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Proceedings of the National Academy of... Mar 2021DNA gyrase, a type II topoisomerase, introduces negative supercoils into DNA using ATP hydrolysis. The highly effective gyrase-targeted drugs, fluoroquinolones (FQs),...
DNA gyrase, a type II topoisomerase, introduces negative supercoils into DNA using ATP hydrolysis. The highly effective gyrase-targeted drugs, fluoroquinolones (FQs), interrupt gyrase by stabilizing a DNA-cleavage complex, a transient intermediate in the supercoiling cycle, leading to double-stranded DNA breaks. MfpA, a pentapeptide-repeat protein in mycobacteria, protects gyrase from FQs, but its molecular mechanism remains unknown. Here, we show that MfpA (MsMfpA) inhibits negative supercoiling by gyrase (Msgyrase) in the absence of FQs, while in their presence, MsMfpA decreases FQ-induced DNA cleavage, protecting the enzyme from these drugs. MsMfpA stimulates the ATPase activity of Msgyrase by directly interacting with the ATPase domain (MsGyrB47), which was confirmed through X-ray crystallography of the MsMfpA-MsGyrB47 complex, and mutational analysis, demonstrating that MsMfpA mimics a T (transported) DNA segment. These data reveal the molecular mechanism whereby MfpA modulates the activity of gyrase and may provide a general molecular basis for the action of other pentapeptide-repeat proteins.
Topics: Adenosine Triphosphatases; Bacterial Proteins; Crystallography, X-Ray; DNA Cleavage; DNA Gyrase; Molecular Mimicry; Monomeric GTP-Binding Proteins; Mycobacterium; Protein Conformation
PubMed: 33836580
DOI: 10.1073/pnas.2016705118 -
Antimicrobial Agents and Chemotherapy Sep 2022Tricyclic pyrrolopyrimidines (TPPs) are a new class of antibacterials inhibiting the ATPase of DNA gyrase. TPP8, a representative of this class, is active against...
Tricyclic pyrrolopyrimidines (TPPs) are a new class of antibacterials inhibiting the ATPase of DNA gyrase. TPP8, a representative of this class, is active against Mycobacterium abscessus . Spontaneous TPP8 resistance mutations mapped to the ATPase domain of M. abscessus DNA gyrase, and the compound inhibited DNA supercoiling activity of recombinant M. abscessus enzyme. Further profiling of TPP8 in macrophage and mouse infection studies demonstrated proof-of-concept activity against M. abscessus and .
Topics: Adenosine Triphosphatases; Animals; Anti-Bacterial Agents; DNA Gyrase; Mice; Microbial Sensitivity Tests; Mycobacterium Infections, Nontuberculous; Mycobacterium abscessus; Nontuberculous Mycobacteria; Pyrimidines; Pyrroles
PubMed: 36005813
DOI: 10.1128/aac.00669-22 -
Nature Chemical Biology Jun 2018Gyrase is an essential bacterial molecular motor that supercoils DNA using a conformational cycle in which chiral wrapping of > 100 base pairs confers directionality...
Gyrase is an essential bacterial molecular motor that supercoils DNA using a conformational cycle in which chiral wrapping of > 100 base pairs confers directionality on topoisomerization. To understand the mechanism of this nucleoprotein machine, global structural transitions must be mapped onto the nucleotide cycle of ATP binding, hydrolysis and product release. Here we investigate coupling mechanisms using single-molecule tracking of DNA rotation and contraction during Escherichia coli gyrase activity under varying nucleotide conditions. We find that ADP must be exchanged for ATP to drive the rate-limiting remodeling transition that generates the chiral wrap. ATP hydrolysis accelerates subsequent duplex strand passage and is required for resetting the enzyme and recapturing transiently released DNA. Our measurements suggest how gyrase coordinates DNA rearrangements with the dynamics of its ATP-driven protein gate, how the motor minimizes futile cycles of ATP hydrolysis and how gyrase may respond to changing cellular energy levels to link gene expression with metabolism.
Topics: Adenosine Diphosphate; Adenosine Triphosphate; DNA; DNA Gyrase; Escherichia coli; Gene Expression Profiling; Gene Expression Regulation; Hydrolysis; Kinetics; Models, Molecular; Nucleic Acid Conformation; Nucleotides; Protein Binding; Protein Conformation; Stereoisomerism
PubMed: 29662209
DOI: 10.1038/s41589-018-0037-0 -
Cell Reports Aug 2021The bacterial DNA gyrase complex (GyrA/GyrB) plays a crucial role during DNA replication and serves as a target for multiple antibiotics, including the fluoroquinolones....
The bacterial DNA gyrase complex (GyrA/GyrB) plays a crucial role during DNA replication and serves as a target for multiple antibiotics, including the fluoroquinolones. Despite it being a valuable antibiotics target, resistance emergence by pathogens including Pseudomonas aeruginosa are proving problematic. Here, we describe Igy, a peptide inhibitor of gyrase, encoded by Pseudomonas bacteriophage LUZ24 and other members of the Bruynoghevirus genus. Igy (5.6 kDa) inhibits in vitro gyrase activity and interacts with the P. aeruginosa GyrB subunit, possibly by DNA mimicry, as indicated by a de novo model of the peptide and mutagenesis. In vivo, overproduction of Igy blocks DNA replication and leads to cell death also in fluoroquinolone-resistant bacterial isolates. These data highlight the potential of discovering phage-inspired leads for antibiotics development, supported by co-evolution, as Igy may serve as a scaffold for small molecule mimicry to target the DNA gyrase complex, without cross-resistance to existing molecules.
Topics: DNA Gyrase; DNA Replication; DNA, Bacterial; Podoviridae; Pseudomonas Phages; Pseudomonas aeruginosa; Topoisomerase II Inhibitors; Viral Proteins
PubMed: 34433028
DOI: 10.1016/j.celrep.2021.109567 -
Journal of Molecular Biology Aug 2019Microcin B17 (MccB17) is an antibacterial peptide produced by strains of Escherichia coli harboring the plasmid-borne mccB17 operon. MccB17 possesses many notable... (Review)
Review
Microcin B17 (MccB17) is an antibacterial peptide produced by strains of Escherichia coli harboring the plasmid-borne mccB17 operon. MccB17 possesses many notable features. It is able to stabilize the transient DNA gyrase-DNA cleavage complex, a very efficient mode of action shared with the highly successful fluoroquinolone drugs. MccB17 stabilizes this complex by a distinct mechanism making it potentially valuable in the fight against bacterial antibiotic resistance. MccB17 was the first compound discovered from the thiazole/oxazole-modified microcins family and the linear azole-containing peptides; these ribosomal peptides are post-translationally modified to convert serine and cysteine residues into oxazole and thiazole rings. These chemical moieties are found in many other bioactive compounds like the vitamin thiamine, the anti-cancer drug bleomycin, the antibacterial sulfathiazole and the antiviral nitazoxanide. Therefore, the biosynthetic machinery that produces these azole rings is noteworthy as a general method to create bioactive compounds. Our knowledge of MccB17 now extends to many aspects of antibacterial-bacteria interactions: production, transport, interaction with its target, and resistance mechanisms; this knowledge has wide potential applicability. After a long time with limited progress on MccB17, recent publications have addressed critical aspects of MccB17 biosynthesis as well as an explosion in the discovery of new related compounds in the thiazole/oxazole-modified microcins/linear azole-containing peptides family. It is therefore timely to summarize the evidence gathered over more than 40 years about this still enigmatic molecule and place it in the wider context of antibacterials.
Topics: Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Bacteria; Bacteriocins; Cinoxacin; DNA Cleavage; DNA Gyrase; Drug Development; Drug Resistance, Microbial; Escherichia coli; Fluoroquinolones; Humans; Mutation; Nitro Compounds; Peptides; Protein Processing, Post-Translational; Thiazoles; Toxins, Biological
PubMed: 31181289
DOI: 10.1016/j.jmb.2019.05.050 -
Biomedicine & Pharmacotherapy =... Feb 2021DNA gyrase and Topoisomerase IV are promising antibacterial drug targets as they regulate bacterial DNA replication and topology. In a quest for novel DNA topoisomerase...
DNA gyrase and Topoisomerase IV are promising antibacterial drug targets as they regulate bacterial DNA replication and topology. In a quest for novel DNA topoisomerase inhibitors, a multidisciplinary approach was adopted that involves computational prediction of binding sites and molecular modelling followed by green synthesis and biological evaluation of antibacterial activity of spirobenzimidazo quinazolines derivatives. Using basic quantum chemistry principles, we evaluated spirobenzimidazo quinazolines derivatives with their pharmacokinetic profiles. Based on the results of the aforesaid in-silico studies, we synthesized a series of titled compounds using green synthetic methodology that were validated as potential antimicrobial agents. Quantum chemoinformatics based predicted activity for the synthesized compounds 9b, 9c, and 9j was concomitant with biological evaluation of broadspectrum antibacterial activity. Biological evaluation revealed that inhibition of biofilm formation was due to their potential antibacterial activity. We believe that the novel spirobenzimidazo quinazolines have the potential to be alternatives to aminocoumarins and classical quinazolines upon detailed target specific biological studies.
Topics: Anti-Bacterial Agents; Bacteria; Benzimidazoles; Binding Sites; Biofilms; Computer-Aided Design; DNA Gyrase; Drug Design; Green Chemistry Technology; Microbial Sensitivity Tests; Molecular Docking Simulation; Molecular Structure; Protein Binding; Quantitative Structure-Activity Relationship; Quinazolines; Topoisomerase II Inhibitors
PubMed: 33360050
DOI: 10.1016/j.biopha.2020.111132 -
European Journal of Medicinal Chemistry Jun 2018The ATP binding site located on the subunit B of DNA gyrase is an attractive target for the development of new antibacterial agents. In recent decades, several...
The ATP binding site located on the subunit B of DNA gyrase is an attractive target for the development of new antibacterial agents. In recent decades, several small-molecule inhibitor classes have been discovered but none has so far reached the market. We present here the discovery of a promising new series of N-phenylpyrrolamides with low nanomolar IC values against DNA gyrase, and submicromolar IC values against topoisomerase IV from Escherichia coli and Staphylococcus aureus. The most potent compound in the series has an IC value of 13 nM against E. coli gyrase. Minimum inhibitory concentrations (MICs) against Gram-positive bacteria are in the low micromolar range. The oxadiazolone derivative 11a, with an IC value of 85 nM against E. coli DNA gyrase displays the most potent antibacterial activity, with MIC values of 1.56 μM against Enterococcus faecalis, and 3.13 μM against wild type S. aureus, methicillin-resistant S. aureus (MRSA) and vancomycin-resistant Enterococcus (VRE). The activity against wild type E. coli in the presence of efflux pump inhibitor phenylalanine-arginine β-naphthylamide (PAβN) is 4.6 μM.
Topics: Amides; Anti-Bacterial Agents; DNA Gyrase; Dose-Response Relationship, Drug; Enterococcus faecalis; Escherichia coli; Microbial Sensitivity Tests; Molecular Structure; Pyrroles; Staphylococcus aureus; Structure-Activity Relationship; Topoisomerase II Inhibitors
PubMed: 29778894
DOI: 10.1016/j.ejmech.2018.05.011 -
Antimicrobial Agents and Chemotherapy Aug 2020Fluoroquinolones, antibiotics that cause DNA damage by inhibiting DNA topoisomerases, are clinically important, but their mechanism of action is not yet fully...
Fluoroquinolones, antibiotics that cause DNA damage by inhibiting DNA topoisomerases, are clinically important, but their mechanism of action is not yet fully understood. In particular, the dynamical response of bacterial cells to fluoroquinolone exposure has hardly been investigated, although the SOS response, triggered by DNA damage, is often thought to play a key role. Here, we investigated the growth inhibition of the bacterium by the fluoroquinolone ciprofloxacin at low concentrations. We measured the long-term and short-term dynamical response of the growth rate and DNA production rate to ciprofloxacin at both the population and single-cell levels. We show that, despite the molecular complexity of DNA metabolism, a simple roadblock-and-kill model focusing on replication fork blockage and DNA damage by ciprofloxacin-poisoned DNA topoisomerase II (gyrase) quantitatively reproduces long-term growth rates in the presence of ciprofloxacin. The model also predicts dynamical changes in the DNA production rate in wild-type and in a recombination-deficient mutant following a step-up of ciprofloxacin. Our work highlights that bacterial cells show a delayed growth rate response following fluoroquinolone exposure. Most importantly, our model explains why the response is delayed: it takes many doubling times to fragment the DNA sufficiently to inhibit gene expression. We also show that the dynamical response is controlled by the timescale of DNA replication and gyrase binding/unbinding to the DNA rather than by the SOS response, challenging the accepted view. Our work highlights the importance of including detailed biophysical processes in biochemical-systems models to quantitatively predict the bacterial response to antibiotics.
Topics: Anti-Bacterial Agents; Ciprofloxacin; DNA; DNA Gyrase; DNA Topoisomerase IV; DNA Topoisomerases, Type II; DNA, Bacterial; Escherichia coli; Fluoroquinolones; Mutation
PubMed: 32601161
DOI: 10.1128/AAC.02487-19 -
Revista Chilena de Infectologia :... Oct 2015Quinolones are a family of synthetic broad-spectrum antimicrobial drugs whose target is the synthesis of DNA. They directly inhibit DNA replication by interacting with... (Review)
Review
Quinolones are a family of synthetic broad-spectrum antimicrobial drugs whose target is the synthesis of DNA. They directly inhibit DNA replication by interacting with two enzymes; DNA gyrase and topoisomerase IV. They have been widely used for the treatment of several community and hospital acquired infections, in the food processing industry and in the agricultural field, making the increasing incidence of quinolone resistance a frequent problem associated with constant exposition to diverse microorganisms. Resistance may be achieved by three non-exclusive mechanisms; through chromosomic mutations in the Quinolone Resistance-Determining Regions of DNA gyrase and topoisomerase IV, by reducing the intracytoplasmic concentrations of quinolones actively or passively and by Plasmid-Mediated Quinolones-Resistance genes, [Qnr determinant genes of resistance to quinolones, variant gene of the aminoglycoside acetyltransferase (AAC(6')-Ib-c)] and encoding genes of efflux pumps (qepA and oqxAB)]. The future of quinolones is uncertain, however, meanwhile they continue to be used in an irrational way, increasing resistance to quinolones should remain as an area of primary priority for research.
Topics: Acetyltransferases; Anti-Bacterial Agents; DNA Gyrase; DNA Topoisomerase IV; Drug Resistance, Bacterial; Enterobacteriaceae; Humans; Quinolones
PubMed: 26633105
DOI: 10.4067/S0716-10182015000600002 -
PLoS Genetics May 2017Movement of the transcription machinery along a template alters DNA topology resulting in the accumulation of supercoils in DNA. The positive supercoils generated ahead...
Movement of the transcription machinery along a template alters DNA topology resulting in the accumulation of supercoils in DNA. The positive supercoils generated ahead of transcribing RNA polymerase (RNAP) and the negative supercoils accumulating behind impose severe topological constraints impeding transcription process. Previous studies have implied the role of topoisomerases in the removal of torsional stress and the maintenance of template topology but the in vivo interaction of functionally distinct topoisomerases with heterogeneous chromosomal territories is not deciphered. Moreover, how the transcription-induced supercoils influence the genome-wide recruitment of DNA topoisomerases remains to be explored in bacteria. Using ChIP-Seq, we show the genome-wide occupancy profile of both topoisomerase I and DNA gyrase in conjunction with RNAP in Mycobacterium tuberculosis taking advantage of minimal topoisomerase representation in the organism. The study unveils the first in vivo genome-wide interaction of both the topoisomerases with the genomic regions and establishes that transcription-induced supercoils govern their recruitment at genomic sites. Distribution profiles revealed co-localization of RNAP and the two topoisomerases on the active transcriptional units (TUs). At a given locus, topoisomerase I and DNA gyrase were localized behind and ahead of RNAP, respectively, correlating with the twin-supercoiled domains generated. The recruitment of topoisomerases was higher at the genomic loci with higher transcriptional activity and/or at regions under high torsional stress compared to silent genomic loci. Importantly, the occupancy of DNA gyrase, sole type II topoisomerase in Mtb, near the Ter domain of the Mtb chromosome validates its function as a decatenase.
Topics: DNA; DNA Gyrase; DNA Topoisomerases, Type I; DNA, Superhelical; DNA-Directed RNA Polymerases; Gene Expression Regulation, Bacterial; Genome, Bacterial; Genomics; Humans; Mycobacterium tuberculosis; Nucleotide Motifs; Signal Transduction; Transcription, Genetic
PubMed: 28463980
DOI: 10.1371/journal.pgen.1006754