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Valakunja Nagaraja - One of the best experts on this subject based on the ideXlab platform.
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yacg from escherichia coli is a specific endogenous inhibitor of DNA Gyrase
Nucleic Acids Research, 2008Co-Authors: Sugopa Sengupta, Valakunja NagarajaAbstract:We assign a function for a small protein, YacG encoded by Escherichia coli genome. The NMR structure of YacG shows the presence of an unusual zinc-finger motif. YacG was predicted to be a part of DNA Gyrase interactome based on protein–protein interaction network. We demonstrate that YacG inhibits all the catalytic activities of DNA Gyrase by preventing its DNA binding. Topoisomerase I and IV activities remain unaltered in the presence of YacG and its action appears to be restricted only to DNA Gyrase. The inhibition of the enzyme activity is due to the binding of YacG to carboxyl terminal domain of GyrB. Overexpression of YacG results in growth inhibition and alteration in DNA topology due to uncontrolled inhibition of Gyrase.
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Inhibition of DNA Gyrase activity by Mycobacterium smegmatis MurI
FEMS microbiology letters, 2008Co-Authors: Sugopa Sengupta, Valakunja NagarajaAbstract:Glutamate racemase (MurI) catalyzes the interconversion of l-glutamate to d-glutamate, one of the essential amino acids present in the peptidoglycan. In addition to this essential enzymatic function, MurI from Escherichia coli, Bacillus subtilis and Mycobacterium tuberculosis inhibit DNA Gyrase activity. A single gene for murI found in the Mycobacterium smegmatis genome was cloned and overexpressed in a homologous expression system to obtain a highly soluble enzyme. In addition to the racemization activity, M. smegmatis MurI inhibits DNA Gyrase activity by preventing DNA binding of Gyrase. The sequestration of the Gyrase by MurI results in inhibition of all reactions catalyzed by DNA Gyrase. More importantly, MurI overexpression in vivo in mycobacterial cells provides protection against the action of ciprofloxacin. The DNA Gyrase-inhibitory property thus appears to be a typical characteristic of MurI and would have probably evolved to either modulate the function of the essential housekeeping enzyme or to provide protection to Gyrase against Gyrase inhibitors, which cause double-strand breaks in the genome.
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A complex of DNA Gyrase and RNA polymerase fosters transcription in Mycobacterium smegmatis
Biochemical and biophysical research communications, 2006Co-Authors: Richa Gupta, Arnab China, U. H. Manjunatha, N.m. Ponnanna, Valakunja NagarajaAbstract:We report here the existence of a complex between RNA polymerase (RNAP) and DNA Gyrase in Mycobacterium smegmatis. The interaction between the two enzymes was detected during our attempts to purify DNA Gyrase from M. smegmatis. RNAP subunits co-eluted along with DNA Gyrase in two different affinity chromatography column procedures employed to purify the latter enzyme. A complex containing both the enzymes was isolated through gel filtration chromatography and sucrose density gradient centrifugation of the cell free extracts. The complex exhibited both DNA supercoiling and transcription activities. Reduction in the transcription activity of the complex in the presence of DNA Gyrase inhibitor indicates a role for DNA Gyrase in stimulating transcription.
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A monoclonal antibody that inhibits mycobacterial DNA Gyrase by a novel mechanism
Nucleic acids research, 2005Co-Authors: U. H. Manjunatha, Anthony Maxwell, Valakunja NagarajaAbstract:DNA Gyrase is a DNA topoisomerase indispensable for cellular functions in bacteria. We describe a novel, hitherto unknown, mechanism of specific inhibition of Mycobacterium smegmatis and Mycobacterium tuberculosis DNA Gyrase by a monoclonal antibody (mAb). Binding of the mAb did not affect either GyrA-GyrB or Gyrase-DNA interactions. More importantly, the ternary complex of Gyrase-DNA-mAb retained the ATPase activity of the enzyme and was competent to catalyse DNA cleavage-religation reactions, implying a new mode of action different from other classes of Gyrase inhibitors. DNA Gyrase purified from fluoroquinolone-resistant strains of M.tuberculosis and M.smegmatis were inhibited by the mAb. The absence of cross-resistance of the drug-resistant enzymes from two different sources to the antibody-mediated inhibition corroborates the new mechanism of inhibition. We suggest that binding of the mAb in the proximity of the primary dimer interface region of GyrA in the heterotetrameric enzyme appears to block the release of the transported segment after strand passage, leading to enzyme inhibition. The specific inhibition of mycobacterial DNA Gyrase with the mAb opens up new avenues for designing novel lead molecules for drug discovery and for probing Gyrase mechanism.
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Functional characterisation of mycobacterial DNA Gyrase: an efficient decatenase
Nucleic acids research, 2002Co-Authors: U. H. Manjunatha, Sandhya S. Visweswariah, Monalisa Chatterji, M. Dalal, Deshpande R. Radha, Valakunja NagarajaAbstract:A rapid single step immunoaffinity purification procedure is described for Mycobacterium smegmatis DNA Gyrase. The mycobacterial enzyme is a 340 kDa heterotetrameric protein comprising two subunits each of GyrA and GyrB, exhibiting subtle differences and similarities to the well-characterised Escherichia coli Gyrase. In contrast to E.coli Gyrase, the M.smegmatis enzyme exhibits strong decatenase activity at physiological Mg2+ concentrations. Further, the enzymes exhibited marked differences in ATPase activity, DNA binding characteristics and susceptibility to fluoroquinolones. The holoenzyme showed very low intrinsic ATPase activity and was stimulated 20-fold in the presence of DNA. The DNA-stimulated ATPase kinetics revealed apparent K0.5 and kcat of 0.68 mM and 0.39 s–1, respectively. The dissociation constant for DNA was found to be 9.2 nM, which is 20 times weaker than that of E.coli DNA Gyrase. The differences between the enzymes were further substantiated as they exhibited varied sensitivity to moxifloxacin and ciprofloxacin. In spite of these differences, mycobacterial DNA Gyrase is a functionally and mechanistically conserved enzyme and the variations in activity seem to reflect functional optimisation for its physiological role during mycobacterial genome replication.
Danijel Kikelj - One of the best experts on this subject based on the ideXlab platform.
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Hybrid Inhibitors of DNA Gyrase A and B: Design, Synthesis and Evaluation.
Pharmaceutics, 2020Co-Authors: Martina Durcik, Žiga Skok, Nace Zidar, Anamarija Zega, Janez Ilaš, Danijel Kikelj, Petra Szili, Gábor Draskovits, Tamás Révész, Ákos NyergesAbstract:The discovery of multi-targeting ligands of bacterial enzymes is an important strategy to combat rapidly spreading antimicrobial resistance. Bacterial DNA Gyrase and topoisomerase IV are validated targets for the development of antibiotics. They can be inhibited at their catalytic sites or at their ATP binding sites. Here we present the design of new hybrids between the catalytic inhibitor ciprofloxacin and ATP-competitive inhibitors that show low nanomolar inhibition of DNA Gyrase and antibacterial activity against Gram-negative pathogens. The most potent hybrid 3a has MICs of 0.5 µg/mL against Klebsiella pneumoniae, 4 µg/mL against Enterobacter cloacae, and 2 µg/mL against Escherichia coli. In addition, inhibition of mutant E. coli strains shows that these hybrid inhibitors interact with both subunits of DNA Gyrase (GyrA, GyrB), and that binding to both of these sites contributes to their antibacterial activity.
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Design, synthesis and biological evaluation of novel DNA Gyrase inhibitors and their siderophore mimic conjugates.
Bioorganic Chemistry, 2020Co-Authors: Andraž Lamut, Cristina D. Cruz, Žiga Skok, Michaela Barančoková, Nace Zidar, Anamarija Zega, Lucija Peterlin Mašič, Janez Ilaš, Päivi Tammela, Danijel KikeljAbstract:Abstract Bacterial DNA Gyrase is an important target for the development of novel antibacterial drugs, which are urgently needed because of high level of antibiotic resistance worldwide. We designed and synthesized new 4,5,6,7-tetrahydrobenzo[d]thiazole-based DNA Gyrase B inhibitors and their conjugates with siderophore mimics, which were introduced to increase the uptake of inhibitors into the bacterial cytoplasm. The most potent conjugate 34 had an IC50 of 58 nM against Escherichia coli DNA Gyrase and displayed MIC of 14 µg/mL against E. coli ΔtolC strain. Only minor improvements in the antibacterial activities against wild-type E. coli in low-iron conditions were seen for DNA Gyrase inhibitor – siderophore mimic conjugates.
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New N-phenyl-4,5-dibromopyrrolamides as DNA Gyrase B inhibitors
MedChemComm, 2019Co-Authors: Nace Zidar, Anamarija Zega, Lucija Peterlin Mašič, Janez Ilaš, Päivi Tammela, Tihomir Tomašič, Helena Macut, Danijel KikeljAbstract:Due to the rapid development of antimicrobial resistance, the discovery of new antibacterials is essential in the fight against potentially lethal infections. The DNA Gyrase B (GyrB) subunit of bacterial DNA Gyrase is an excellent target for the design of antibacterials, as it has been clinically validated by novobiocin. However, there are currently no drugs in clinical use that target GyrB. We prepared a new series of N-phenyl-4,5-dibromopyrrolamides and evaluated them against DNA Gyrase and against the structurally and functionally similar enzyme, topoisomerase IV. The most active compound, 28, had an IC50 of 20 nM against Escherichia coli DNA Gyrase. The IC50 values of 28 against Staphylococcus aureus DNA Gyrase, and E. coli and S. aureus topoisomerase IV were in the low micromolar range. However, the compounds evaluated did not show significant antibacterial activities against selected Gram-positive and Gram-negative bacteria. Our results indicate that for potent inhibition of DNA Gyrase, a combination of polar groups on the carboxylic end of the molecule and substituents that reach into the ‘lipophilic floor’ of the enzyme is required.
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Synthesis and Evaluation of N‐Phenylpyrrolamides as DNA Gyrase B Inhibitors
ChemMedChem, 2018Co-Authors: Martina Durcik, Michaela Barančoková, Janez Ilaš, Päivi Tammela, Danijel Kikelj, Tihomir Tomašič, Nace ZidarAbstract:ATP-competitive inhibitors of DNA Gyrase and topoisomerase IV are among the most interesting classes of antibacterial drugs that are unrepresented in the antibacterial pipeline. We developed 32 new N-phenylpyrrolamides and evaluated them against DNA Gyrase and topoisomerase IV from E. coli and Staphylococcus aureus. Antibacterial activities were studied against Gram-positive and Gram-negative bacterial strains. The most potent compound displayed an IC50 of 47micro nm against E.coli DNA Gyrase, and a minimum inhibitory concentration (MIC) of 12.5 microm against the Gram-positive Enterococcus faecalis. Some compounds displayed good antibacterial activities against an efflux-pump-deficient E. coli strain (MIC=6.25 microm) and against wild-type E. coli in the presence of efflux pump inhibitor PAbetaN (MIC=3.13 microm). Here we describe new findings regarding the structure-activity relationships of N-phenylpyrrolamide DNA Gyrase B inhibitors and investigate the factors that are important for the antibacterial activity of this class of compounds.
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Discovery of substituted oxadiazoles as a novel scaffold for DNA Gyrase inhibitors.
European Journal of Medicinal Chemistry, 2017Co-Authors: Žiga Jakopin, Michaela Barančoková, Janez Ilaš, Päivi Tammela, Tihomir Tomašič, Matjaž Brvar, Marija Sollner Dolenc, Danijel KikeljAbstract:DNA Gyrase and topoisomerase IV are type IIa topoisomerases that are essential bacterial enzymes required to oversee the topological state of DNA during transcription and replication processes. Their ATPase domains, GyrB and ParE, respectively, are recognized as viable targets for small molecule inhibitors, however, no synthetic or natural product GyrB/ParE inhibitors have so far reached the clinic for use as novel antibacterial agents, except for novobiocin which was withdrawn from the market. In the present study, a series of substituted oxadiazoles have been designed and synthesized as potential DNA Gyrase inhibitors. Structure-based optimization resulted in the identification of compound 35, displaying an IC50 of 1.2 μM for Escherichia coli DNA Gyrase, while also exhibiting a balanced low micromolar inhibition of E. coli topoisomerase IV and of the respective Staphylococcus aureus homologues. The most promising inhibitors identified from each series were ultimately evaluated against selected Gram-positive and Gram-negative bacterial strains, of which compound 35 inhibited Enterococcus faecalis with a MIC90 of 75 μM. Our study thus provides further insight into the structural requirements of substituted oxadiazoles for dual inhibition of DNA Gyrase and topoisomerase IV.
Patrick Forterre - One of the best experts on this subject based on the ideXlab platform.
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Mycobacterium tuberculosis DNA Gyrase possesses two functional GyrA-boxes.
Biochemical Journal, 2013Co-Authors: Aurélie Bouige, Patrick Forterre, Amélie Darmon, Jérémie Piton, Mélanie Roue, Stéphanie Petrella, Estelle Capton, Alexandra Aubry, Claudine MayerAbstract:In contrast with most bacteria which possess two type II topoisomerases (topoisomerase IV and DNA Gyrase), Mycobacterium tuberculosis possesses only one, DNA Gyrase, which is functionally a hybrid enzyme. Functional differences between the two type IIA topoisomerases are thought to be specified by a CTD (C-terminal DNA-binding domain), which controls DNA recognition. To explore the molecular mechanism responsible for the hybrid functions of the M. tuberculosis DNA Gyrase, we conducted a series of sequence analyses and structural and biochemical experiments with the isolated GyrA CTD and the holoenzyme. Although the CTD displayed a global structure similar to that of bona fide GyrA and ParC paralogues, it harbours a second key motif similar in all respects to that of the conserved GyrA-box sequence motif. Biochemical assays showed that the GyrA-box is responsible for DNA supercoiling, whereas the second GyrA-box-l (GyrA-box-like motif) is responsible for the enhanced decatenation activity, suggesting that the mechanistic originality of M. tuberculosis DNA Gyrase depends largely on the particular DNA path around the CTD allowed for by the presence of GyrA-box-l. The results of the present study also provide, through phylogenetic exploration of the entire Corynebacterineae suborder, a new and broader insight into the functional diversity of bacterial type IIA topoisomerases.
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[13] DNA Gyrase from Thermotoga maritima
Methods in enzymology, 2001Co-Authors: Olivier Guipaud, Patrick ForterreAbstract:Publisher Summary DNA Gyrases are members of the Topo IIA family of type II DNA topoisomerases. They are unique among these enzymes in their ability to introduce negative supercoiling in a covalently closed circular DNA at the expense of ATP. DNA Gyrase is a heterotetramer composed of two subunits, GyrA and GyrB. This chapter describes the purification of DNA Gyrase from T. maritima , a hyperthermophilic anaeorobic bacterium from the order of Thermotogales , with an optimal growth temperature of 80°. This hyperthermophile contains both a DNA Gyrase and a reverse Gyrase. Also, the plasmid pRQ7, from another Thermotoga species, has been found to be negatively supercoiled. This indicates that when the two enzymes are present in the same organism, DNA Gyrase activity predominates over that of the reverse Gyrase in vivo .
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Both DNA Gyrase and reverse Gyrase are present in the hyperthermophilic bacterium Thermotoga maritima
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Olivier Guipaud, Evelyne Marguet, Kenneth M. Noll, Claire Bouthier De La Tour, Patrick ForterreAbstract:Like all hyperthermophiles yet tested, the bacterium Thermotoga maritima contains a reverse Gyrase. Here we show that it contains also a DNA Gyrase. The genes top2A and top2B encoding the two subunits of a DNA Gyrase-like enzyme have been cloned and sequenced. The Top2A (type II DNA topoisomerase A protein) is more similar to GyrA (DNA Gyrase A protein) than to ParC [topoisomerase IV (Topo IV) C protein]. The difference is especially striking at the C-terminal domain, which differentiates DNA Gyrases from Topo IV. DNA Gyrase activity was detected in T. maritima and purified to homogeneity using a novobiocin-Sepharose column. This hyperhermophilic DNA Gyrase has an optimal activity around 82–86°C. In contrast to plasmids from hyperthermophilic archaea, which are from relaxed to positively supercoiled, we found that the plasmid pRQ7 from Thermotoga sp. RQ7 is negatively supercoiled. pRQ7 became positively supercoiled after addition of novobiocin to cell cultures, indicating that its negative supercoiling is due to the DNA Gyrase of the host strain. The findings concerning DNA Gyrase and negative supercoiling in Thermotogales put into question the role of reverse Gyrase in hyperthermophiles.
Claudine Mayer - One of the best experts on this subject based on the ideXlab platform.
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Mycobacterium tuberculosis DNA Gyrase possesses two functional GyrA-boxes.
Biochemical Journal, 2013Co-Authors: Aurélie Bouige, Patrick Forterre, Amélie Darmon, Jérémie Piton, Mélanie Roue, Stéphanie Petrella, Estelle Capton, Alexandra Aubry, Claudine MayerAbstract:In contrast with most bacteria which possess two type II topoisomerases (topoisomerase IV and DNA Gyrase), Mycobacterium tuberculosis possesses only one, DNA Gyrase, which is functionally a hybrid enzyme. Functional differences between the two type IIA topoisomerases are thought to be specified by a CTD (C-terminal DNA-binding domain), which controls DNA recognition. To explore the molecular mechanism responsible for the hybrid functions of the M. tuberculosis DNA Gyrase, we conducted a series of sequence analyses and structural and biochemical experiments with the isolated GyrA CTD and the holoenzyme. Although the CTD displayed a global structure similar to that of bona fide GyrA and ParC paralogues, it harbours a second key motif similar in all respects to that of the conserved GyrA-box sequence motif. Biochemical assays showed that the GyrA-box is responsible for DNA supercoiling, whereas the second GyrA-box-l (GyrA-box-like motif) is responsible for the enhanced decatenation activity, suggesting that the mechanistic originality of M. tuberculosis DNA Gyrase depends largely on the particular DNA path around the CTD allowed for by the presence of GyrA-box-l. The results of the present study also provide, through phylogenetic exploration of the entire Corynebacterineae suborder, a new and broader insight into the functional diversity of bacterial type IIA topoisomerases.
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Structural insights into the quinolone resistance mechanism of Mycobacterium tuberculosis DNA Gyrase.
PLoS ONE, 2010Co-Authors: Jérémie Piton, Stéphanie Petrella, Alexandra Aubry, Marc Delarue, Gwénaëlle André-leroux, Vincent Jarlier, Claudine MayerAbstract:Mycobacterium tuberculosis DNA Gyrase, an indispensable nanomachine involved in the regulation of DNA topology, is the only type II topoisomerase present in this organism and is hence the sole target for quinolone action, a crucial drug active against multidrug-resistant tuberculosis. To understand at an atomic level the quinolone resistance mechanism, which emerges in extensively drug resistant tuberculosis, we performed combined functional, biophysical and structural studies of the two individual domains constituting the catalytic DNA Gyrase reaction core, namely the Toprim and the breakage-reunion domains. This allowed us to produce a model of the catalytic reaction core in complex with DNA and a quinolone molecule, identifying original mechanistic properties of quinolone binding and clarifying the relationships between amino acid mutations and resistance phenotype of M. tuberculosis DNA Gyrase. These results are compatible with our previous studies on quinolone resistance. Interestingly, the structure of the entire breakage-reunion domain revealed a new interaction, in which the Quinolone-Binding Pocket (QBP) is blocked by the N-terminal helix of a symmetry-related molecule. This interaction provides useful starting points for designing peptide based inhibitors that target DNA Gyrase to prevent its binding to DNA.
Olivier Guipaud - One of the best experts on this subject based on the ideXlab platform.
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[13] DNA Gyrase from Thermotoga maritima
Methods in enzymology, 2001Co-Authors: Olivier Guipaud, Patrick ForterreAbstract:Publisher Summary DNA Gyrases are members of the Topo IIA family of type II DNA topoisomerases. They are unique among these enzymes in their ability to introduce negative supercoiling in a covalently closed circular DNA at the expense of ATP. DNA Gyrase is a heterotetramer composed of two subunits, GyrA and GyrB. This chapter describes the purification of DNA Gyrase from T. maritima , a hyperthermophilic anaeorobic bacterium from the order of Thermotogales , with an optimal growth temperature of 80°. This hyperthermophile contains both a DNA Gyrase and a reverse Gyrase. Also, the plasmid pRQ7, from another Thermotoga species, has been found to be negatively supercoiled. This indicates that when the two enzymes are present in the same organism, DNA Gyrase activity predominates over that of the reverse Gyrase in vivo .
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Both DNA Gyrase and reverse Gyrase are present in the hyperthermophilic bacterium Thermotoga maritima
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Olivier Guipaud, Evelyne Marguet, Kenneth M. Noll, Claire Bouthier De La Tour, Patrick ForterreAbstract:Like all hyperthermophiles yet tested, the bacterium Thermotoga maritima contains a reverse Gyrase. Here we show that it contains also a DNA Gyrase. The genes top2A and top2B encoding the two subunits of a DNA Gyrase-like enzyme have been cloned and sequenced. The Top2A (type II DNA topoisomerase A protein) is more similar to GyrA (DNA Gyrase A protein) than to ParC [topoisomerase IV (Topo IV) C protein]. The difference is especially striking at the C-terminal domain, which differentiates DNA Gyrases from Topo IV. DNA Gyrase activity was detected in T. maritima and purified to homogeneity using a novobiocin-Sepharose column. This hyperhermophilic DNA Gyrase has an optimal activity around 82–86°C. In contrast to plasmids from hyperthermophilic archaea, which are from relaxed to positively supercoiled, we found that the plasmid pRQ7 from Thermotoga sp. RQ7 is negatively supercoiled. pRQ7 became positively supercoiled after addition of novobiocin to cell cultures, indicating that its negative supercoiling is due to the DNA Gyrase of the host strain. The findings concerning DNA Gyrase and negative supercoiling in Thermotogales put into question the role of reverse Gyrase in hyperthermophiles.