New insights into the binding mode of pyridine-3-carboxamide inhibitors of E. coli DNA gyrase

Staphylococcus aureus gyrA and gyrB genes encoding DNA gyrase subunits were cloned and coexpressed in Escherichia coli under the control of the T7 promoter-T7 RNA polymerase system, leading to soluble gyrase which was purified to homogeneity. Purified gyrase was catalytically indistinguishable from the gyrase purified from S. aureus and did not contain detectable amounts of topoisomerases from the E. coli host. Topoisomerase IV subunits GrlA and GrlB from S. aureus were also expressed in E. coli and were separately purified to apparent homogeneity. Topoisomerase IV, which was reconstituted by mixing equimolar amounts of GrlA and GrlB, had both ATP-dependent decatenation and DNA relaxation activities in vitro. This enzyme was more sensitive than gyrase to inhibition by typical fluoroquinolone antimicrobial agents such as ciprofloxacin or sparfloxacin, adding strong support to genetic studies which indicate that topoisomerase IV is the primary target of fluoroquinolones in S. aureus. The results obtained with ofloxacin suggest that this fluoroquinolone could also primarily target gyrase. No cleavable complex could be detected with S. aureus gyrase upon incubation with ciprofloxacin or sparfloxacin at concentrations which fully inhibit DNA supercoiling. This suggests that these drugs do not stabilize the open DNA-gyrase complex, at least under standard in vitro incubation conditions, but are more likely to interfere primarily with the DNA breakage step, contrary to what has been reported with E. coli gyrase. Both S. aureus gyrase-catalyzed DNA supercoiling and S. aureus topoisomerase IV-catalyzed decatenation were dramatically stimulated by potassium glutamate or aspartate (500- and 50-fold by 700 and 350 mM glutamate, respectively), whereas topoisomerase IV-dependent DNA relaxation was inhibited 3-fold by 350 mM glutamate. The relevance of the effect of dicarboxylic amino acids on the activities of type II topoisomerases is discussed with regard to the intracellular osmolite composition of S. aureus.

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Streptococcus pneumoniae DNA Gyrase and Topoisomerase IV: Overexpression, Purification, and Differential Inhibition by Fluoroquinolones

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.5.1129 ◽

1999 ◽

Vol 43 (5) ◽

pp. 1129-1136 ◽

Cited By ~ 85

Author(s):

Xiao-Su Pan ◽

L. Mark Fisher

Keyword(s):

Streptococcus Pneumoniae ◽

Dna Gyrase ◽

Dna Supercoiling ◽

Topoisomerase Iv ◽

T7 Promoter ◽

Bacterial Killing ◽

E Coli ◽

Apparent Homogeneity ◽

Comparable Activity

ABSTRACT Streptococcus pneumoniae gyrA and gyrBgenes specifying the DNA gyrase subunits have been cloned into pET plasmid vectors under the control of an inducible T7 promoter and have been separately expressed in Escherichia coli. Soluble 97-kDa GyrA and 72-kDa GyrB proteins bearing polyhistidine tags at their respective C-terminal and N-terminal ends were purified to apparent homogeneity by one-step nickel chelate column chromatography and were free of host E. coli topoisomerase activity. Equimolar amounts of the gyrase subunits reconstituted ATP-dependent DNA supercoiling with comparable activity to gyrase of E. coli and Staphylococcus aureus. In parallel, S. pneumoniae topoisomerase IV ParC and ParE subunits were similarly expressed in E. coli, purified to near homogeneity as 93- and 73-kDa proteins, and shown to generate efficient ATP-dependent DNA relaxation and DNA decatenation activities. Using the purified enzymes, we examined the inhibitory effects of three paradigm fluoroquinolones—ciprofloxacin, sparfloxacin, and clinafloxacin—which previous genetic studies with S. pneumoniae suggested act preferentially through topoisomerase IV, through gyrase, and through both enzymes, respectively. Surprisingly, all three quinolones were more active in inhibiting purified topoisomerase IV than gyrase, with clinafloxacin showing the greatest inhibitory potency. Moreover, the tested agents were at least 25-fold more effective in stabilizing a cleavable complex (the relevant cytotoxic lesion) with topoisomerase IV than with gyrase, with clinafloxacin some 10- to 32-fold more potent against either enzyme, in line with its superior activity againstS. pneumoniae. The uniform target preference of the three fluoroquinolones for topoisomerase IV in vitro is in apparent contrast to the genetic data. We interpret these results in terms of a model for bacterial killing by quinolones in which cellular factors can modulate the effects of target affinity to determine the cytotoxic pathway.

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Quinolone-resistant mutants of escherichia coli DNA topoisomerase IV parC gene.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.3.710 ◽

1996 ◽

Vol 40 (3) ◽

pp. 710-714 ◽

Cited By ~ 53

Author(s):

Y Kumagai ◽

J I Kato ◽

K Hoshino ◽

T Akasaka ◽

K Sato ◽

...

Keyword(s):

Escherichia Coli ◽

Dna Gyrase ◽

Mutant Gene ◽

Missense Mutations ◽

Topoisomerase Iv ◽

Dna Topoisomerase ◽

E Coli ◽

A Subunit ◽

Resistant Strains ◽

Mutant Genes

Escherichia coli quinolone-resistant strains with mutations of the parC gene, which codes for a subunit of topoisomerase IV, were isolated from a quinolone-resistant gyrA mutant of DNA gyrase. Quinolone-resistant parC mutants were also identified among the quinolone-resistant clinical strains. The parC mutants became susceptible to quinolones by introduction of a parC+ plasmid. Introduction of the multicopy plasmids carrying the quinolone-resistant parC mutant gene resulted in an increase in MICs of quinolones for the parC+ and quinolone-resistant gyrA strain. Nucleotide sequences of the quinolone-resistant parC mutant genes were determined, and missense mutations at position Gly-78, Ser-80, or Glu-84, corresponding to those in the quinolone-resistance-determining region of DNA gyrase, were identified. These results indicate that topoisomerase IV is a target of quinolones in E. coli and suggest that the susceptibility of E. coli cells to quinolones is determined by sensitivity of the targets, DNA gyrase and topoisomerase IV.

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Decoding Antibacterial and Antibiofilm Properties of Cinnamon and Cardamom Essential Oils: A Combined Molecular Docking and Experimental Study

10.21203/rs.3.rs-955241/v1 ◽

2021 ◽

Author(s):

Elahe Pourkhosravani ◽

fatemeh dehghan nayeri ◽

Mitra Mohammadi Bazargani

Keyword(s):

Cell Attachment ◽

Antibacterial Properties ◽

Binding Mode ◽

Dilution Method ◽

Antibiofilm Activity ◽

Bacterial Strains ◽

Biofilm Growth ◽

Computational Docking ◽

E Coli ◽

Industrial Strains

Abstract This study sets out to compare the antibacterial and antibiofilm profiles of Ci/Ca EOs alone and in combination together against infectious bacterial strains. MIC assay was carried out to survey the effectiveness of prepared EOs by two-fold serial dilution method and MTT evaluation. Synergic antibacterial properties of EOs against target strains were studied by using checkerboard titration method. Biofilm growth and development were evaluated using CV and XTT reduction assays. Antibacterial activity was observed for EOs against both bacterial strains with stronger activity for CiEO against both bacteria. The synergistic antibacterial effect was observed only against B. subtilis. Based on the FIC index, combinations could not inhibit the growth of E. coli. The pure EOs and their combination inhibited cell attachment for both studied bacteria with stronger effect on E. coli. CV and XTT reduction assays results showed that Ci EO and its combination with CaEO had the highest antibiofilm activity at lowest MIC value 0.08% and 0.04/0.02% against biofilm formed by E. coli and B. subtilis respectively, indicating a high antibiofilm potential. Computational docking analyses also postulated that the active constituents of evaluated EOs have the potential to interact with different bacterial targets, suggested binding mode of action of EOs metabolites. By and large, synergistic anti-biofilm properties of EOs may provide further options for developing novel formula to inhibit a variety of infectious clinical and industrial strains without (or less) toxicity effects on human body.

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Inhibitory Activities of Gatifloxacin (AM-1155), a Newly Developed Fluoroquinolone, against Bacterial and Mammalian Type II Topoisomerases

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.10.2678 ◽

1998 ◽

Vol 42 (10) ◽

pp. 2678-2681 ◽

Cited By ~ 22

Author(s):

Masaya Takei ◽

Hideyuki Fukuda ◽

Tokutaro Yasue ◽

Masaki Hosaka ◽

Yasuo Oomori

Keyword(s):

Hela Cell ◽

Topoisomerase Ii ◽

Dna Gyrase ◽

Antibacterial Activities ◽

Type Ii ◽

Topoisomerase Iv ◽

E Coli ◽

Bacterial Enzyme ◽

Inhibitory Activities ◽

Bacterial Type

ABSTRACT We determined the inhibitory activities of gatifloxacin againstStaphylococcus aureus topoisomerase IV,Escherichia coli DNA gyrase, and HeLa cell topoisomerase II and compared them with those of several quinolones. The inhibitory activities of quinolones against these type II topoisomerases significantly correlated with their antibacterial activities or cytotoxicities (correlation coefficient [r] = 0.926 forS. aureus, r = 0.972 for E. coli, and r = 0.648 for HeLa cells). Gatifloxacin possessed potent inhibitory activities against bacterial type II topoisomerases (50% inhibitory concentration [IC50] = 13.8 μg/ml for S. aureustopoisomerase IV; IC50 = 0.109 μg/ml for E. coli DNA gyrase) but the lowest activity against HeLa cell topoisomerase II (IC50 = 265 μg/ml) among the quinolones tested. There was also a significant correlation between the inhibitory activities of quinolones against S. aureustopoisomerase IV and those against E. coli DNA gyrase (r = 0.969). However, the inhibitory activity against HeLa cell topoisomerase II did not correlate with that against either bacterial enzyme. The IC50 of gatifloxacin for HeLa cell topoisomerase II was 19 and was more than 2,400 times higher than that for S. aureus topoisomerase IV and that for E. coli DNA gyrase. These ratios were higher than those for other quinolones, indicating that gatifloxacin possesses a higher selectivity for bacterial type II topoisomerases.

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Cloning, Expression, and Enzymatic Characterization of Pseudomonas aeruginosaTopoisomerase IV

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.3.530 ◽

1999 ◽

Vol 43 (3) ◽

pp. 530-536 ◽

Cited By ~ 14

Author(s):

Takaaki Akasaka ◽

Yoshikuni Onodera ◽

Mayumi Tanaka ◽

Kenichi Sato

Keyword(s):

Dna Gyrase ◽

Fusion System ◽

Enzymatic Characterization ◽

Wild Type ◽

Inhibitory Effects ◽

Topoisomerase Iv ◽

Upstream Gene ◽

E Coli ◽

New Quinolones

ABSTRACT The topoisomerase IV subunit A gene, parC homolog, has been cloned and sequenced from Pseudomonas aeruginosa PAO1, with cDNA encoding the N-terminal region of Escherichia coli parC used as a probe. The homolog and its upstream gene were presumed to be parC and parE through sequence homology with the parC and parE genes of other organisms. The deduced amino acid sequence of ParC and ParE showed 33 and 32% identity with that of the P. aeruginosa DNA gyrase subunits, GyrA and GyrB, respectively, and 69 and 75% identity with that of E. coli ParC and ParE, respectively. The putative ParC and ParE proteins were overexpressed and separately purified by use of a fusion system with a maltose-binding protein, and their enzymatic properties were examined. The reconstituted enzyme had ATP-dependent decatenation activity, which is the main catalytic activity of bacterial topoisomerase IV, and relaxing activities but had no supercoiling activity. So, the cloned genes were identified asP. aeruginosa topoisomerase IV genes. The inhibitory effects of quinolones on the activities of topoisomerase IV and DNA gyrase were compared. The 50% inhibitory concentrations of quinolones for the decatenation activity of topoisomerase IV were from five to eight times higher than those for the supercoiling activities ofP. aeruginosa DNA gyrase. These results confirmed that topoisomerase IV is less sensitive to fluoroquinolones than is DNA gyrase and may be a secondary target of new quinolones in wild-typeP. aeruginosa.

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Molecular Cloning of the gyrA andgyrB Genes of Bacteroides fragilis Encoding DNA Gyrase

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.10.2423 ◽

1999 ◽

Vol 43 (10) ◽

pp. 2423-2429 ◽

Cited By ~ 19

Author(s):

Yoshikuni Onodera ◽

Kenichi Sato

Keyword(s):

Hot Spot ◽

Bacteroides Fragilis ◽

Dna Gyrase ◽

E Coli ◽

Important Target ◽

Mutant Strains ◽

Genes Encoding ◽

Gyrase A ◽

Selection For

ABSTRACT The genes encoding the DNA gyrase A and B subunits ofBacteroides fragilis were cloned and sequenced. ThegyrA and gyrB genes code for proteins of 845 and 653 amino acids, respectively. These proteins were expressed inEscherichia coli, and the combination of GyrA and GyrB exhibited ATP-dependent supercoiling activity. To analyze the role of DNA gyrase in quinolone resistance of B. fragilis, we isolated mutant strains by stepwise selection for resistance to increasing concentrations of levofloxacin. We analyzed the resistant mutants and showed that Ser-82 of GyrA, equivalent to resistance hot spot Ser-83 of GyrA in E. coli, was in each case replaced with Phe. These results suggest that DNA gyrase is an important target for quinolones in B. fragilis.

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1,2H hyperfine spectroscopy and DFT modeling unveil the demethylmenasemiquinone binding mode to E. coli nitrate reductase A (NarGHI)

Biochimica et Biophysica Acta (BBA) - Bioenergetics ◽

10.1016/j.bbabio.2020.148203 ◽

2020 ◽

Vol 1861 (8) ◽

pp. 148203

Author(s):

Maryam Seif Eddine ◽

Frédéric Biaso ◽

Julia Rendon ◽

Eric Pilet ◽

Bruno Guigliarelli ◽

...

Keyword(s):

Nitrate Reductase ◽

Binding Mode ◽

E Coli ◽

Dft Modeling ◽

Hyperfine Spectroscopy

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Molecular Basis for the Differential Quinolone Susceptibility of Mycobacterial DNA Gyrase

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01958-13 ◽

2014 ◽

Vol 58 (4) ◽

pp. 2013-2020 ◽

Cited By ~ 14

Author(s):

Rupesh Kumar ◽

Bhavani Shankar Madhumathi ◽

Valakunja Nagaraja

Keyword(s):

Differential Susceptibility ◽

Dna Gyrase ◽

Drug Binding ◽

Double Strand Breaks ◽

Type Ii ◽

Content Type ◽

Strand Breaks ◽

E Coli ◽

Dna Complex ◽

Negative Supercoils

ABSTRACTDNA gyrase is a type II topoisomerase that catalyzes the introduction of negative supercoils in the genomes of eubacteria. Fluoroquinolones (FQs), successful as drugs clinically, target the enzyme to trap the gyrase-DNA complex, leading to the accumulation of double-strand breaks in the genome. Mycobacteria are less susceptible to commonly used FQs. However, an 8-methoxy-substituted FQ, moxifloxacin (MFX), is a potent antimycobacterial, and a higher susceptibility of mycobacterial gyrase to MFX has been demonstrated. Although several models explain the mechanism of FQ action and gyrase-DNA-FQ interaction, the basis for the differential susceptibility of mycobacterial gyrase to various FQs is not understood. We have addressed the basis of the differential susceptibility of the gyrase and revisited the mode of action of FQs. We demonstrate that FQs bind bothEscherichia coliandMycobacterium tuberculosisgyrases in the absence of DNA and that the addition of DNA enhances the drug binding. The FQs bind primarily to the GyrA subunit of mycobacterial gyrase, while inE. coliholoenzyme is the target. The binding of MFX to GyrA ofM. tuberculosiscorrelates with its effectiveness as a better inhibitor of the enzyme and its efficacy in cell killing.

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Active-Site Residues of Escherichia coli DNA Gyrase Required in Coupling ATP Hydrolysis to DNA Supercoiling and Amino Acid Substitutions Leading to Novobiocin Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.3.1037-1046.2003 ◽

2003 ◽

Vol 47 (3) ◽

pp. 1037-1046 ◽

Cited By ~ 47

Author(s):

Christian H. Gross ◽

Jonathan D. Parsons ◽

Trudy H. Grossman ◽

Paul S. Charifson ◽

Steven Bellon ◽

...

Keyword(s):

Amino Acid ◽

Atp Hydrolysis ◽

Dna Gyrase ◽

Dna Supercoiling ◽

Wild Type ◽

Wild Type Enzyme ◽

Mutant Proteins ◽

E Coli ◽

Novobiocin Resistance

ABSTRACT DNA gyrase is a bacterial type II topoisomerase which couples the free energy of ATP hydrolysis to the introduction of negative supercoils into DNA. Amino acids in proximity to bound nonhydrolyzable ATP analog (AMP · PNP) or novobiocin in the gyrase B (GyrB) subunit crystal structures were examined for their roles in enzyme function and novobiocin resistance by site-directed mutagenesis. Purified Escherichia coli GyrB mutant proteins were complexed with the gyrase A subunit to form the functional A2B2 gyrase enzyme. Mutant proteins with alanine substitutions at residues E42, N46, E50, D73, R76, G77, and I78 had reduced or no detectable ATPase activity, indicating a role for these residues in ATP hydrolysis. Interestingly, GyrB proteins with P79A and K103A substitutions retained significant levels of ATPase activity yet demonstrated no DNA supercoiling activity, even with 40-fold more enzyme than the wild-type enzyme, suggesting that these amino acid side chains have a role in the coupling of the two activities. All enzymes relaxed supercoiled DNA to the same extent as the wild-type enzyme did, implying that only ATP-dependent reactions were affected. Mutant genes were examined in vivo for their abilities to complement a temperature-sensitive E. coli gyrB mutant, and the activities correlated well with the in vitro activities. We show that the known R136 novobiocin resistance mutations bestow a significant loss of inhibitor potency in the ATPase assay. Four new residues (D73, G77, I78, and T165) that, when changed to the appropriate amino acid, result in both significant levels of novobiocin resistance and maintain in vivo function were identified in E. coli.

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