while nalidixic acid targeted gyrase and inhibited replication rapidly. Ciprofloxacin exhibited an intermediate response, consistent with both enzymes being targeted. The absence of RecA had little influence on target choice by this assay, indicating that differences in rebound (repair) DNA synthesis were not responsible for the results. At saturating drug concentrations, norfloxacin and a gyrA mutant were used to show that Topoisomerase IV-norfloxacin-cleaved DNA complexes are distributed on the S. aureus chromosome at intervals of about 30 kbp. If cleaved complexes block DNA replication, as indicated by previous work, such close spacing of Topoisomerase-quinolone-DNA complexes should block replication rapidly (replication forks are likely to encounter a cleaved complex within a minute). Thus, the slow inhibition of DNA synthesis at growth-inhibitory concentrations suggests that a subset of more distantly distributed complexes is physiologically relevant for drug action and is unlikely to be located immediately in front of the DNA replication fork.
L M Fisher - One of the best experts on this subject based on the ideXlab platform.
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targeting of dna gyrase in streptococcus pneumoniae by sparfloxacin selectIVe targeting of gyrase or Topoisomerase IV by quinolones
Antimicrobial Agents and Chemotherapy, 1997Co-Authors: Xiaosu Pan, L M FisherAbstract: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.
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involvement of Topoisomerase IV and dna gyrase as ciprofloxacin targets in streptococcus pneumoniae
Antimicrobial Agents and Chemotherapy, 1996Co-Authors: Xiaosu Pan, J Ambler, S Mehtar, L M FisherAbstract: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.
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Cloning and characterization of the parC and parE genes of Streptococcus pneumoniae encoding DNA Topoisomerase IV: role in fluoroquinolone resistance.
Journal of bacteriology, 1996Co-Authors: X S Pan, L M FisherAbstract: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.
Keir C Neuman - One of the best experts on this subject based on the ideXlab platform.
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Bimodal Actions of a Naphthyridone/Aminopiperidine-Based Antibacterial That Targets Gyrase and Topoisomerase IV
Biochemistry, 2019Co-Authors: Elizabeth G. Gibson, Keir C Neuman, Alexandria A. Oviatt, Monica Cacho, Pan F. Chan, Neil OsheroffAbstract:Gyrase and Topoisomerase IV are the targets of fluoroquinolone antibacterials. However, the rise in antimicrobial resistance has undermined the clinical use of this important drug class. Therefore,...
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bimodal actions of a naphthyridone aminopiperidine based antibacterial that targets gyrase and Topoisomerase IV
Biochemistry, 2019Co-Authors: Elizabeth G. Gibson, Keir C Neuman, Alexandria A. Oviatt, Monica Cacho, Pan F. Chan, Neil OsheroffAbstract:Gyrase and Topoisomerase IV are the targets of fluoroquinolone antibacterials. However, the rise in antimicrobial resistance has undermined the clinical use of this important drug class. Therefore,...
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actIVities of gyrase and Topoisomerase IV on positIVely supercoiled dna
Nucleic Acids Research, 2017Co-Authors: Rachel E Ashley, Charles L Turnbough, Keir C Neuman, Sylvia A. Mcpherson, Andrew Dittmore, Neil OsheroffAbstract:: Although bacterial gyrase and Topoisomerase IV have critical interactions with positIVely supercoiled DNA, little is known about the actions of these enzymes on overwound substrates. Therefore, the abilities of Bacillus anthracis and Escherichia coli gyrase and Topoisomerase IV to relax and cleave positIVely supercoiled DNA were analyzed. Gyrase removed positIVe supercoils ∼10-fold more rapidly and more processIVely than it introduced negatIVe supercoils into relaxed DNA. In time-resolved single-molecule measurements, gyrase relaxed overwound DNA with burst rates of ∼100 supercoils per second (average burst size was 6.2 supercoils). Efficient positIVe supercoil removal required the GyrA-box, which is necessary for DNA wrapping. Topoisomerase IV also was able to distinguish DNA geometry during strand passage and relaxed positIVely supercoiled substrates ∼3-fold faster than negatIVely supercoiled molecules. Gyrase maintained lower levels of cleavage complexes with positIVely supercoiled (compared with negatIVely supercoiled) DNA, whereas Topoisomerase IV generated similar levels with both substrates. Results indicate that gyrase is better suited than Topoisomerase IV to safely remove positIVe supercoils that accumulate ahead of replication forks. They also suggest that the wrapping mechanism of gyrase may have evolved to promote rapid removal of positIVe supercoils, rather than induction of negatIVe supercoils.
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Role of the water-metal ion bridge in mediating interactions between quinolones and Escherichia coli Topoisomerase IV.
Biochemistry, 2014Co-Authors: Katie J. Aldred, Keir C Neuman, Robert J. Kerns, Erin J. Breland, Vladislava Vlčková, Marie-paule Strub, Neil OsheroffAbstract: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.
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The tail that wags the dog: Topoisomerase IV ParC C-terminal domain controls strand passage actIVity through multipartite topology-dependent interactions with DNA.
Journal of molecular biology, 2013Co-Authors: Keir C NeumanAbstract:Type IIA Topoisomerases are essential enzymes that contribute to chromosomal integrity by controlling the degree of supercoiling and segregating newly replicated chromosomes1. They share a conserved core mechanism in which a double-stranded segment of DNA is passed through a transient double-stranded break in a second segment of DNA (Figure 1). Remarkably, this conserved strand passage reaction results in a range of distinct actIVities catalyzed by different but related type IIA Topoisomerases2. At one extreme is prokaryotic DNA gyrase that negatIVely supercoils DNA, but is a poor decatenase. At the other extreme is the closely related prokaryotic Topoisomerase IV that is an efficient decatenase and relaxes positIVe supercoils more efficiently than negatIVe supercoils1. Eukaryotic type IIA Topoisomerases display similar, though less extreme, topologically-dependent differences in actIVity3. For example, human Topoisomerase IIα preferentially relaxes positIVely supercoiled DNA, whereas the other human isoform, Topoisomerase IIβ, relaxes positIVely and negatIVely supercoiled DNA with equal efficiency. The differences in actIVity among these enzymes can largely be attributed to differences in the poorly conserved C-terminal domains (CTDs), which contain a highly positIVely charged DNA binding surface4. Deletion of the gyrase GyrA CTD or Topoisomerase IV ParC CTD results in a type IIA Topoisomerase lacking chiral preference in relaxation and which no longer introduces negatIVe supercoils4; 5. Indeed, chiral discrimination by Topoisomerase IIA enzymes appears to be entirely dictated by the CTD domain. The chirality-dependent actIVity of chimeras between the human Topoisomerase II α and β core enzymes with the alternatIVe CTDs is almost entirely dictated by the CTDs rather than the core enzyme6. In addition, subtle mutations in a gyrase GyrA CTD result in an enzyme that exhibits Topoisomerase IV-like chirality-dependent strand passage actIVity7. Whereas the domain responsible for chiral discrimination has been identified, the mechanistic basis for chiral discrimination by type IIA Topoisomerases remains under debate. Proposed mechanisms include discrimination based on DNA crossing geometry and chirality-dependent differences in processIVity4; 8; 9; 10; 11. Crystal structures of bacterial GyrA CTDs and ParC CTDs have not provided a great deal of additional insight. The GyrA and ParC CTDs are multi-bladed structures that contain a highly positIVely charged region that binds DNA 12. The similarity of the fIVe blades found in E. coli ParC CTD suggests that it may simply provide an extended high-affinity DNA binding surface4; 12. Thus, despite determining the structure of the domain responsible for chiral discrimination by Topoisomerase IV, the relationship between the structure and mechanism was not immediately clear. Figure 1 Topoisomerase IIA strand passage reaction. Topoisomerase binds gate segment DNA (blue) followed by binding of transfer segment DNA (yellow) and ATP that closes the N-gate (light blue). The gate segment is cleaved and the C-gate (light green) opens allowing ... In this issue, Vos, Lee, and Berger present the results of a systematic dissection of the role of key residues in each of the fIVe blades of the Topoisomerase IV Par-C CTD. By measuring the chirality-dependent relaxation and unlinking actIVity of point mutations of highly conserved basic residues in each blade, complemented by binding and bending assays of the intact enzyme as well as the isolated CTD, the authors made a number of striking discoveries. Rather than being a monolithic DNA binding element, each blade in the CTD appears to affect different aspects of Topoisomerase IV actIVity in dramatically different manners. These results provide an important connection between the structure of the CTD and chirality-dependent modulation of strand passage actIVity. Remarkably, interactions between the DNA and the Par-C CTD appear to both stimulate and inhibit specific topology and substrate-dependent actIVities of Topoisomerase IV. Perhaps the most interesting finding is that residues in blade 1, which is proximal to the N-terminal portion of the ParC domain, contribute to bending of the G-segment DNA bound by the core enzyme. Disruption of this residue leads to a severe decrease in actIVity across all measurements, consistent with the growing body of evidence establishing the importance of DNA bending for Topoisomerase IIA actIVity13; 14. However, this is the first evidence that the ParC CTD participates in G-segment DNA interactions and it explains the dramatic decrease in actIVity observed in the Topoisomerase IV ParC CTD deletion mutant4. The remaining blades modulate the actIVity of Topoisomerase IV in distinct DNA substrate-dependent manners. Conserved basic residues in blades 2–4 modulate the actIVity, rate, and processIVity of positIVe supercoil relaxation. Mutations of these residues decrease the overall actIVity of Topoisomerase IV in relaxing positIVe supercoils. Paradoxically, these mutations slightly increase the relaxation rate while decreasing the processIVity of positIVe supercoil relaxation. Mutations in blade 5 have little overall effect on the relaxation of positIVe supercoils. Remarkably, blade 5 appears to specifically inhibit negatIVe supercoil relaxation as both the overall actIVity and rate increase when interactions between this blade and the DNA are disrupted. NegatIVe supercoil relaxation appears to be governed by blades 2 and 3 as mutations in these blades decrease the overall actIVity, processIVity, and rate of Topoisomerase IV in relaxing negatIVe supercoils. Mutations in blade 4 have virtually no effect on negatIVe supercoil relaxation. Similar to negatIVe supercoil relaxation, decatenation appears to be inhibited through the interaction of DNA with blade 5, but only blade 3 appears to be important for decatenation. Mutations in blades 2 and 4 have essentially no effect on decatenation actIVity or rate. The final surprising discovery by Vos and co-workers is that the effects of disrupting DNA binding at each blade did not uniformly decrease the overall binding affinity of the isolated CTD. Blades 1 and 2 appear to have little effect on DNA binding, whereas blades 3, 4, and 5 appear to play important roles in DNA binding, with mutations in blade 4 showing the strongest effect.