scholarly journals Basic residues at the C-gate of DNA gyrase are involved in DNA supercoiling

2021 ◽  
pp. 101000
Author(s):  
Eric M. Smith ◽  
Alfonso Mondragón
Keyword(s):  
Dna Gyrase ◽  
Dna Supercoiling

scholarly journals Differential behaviors of Staphylococcus aureus and Escherichia coli type II DNA topoisomerases.

1996 ◽  
Vol 40 (12) ◽  
pp. 2714-2720 ◽  
Author(s):  
F Blanche ◽  
B Cameron ◽  
F X Bernard ◽  
L Maton ◽  
B Manse ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Strong Support ◽  
Dna Gyrase ◽  
Dna Supercoiling ◽  
Type Ii ◽  
Topoisomerase Iv ◽  
E Coli ◽  
Dna Relaxation

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.

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

1999 ◽  
Vol 43 (5) ◽  
pp. 1129-1136 ◽  
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.

scholarly journals Structural insight into negative DNA supercoiling by DNA gyrase, a bacterial type 2A DNA topoisomerase

2013 ◽  
Vol 41 (16) ◽  
pp. 7815-7827 ◽  
Author(s):  
Julie Papillon ◽  
Jean-François Ménétret ◽  
Claire Batisse ◽  
Reynald Hélye ◽  
Patrick Schultz ◽  
...  
Keyword(s):  
Dna Gyrase ◽  
Dna Supercoiling ◽  
Dna Topoisomerase ◽  
Gyrase A ◽  
Structural Insight ◽  
Bacterial Type ◽  
Insight Into

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

2003 ◽  
Vol 47 (3) ◽  
pp. 1037-1046 ◽  
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.

scholarly journals Extensive regulation compromises the extent to which DNA gyrase controls DNA supercoiling and growth rate ofEscherichia coli

1999 ◽  
Vol 266 (3) ◽  
pp. 865-877 ◽  
Author(s):  
Peter Ruhdal Jensen ◽  
Coen C. van der Weijden ◽  
Lars Bogø Jensen ◽  
Hans V. Westerhoff ◽  
Jacky L. Snoep
Keyword(s):  
Growth Rate ◽  
Dna Gyrase ◽  
Dna Supercoiling ◽  
Ofescherichia Coli

scholarly journals Monoclonal antibodies to mycobacterial DNA gyrase A inhibit DNA supercoiling activity

2001 ◽  
Vol 268 (7) ◽  
pp. 2038-2046 ◽  
Author(s):  
U. H. Manjunatha ◽  
S. Mahadevan ◽  
Sandhya S. Visweswariah ◽  
V. Nagaraja
Keyword(s):  
Monoclonal Antibodies ◽  
Dna Gyrase ◽  
Dna Supercoiling ◽  
Gyrase A

scholarly journals Discovery of Novel DNA Gyrase Inhibitors by High-Throughput Virtual Screening

2007 ◽  
Vol 51 (10) ◽  
pp. 3688-3698 ◽  
Author(s):  
David A. Ostrov ◽  
José A. Hernández Prada ◽  
Patrick E. Corsino ◽  
Kathryn A. Finton ◽  
Nhan Le ◽  
...  
Keyword(s):  
Small Molecules ◽  
Dna Gyrase ◽  
Binding Pocket ◽  
Small Region ◽  
Dna Supercoiling ◽  
Resistant Bacteria ◽  
Topoisomerase Iv ◽  
Antimicrobial Drugs ◽  
Drug Resistant Bacteria ◽  
Gyrase Inhibitors

ABSTRACT The bacterial type II topoisomerases DNA gyrase and topoisomerase IV are validated targets for clinically useful quinolone antimicrobial drugs. A significant limitation to widely utilized quinolone inhibitors is the emergence of drug-resistant bacteria due to an altered DNA gyrase. To address this problem, we have used structure-based molecular docking to identify novel drug-like small molecules that target sites distinct from those targeted by quinolone inhibitors. A chemical ligand database containing approximately 140,000 small molecules (molecular weight, <500) was molecularly docked onto two sites of Escherichia coli DNA gyrase targeting (i) a previously unexplored structural pocket formed at the dimer interface of subunit A and (ii) a small region of the ATP binding pocket on subunit B overlapping the site targeted by coumarin and cyclothialidine drugs. This approach identified several small-molecule compounds that inhibited the DNA supercoiling activity of purified E. coli DNA gyrase. These compounds are structurally unrelated to previously identified gyrase inhibitors and represent potential scaffolds for the optimization of novel antibacterial agents that act on fluoroquinolone-resistant strains.

scholarly journals Structural and mechanistic analysis of ATPase inhibitors targeting mycobacterial DNA gyrase

2020 ◽  
Vol 75 (10) ◽  
pp. 2835-2842
Author(s):  
Sara R Henderson ◽  
Clare E M Stevenson ◽  
Brandon Malone ◽  
Yelyzaveta Zholnerovych ◽  
Lesley A Mitchenall ◽  
...  
Keyword(s):  
Mycobacterium Smegmatis ◽  
Dna Gyrase ◽  
Binding Pocket ◽  
Dna Supercoiling ◽  
X Ray Crystallography ◽  
Mechanistic Analysis ◽  
Target Dna ◽  
Atpase Inhibitors ◽  
A Site ◽  
Made In

Abstract Objectives To evaluate the efficacy of two novel compounds against mycobacteria and determine the molecular basis of their action on DNA gyrase using structural and mechanistic approaches. Methods Redx03863 and Redx04739 were tested in antibacterial assays, and also against their target, DNA gyrase, using DNA supercoiling and ATPase assays. X-ray crystallography was used to determine the structure of the gyrase B protein ATPase sub-domain from Mycobacterium smegmatis complexed with the aminocoumarin drug novobiocin, and structures of the same domain from Mycobacterium thermoresistibile complexed with novobiocin, and also with Redx03863. Results Both compounds, Redx03863 and Redx04739, were active against selected Gram-positive and Gram-negative species, with Redx03863 being the more potent, and Redx04739 showing selectivity against M. smegmatis. Both compounds were potent inhibitors of the supercoiling and ATPase reactions of DNA gyrase, but did not appreciably affect the ATP-independent relaxation reaction. The structure of Redx03863 bound to the gyrase B protein ATPase sub-domain from M. thermoresistibile shows that it binds at a site adjacent to the ATP- and novobiocin-binding sites. We found that most of the mutations that we made in the Redx03863-binding pocket, based on the structure, rendered gyrase inactive. Conclusions Redx03863 and Redx04739 inhibit gyrase by preventing the binding of ATP. The fact that the Redx03863-binding pocket is distinct from that of novobiocin, coupled with the lack of activity of resistant mutants, suggests that such compounds could have potential to be further exploited as antibiotics.

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