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International Journal of Molecular... May 2018Gyrase is a type IIA topoisomerase that catalyzes negative supercoiling of DNA. The enzyme consists of two GyrA and two GyrB subunits. It is believed to introduce... (Review)
Review
Gyrase is a type IIA topoisomerase that catalyzes negative supercoiling of DNA. The enzyme consists of two GyrA and two GyrB subunits. It is believed to introduce negative supercoils into DNA by converting a positive DNA node into a negative node through strand passage: First, it cleaves both DNA strands of a double-stranded DNA, termed the G-segment, and then it passes a second segment of the same DNA molecule, termed the T-segment, through the gap created. As a two-fold symmetric enzyme, gyrase contains two copies of all elements that are key for the supercoiling reaction: The GyrB subunits provide two active sites for ATP binding and hydrolysis. The GyrA subunits contain two C-terminal domains (CTDs) for DNA binding and wrapping to stabilize the positive DNA node, and two catalytic tyrosines for DNA cleavage. While the presence of two catalytic tyrosines has been ascribed to the necessity of cleaving both strands of the G-segment to enable strand passage, the role of the two ATP hydrolysis events and of the two CTDs has been less clear. This review summarizes recent results on the role of these duplicate elements for individual steps of the supercoiling reaction, and discusses the implications for the mechanism of DNA supercoiling.
Topics: Animals; DNA; DNA Gyrase; DNA Topoisomerases, Type II; Humans; Nucleic Acid Conformation; Protein Subunits; Structure-Activity Relationship
PubMed: 29772727
DOI: 10.3390/ijms19051489 -
Molecular Microbiology Jan 2023Transcription is a noisy and stochastic process that produces sibling-to-sibling variations in physiology across a population of genetically identical cells. This... (Review)
Review
Transcription is a noisy and stochastic process that produces sibling-to-sibling variations in physiology across a population of genetically identical cells. This pattern of diversity reflects, in part, the burst-like nature of transcription. Transcription bursting has many causes and a failure to remove the supercoils that accumulate in DNA during transcription elongation is an important contributor. Positive supercoiling of the DNA ahead of the transcription elongation complex can result in RNA polymerase stalling if this DNA topological roadblock is not removed. The relaxation of these positive supercoils is performed by the ATP-dependent type II topoisomerases DNA gyrase and topoisomerase IV. Interference with the action of these topoisomerases involving, inter alia, topoisomerase poisons, fluctuations in the [ATP]/[ADP] ratio, and/or the intervention of nucleoid-associated proteins with GapR-like or YejK-like activities, may have consequences for the smooth operation of the transcriptional machinery. Antibiotic-tolerant (but not resistant) persister cells are among the phenotypic outliers that may emerge. However, interference with type II topoisomerase activity can have much broader consequences, making it an important epigenetic driver of physiological diversity in the bacterial population.
Topics: DNA; DNA Gyrase; DNA Topoisomerase IV; Bacteria; DNA Topoisomerases, Type I; Adenosine Triphosphate; Epigenesis, Genetic; DNA, Superhelical; DNA, Bacterial
PubMed: 36565252
DOI: 10.1111/mmi.15014 -
Biochemistry Jan 2006The pentapeptide repeat protein (PRP) family has more than 500 members in the prokaryotic and eukaryotic kingdoms. These proteins are composed of, or contain domains...
The pentapeptide repeat protein (PRP) family has more than 500 members in the prokaryotic and eukaryotic kingdoms. These proteins are composed of, or contain domains composed of, tandemly repeated amino acid sequences with a consensus sequence of [S,T,A,V][D,N][L,F][S,T,R][G]. The biochemical function of the vast majority of PRP family members is unknown. The three-dimensional structure of the first member of the PRP family was determined for the fluoroquinolone resistance protein (MfpA) from Mycobacterium tuberculosis. The structure revealed that the pentapeptide repeats encode the folding of a novel right-handed quadrilateral beta-helix. MfpA binds to DNA gyrase and inhibits its activity. The rod-shaped, dimeric protein exhibits remarkable similarity in size, shape, and electrostatics to DNA.
Topics: Bacterial Proteins; Base Sequence; DNA Gyrase; Dimerization; Drug Resistance, Microbial; Fluoroquinolones; Monomeric GTP-Binding Proteins; Mycobacterium tuberculosis; Oligopeptides; Protein Conformation; Static Electricity; Structure-Activity Relationship
PubMed: 16388575
DOI: 10.1021/bi052130w -
Molecules (Basel, Switzerland) Nov 2021Broad antibacterial spectrum, high oral bioavailability and excellent tissue penetration combined with safety and few, yet rare, unwanted effects, have made the... (Review)
Review
Broad antibacterial spectrum, high oral bioavailability and excellent tissue penetration combined with safety and few, yet rare, unwanted effects, have made the quinolones class of antimicrobials one of the most used in inpatients and outpatients. Initially discovered during the search for improved chloroquine-derivative molecules with increased anti-malarial activity, today the quinolones, intended as antimicrobials, comprehend four generations that progressively have been extending antimicrobial spectrum and clinical use. The quinolone class of antimicrobials exerts its antimicrobial actions through inhibiting DNA gyrase and Topoisomerase IV that in turn inhibits synthesis of DNA and RNA. Good distribution through different tissues and organs to treat Gram-positive and Gram-negative bacteria have made quinolones a good choice to treat disease in both humans and animals. The extensive use of quinolones, in both human health and in the veterinary field, has induced a rise of resistance and menace with leaving the quinolones family ineffective to treat infections. This review revises the evolution of quinolones structures, biological activity, and the clinical importance of this evolving family. Next, updated information regarding the mechanism of antimicrobial activity is revised. The veterinary use of quinolones in animal productions is also considered for its environmental role in spreading resistance. Finally, considerations for the use of quinolones in human and veterinary medicine are discussed.
Topics: Anti-Infective Agents; Bacterial Infections; DNA Gyrase; DNA Topoisomerase IV; DNA, Bacterial; Gram-Negative Bacteria; Gram-Positive Bacteria; Humans; Quinolones; RNA, Bacterial; Topoisomerase II Inhibitors
PubMed: 34885734
DOI: 10.3390/molecules26237153 -
Microbiology Spectrum Aug 2014The fluoroquinolones (FQs) are synthetic antibiotics effectively used for curing patients with multidrug-resistant tuberculosis (TB). When a multidrug-resistant strain... (Review)
Review
The fluoroquinolones (FQs) are synthetic antibiotics effectively used for curing patients with multidrug-resistant tuberculosis (TB). When a multidrug-resistant strain develops resistance to the FQs, as in extensively drug-resistant strains, obtaining a cure is much more difficult, and molecular methods can help by rapidly identifying resistance-causing mutations. The only mutations proven to confer FQ resistance in M. tuberculosis occur in the FQ target, the DNA gyrase, at critical amino acids from both the gyrase A and B subunits that form the FQ binding pocket. GyrA substitutions are much more common and generally confer higher levels of resistance than those in GyrB. Molecular techniques to detect resistance mutations have suboptimal sensitivity because gyrase mutations are not detected in a variable percentage of phenotypically resistant strains. The inability to find gyrase mutations may be explained by heteroresistance: bacilli with a resistance-conferring mutation are present only in a minority of the bacterial population (>1%) and are therefore detected by the proportion method, but not in a sufficient percentage to be reliably detected by molecular techniques. Alternative FQ resistance mechanisms in other bacteria--efflux pumps, pentapeptide proteins, or enzymes that inactivate the FQs--have not yet been demonstrated in FQ-resistant M. tuberculosis but may contribute to intrinsic levels of resistance to the FQs or induced tolerance leading to more frequent gyrase mutations. Moxifloxacin is currently the best anti-TB FQ and is being tested for use with other new drugs in shorter first-line regimens to cure drug-susceptible TB.
Topics: Antitubercular Agents; DNA Gyrase; Drug Resistance, Bacterial; Drug Therapy, Combination; Fluoroquinolones; Humans; Moxifloxacin; Mutant Proteins; Mutation, Missense; Mycobacterium tuberculosis; Tuberculosis
PubMed: 26104201
DOI: 10.1128/microbiolspec.MGM2-0009-2013 -
PLoS Genetics Oct 2020DNA supercoiling is essential for all living cells because it controls all processes involving DNA. In bacteria, global DNA supercoiling results from the opposing...
DNA supercoiling is essential for all living cells because it controls all processes involving DNA. In bacteria, global DNA supercoiling results from the opposing activities of topoisomerase I, which relaxes DNA, and DNA gyrase, which compacts DNA. These enzymes are widely conserved, sharing >91% amino acid identity between the closely related species Escherichia coli and Salmonella enterica serovar Typhimurium. Why, then, do E. coli and Salmonella exhibit different DNA supercoiling when experiencing the same conditions? We now report that this surprising difference reflects disparate activation of their DNA gyrases by the polyamine spermidine and its precursor putrescine. In vitro, Salmonella DNA gyrase activity was sensitive to changes in putrescine concentration within the physiological range, whereas activity of the E. coli enzyme was not. In vivo, putrescine activated the Salmonella DNA gyrase and spermidine the E. coli enzyme. High extracellular Mg2+ decreased DNA supercoiling exclusively in Salmonella by reducing the putrescine concentration. Our results establish the basis for the differences in global DNA supercoiling between E. coli and Salmonella, define a signal transduction pathway regulating DNA supercoiling, and identify potential targets for antibacterial agents.
Topics: DNA Gyrase; DNA Topoisomerases, Type I; DNA, Superhelical; Escherichia coli; Magnesium; Putrescine; Salmonella typhimurium; Spermidine
PubMed: 33125364
DOI: 10.1371/journal.pgen.1009085 -
Molecules (Basel, Switzerland) May 2022The increased use of polyphenols nowadays poses the need for identification of their new pharmacological targets. Recently, structure similarity-based virtual screening...
The increased use of polyphenols nowadays poses the need for identification of their new pharmacological targets. Recently, structure similarity-based virtual screening of DrugBank outlined pseudopurpurin, a hydroxyanthraquinone from spp., as similar to gatifloxacin, a synthetic antibacterial agent. This suggested the bacterial DNA gyrase and DNA topoisomerase IV as potential pharmacological targets of pseudopurpurin. In this study, estimation of structural similarity to referent antibacterial agents and molecular docking in the DNA gyrase and DNA topoisomerase IV complexes were performed for a homologous series of four hydroxyanthraquinones. Estimation of shape- and chemical feature-based similarity with (S)-gatifloxacin, a DNA gyrase inhibitor, and (S)-levofloxacin, a DNA topoisomerase IV inhibitor, outlined pseudopurpurin and munjistin as the most similar structures. The docking simulations supported the hypothesis for a plausible antibacterial activity of hydroxyanthraquinones. The predicted docking poses were grouped into 13 binding modes based on spatial similarities in the active site. The simultaneous presence of 1-OH and 3-COOH substituents in the anthraquinone scaffold were emphasized as relevant features for the binding modes' variability and ability of the compounds to strongly bind in the DNA-enzyme complexes. The results reveal new potential pharmacological targets of the studied polyphenols and help in their prioritization as drug candidates and dietary supplements.
Topics: Anti-Bacterial Agents; DNA Gyrase; DNA Topoisomerase IV; Gatifloxacin; Molecular Docking Simulation; Polyphenols; Rubia
PubMed: 35630751
DOI: 10.3390/molecules27103274 -
Structure (London, England : 1993) May 2020Most biological processes involve formation of transient complexes where binding of a ligand allosterically modulates function. The ccd toxin-antitoxin system is...
Most biological processes involve formation of transient complexes where binding of a ligand allosterically modulates function. The ccd toxin-antitoxin system is involved in plasmid maintenance and bacterial persistence. The CcdA antitoxin accelerates dissociation of CcdB from its complex with DNA gyrase, binds and neutralizes CcdB, but the mechanistic details are unclear. Using a series of experimental and computational approaches, we demonstrate the formation of transient ternary and quaternary CcdA:CcdB:gyrase complexes and delineate the molecular steps involved in the rejuvenation process. Binding of region 61-72 of CcdA to CcdB induces the vital structural and dynamic changes required to facilitate dissociation from gyrase, region 50-60 enhances the dissociation process through additional allosteric effects, and segment 37-49 prevents gyrase rebinding. This study provides insights into molecular mechanisms responsible for recovery of CcdB-poisoned cells from a persister-like state. Similar methodology can be used to characterize other important transient, macromolecular complexes.
Topics: Bacterial Proteins; Bacterial Toxins; Binding Sites; Cysteine; DNA Gyrase; Fluorescence Resonance Energy Transfer; Models, Molecular; Multiprotein Complexes; Mutation; Surface Plasmon Resonance
PubMed: 32294467
DOI: 10.1016/j.str.2020.03.006 -
International Journal of Molecular... Jul 2023Novel bacterial topoisomerase inhibitors (NBTIs) are an emerging class of antibacterials that target gyrase and topoisomerase IV. A hallmark of NBTIs is their ability to...
Novel bacterial topoisomerase inhibitors (NBTIs) are an emerging class of antibacterials that target gyrase and topoisomerase IV. A hallmark of NBTIs is their ability to induce gyrase/topoisomerase IV-mediated single-stranded DNA breaks and suppress the generation of double-stranded breaks. However, a previous study reported that some dioxane-linked amide NBTIs induced double-stranded DNA breaks mediated by gyrase. To further explore the ability of this NBTI subclass to increase double-stranded DNA breaks, we examined the effects of OSUAB-185 on DNA cleavage mediated by gyrase and topoisomerase IV. OSUAB-185 induced single-stranded and suppressed double-stranded DNA breaks mediated by gyrase. However, the compound stabilized both single- and double-stranded DNA breaks mediated by topoisomerase IV. The induction of double-stranded breaks does not appear to correlate with the binding of a second OSUAB-185 molecule and extends to fluoroquinolone-resistant topoisomerase IV, as well as type II enzymes from other bacteria and humans. The double-stranded DNA cleavage activity of OSUAB-185 and other dioxane-linked NBTIs represents a paradigm shift in a hallmark characteristic of NBTIs and suggests that some members of this subclass may have alternative binding motifs in the cleavage complex.
Topics: Humans; DNA Topoisomerase IV; Neisseria gonorrhoeae; DNA Gyrase; DNA Breaks, Double-Stranded; Topoisomerase II Inhibitors
PubMed: 37569485
DOI: 10.3390/ijms241512107 -
Microbiology Spectrum Jun 2023Mycobacterium avium, a member of the M. avium complex (MAC), is the major pathogen contributing to nontuberculous mycobacteria (NTM) infections worldwide....
Mycobacterium avium, a member of the M. avium complex (MAC), is the major pathogen contributing to nontuberculous mycobacteria (NTM) infections worldwide. Fluoroquinolones (FQs) are recommended for the treatment of macrolide-resistant MACs. The association of FQ resistance and mutations in the quinolone resistance-determining region (QRDR) of of M. avium is not yet clearly understood, as many FQ-resistant clinical M. avium isolates do not have such mutations. This study aimed to elucidate the role of amino acid substitution in the QRDR of M. avium GyrA in the development of FQ resistance. We found four clinical M. avium subsp. isolates with Asp-to-Gly change at position 95 (Asp95Gly) and Asp95Tyr mutations in that were highly resistant to FQs and had 2- to 32-fold-higher MICs than the wild-type (WT) isolates. To clarify the contribution of amino acid substitutions to FQ resistance, we produced recombinant WT GyrA, GyrB, and four GyrA mutant proteins (Ala91Val, Asp95Ala, Asp95Gly, and Asp95Tyr) to elucidate their potential role in FQ resistance, using them to perform FQ-inhibited DNA supercoiling assays. While all the mutant GyrAs contributed to the higher (1.3- to 35.6-fold) FQ 50% inhibitory concentration (IC) than the WT, Asp95Tyr was the most resistant mutant, with an IC 15- to 35.6-higher than that of the WT, followed by the Asp95Gly mutant, with an IC 12.5- to 17.6-fold higher than that of the WT, indicating that these amino acid substitutions significantly reduced the inhibitory activity of FQs. Our results showed that amino acid substitutions in the of M. avium contribute to FQ resistance. The emergence of fluoroquinolone (FQ) resistance has further compounded the control of emerging Mycobacterium avium-associated nontuberculous mycobacteria infections worldwide. For M. avium, the association of FQ resistance and mutations in the quinolone resistance-determining region (QRDR) of is not yet clearly understood. Here, we report that four clinical M. avium isolates with a mutation in the QRDR of were highly resistant to FQs. We further clarified the impact of mutations in the QRDR of GyrA proteins by performing FQ-inhibited DNA supercoiling assays. These results confirmed that, like in Mycobacterium tuberculosis, mutations in the QRDR of also strongly contribute to FQ resistance in M. avium. Since many FQ-resistant M. avium isolates do have these mutations, the detailed molecular mechanism of FQ resistance in M. avium needs further exploration.
Topics: Fluoroquinolones; Amino Acid Substitution; DNA Gyrase; Mycobacterium avium; Anti-Bacterial Agents; Mutation; Mycobacterium tuberculosis; Microbial Sensitivity Tests; Drug Resistance, Bacterial
PubMed: 37067420
DOI: 10.1128/spectrum.05088-22