RESUMO
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 Mycobacterium smegmatis MfpA (MsMfpA) inhibits negative supercoiling by M. smegmatis 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.
Assuntos
Proteínas de Bactérias/metabolismo , DNA Girase/metabolismo , Mimetismo Molecular , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Mycobacterium/enzimologia , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/química , Cristalografia por Raios X , Clivagem do DNA , Proteínas Monoméricas de Ligação ao GTP/química , Conformação ProteicaRESUMO
In the cell, DNA is arranged into highly-organised and topologically-constrained (supercoiled) structures. It remains unclear how this supercoiling affects the detailed double-helical structure of DNA, largely because of limitations in spatial resolution of the available biophysical tools. Here, we overcome these limitations, by a combination of atomic force microscopy (AFM) and atomistic molecular dynamics (MD) simulations, to resolve structures of negatively-supercoiled DNA minicircles at base-pair resolution. We observe that negative superhelical stress induces local variation in the canonical B-form DNA structure by introducing kinks and defects that affect global minicircle structure and flexibility. We probe how these local and global conformational changes affect DNA interactions through the binding of triplex-forming oligonucleotides to DNA minicircles. We show that the energetics of triplex formation is governed by a delicate balance between electrostatics and bonding interactions. Our results provide mechanistic insight into how DNA supercoiling can affect molecular recognition, that may have broader implications for DNA interactions with other molecular species.
Assuntos
Pareamento de Bases/genética , DNA Super-Helicoidal/química , Conformação de Ácido Nucleico , Oligonucleotídeos/química , DNA Circular/química , Microscopia de Força Atômica , Simulação de Dinâmica MolecularRESUMO
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.
Assuntos
Adenosina Trifosfatases , DNA Girase , Mycobacterium , Adenosina Trifosfatases/efeitos dos fármacos , Mycobacteriaceae , Novobiocina/farmacologia , Inibidores da Topoisomerase II/farmacologiaRESUMO
Bacterial DNA gyrase introduces negative supercoils into chromosomal DNA and relaxes positive supercoils introduced by replication and transiently by transcription. Removal of these positive supercoils is essential for replication fork progression and for the overall unlinking of the two duplex DNA strands, as well as for ongoing transcription. To address how gyrase copes with these topological challenges, we used high-speed single-molecule fluorescence imaging in live Escherichia coli cells. We demonstrate that at least 300 gyrase molecules are stably bound to the chromosome at any time, with â¼12 enzymes enriched near each replication fork. Trapping of reaction intermediates with ciprofloxacin revealed complexes undergoing catalysis. Dwell times of â¼2 s were observed for the dispersed gyrase molecules, which we propose maintain steady-state levels of negative supercoiling of the chromosome. In contrast, the dwell time of replisome-proximal molecules was â¼8 s, consistent with these catalyzing processive positive supercoil relaxation in front of the progressing replisome.
Assuntos
DNA Girase/química , DNA Super-Helicoidal/química , Proteínas de Ligação a DNA/química , Escherichia coli/enzimologia , Catálise , DNA Girase/genética , DNA Girase/isolamento & purificação , DNA Super-Helicoidal/genética , DNA Super-Helicoidal/isolamento & purificação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Ligação Proteica , Imagem Individual de MoléculaRESUMO
The search for compounds with biological activity for many diseases is turning increasingly to drug repurposing. In this study, we have focused on the European Union-approved antimalarial pyronaridine which was found to have in vitro activity against Mycobacterium tuberculosis (MIC 5⯵g/mL). In macromolecular synthesis assays, pyronaridine resulted in a severe decrease in incorporation of 14C-uracil and 14C-leucine similar to the effect of rifampicin, a known inhibitor of M. tuberculosis RNA polymerase. Surprisingly, the co-administration of pyronaridine (2.5⯵g/ml) and rifampicin resulted in in vitro synergy with an MIC 0.0019-0.0009⯵g/mL. This was mirrored in a THP-1 macrophage infection model, with a 16-fold MIC reduction for rifampicin when the two compounds were co-administered versus rifampicin alone. Docking pyronaridine in M. tuberculosis RNA polymerase suggested the potential for it to bind outside of the RNA polymerase rifampicin binding pocket. Pyronaridine was also found to have activity against a M. tuberculosis clinical isolate resistant to rifampicin, and when combined with rifampicin (10% MIC) was able to inhibit M. tuberculosis RNA polymerase in vitro. All these findings, and in particular the synergistic behavior with the antitubercular rifampicin, inhibition of RNA polymerase in combination in vitro and its current use as a treatment for malaria, may suggest that pyronaridine could also be used as an adjunct for treatment against M. tuberculosis infection. Future studies will test potential for in vivo synergy, clinical utility and attempt to develop pyronaridine analogs with improved potency against M. tuberculosis RNA polymerase when combined with rifampicin.
Assuntos
Antibióticos Antituberculose/farmacologia , Antimaláricos/farmacologia , Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Mycobacterium tuberculosis/efeitos dos fármacos , Naftiridinas/farmacologia , Rifampina/farmacologia , Antimaláricos/química , Antituberculosos/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Reposicionamento de Medicamentos , Farmacorresistência Bacteriana , Sinergismo Farmacológico , Quimioterapia Combinada , Humanos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Naftiridinas/química , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Células THP-1RESUMO
DNA in intracellular Salmonella enterica serovar Typhimurium relaxes during growth in the acidified (pH 4-5) macrophage vacuole and DNA relaxation correlates with the upregulation of Salmonella genes involved in adaptation to the macrophage environment. Bacterial ATP levels did not increase during adaptation to acid pH unless the bacterium was deficient in MgtC, a cytoplasmic-membrane-located inhibitor of proton-driven F1 F0 ATP synthase activity. Inhibiting ATP binding by DNA gyrase and topo IV with novobiocin enhanced the effect of low pH on DNA relaxation. Bacteria expressing novobiocin-resistant (NovR ) derivatives of gyrase or topo IV also exhibited DNA relaxation at acid pH, although further relaxation with novobiocin was not seen in the strain with NovR gyrase. Thus, inhibition of the negative supercoiling activity of gyrase was the primary cause of enhanced DNA relaxation in drug-treated bacteria. The Salmonella cytosol reaches pH 5-6 in response to an external pH of 4-5: the ATP-dependent DNA supercoiling activity of purified gyrase was progressively inhibited by lowering the pH in this range, as was the ATP-dependent DNA relaxation activity of topo IV. We propose that DNA relaxation in Salmonella within macrophage is due to acid-mediated impairment of the negative supercoiling activity of gyrase.
Assuntos
DNA Girase/metabolismo , DNA Super-Helicoidal/metabolismo , Salmonella typhi/genética , Inibidores da Topoisomerase II/metabolismo , Adaptação Fisiológica/genética , DNA Girase/genética , DNA Topoisomerase IV/genética , DNA Topoisomerases Tipo I , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA Super-Helicoidal/genética , Concentração de Íons de Hidrogênio , Novobiocina/farmacologia , Conformação de Ácido Nucleico , Salmonella typhi/metabolismo , Salmonella typhimurium/genética , Estresse Fisiológico/genéticaRESUMO
OBJECTIVE: Agarose gel electrophoresis has been the mainstay technique for the analysis of DNA samples of moderate size. In addition to separating linear DNA molecules, it can also resolve different topological forms of plasmid DNAs, an application useful for the analysis of the reactions of DNA topoisomerases. However, gel electrophoresis is an intrinsically low-throughput technique and suffers from other potential disadvantages. We describe the application of the QIAxcel Advanced System, a high-throughput capillary electrophoresis system, to separate DNA topoisomers, and compare this technique with gel electrophoresis. RESULTS: We prepared a range of topoisomers of plasmids pBR322 and pUC19, and a 339 bp DNA minicircle, and compared their separation by gel electrophoresis and the QIAxcel System. We found superior resolution with the QIAxcel System, and that quantitative analysis of topoisomer distributions was straightforward. We show that the QIAxcel system has advantages in terms of speed, resolution and cost, and can be applied to DNA circles of various sizes. It can readily be adapted for use in compound screening against topoisomerase targets.
Assuntos
DNA Super-Helicoidal/análise , DNA/análise , Eletroforese em Gel de Ágar/métodos , Eletroforese Capilar/métodos , DNA/genética , DNA Girase/metabolismo , DNA Topoisomerases/metabolismo , DNA Super-Helicoidal/genética , DNA Super-Helicoidal/metabolismo , Plasmídeos/genética , Reprodutibilidade dos TestesRESUMO
Objectives: Cross-resistance between antibiotics and biocides is a potentially important driver of MDR. A relationship between susceptibility of Salmonella to quinolones and triclosan has been observed. This study aimed to: (i) investigate the mechanism underpinning this; (ii) determine whether the phenotype is conserved in Escherichia coli; and (iii) evaluate the potential for triclosan to select for quinolone resistance. Methods: WT E. coli, Salmonella enterica serovar Typhimurium and gyrA mutants were used. These were characterized by determining antimicrobial susceptibility, DNA gyrase activity and sensitivity to inhibition. Expression of stress response pathways (SOS, RpoS, RpoN and RpoH) was measured, as was the fitness of mutants. The potential for triclosan to select for quinolone resistance was determined. Results: All gyrase mutants showed increased triclosan MICs and altered supercoiling activity. There was no evidence for direct interaction between triclosan and gyrase. Identical substitutions in GyrA had different impacts on supercoiling in the two species. For both, there was a correlation between altered supercoiling and expression of stress responses. This was more marked in E. coli, where an Asp87Gly GyrA mutant demonstrated greatly increased fitness in the presence of triclosan. Exposure of parental strains to low concentrations of triclosan did not select for quinolone resistance. Conclusions: Our data suggest gyrA mutants are less susceptible to triclosan due to up-regulation of stress responses. The impact of gyrA mutation differs between E. coli and Salmonella. The impacts of gyrA mutation beyond quinolone resistance have implications for the fitness and selection of gyrA mutants in the presence of non-quinolone antimicrobials.
Assuntos
Antibacterianos/farmacologia , DNA Girase/genética , Desinfetantes/farmacologia , Mutação/efeitos dos fármacos , Quinolonas/farmacologia , Triclosan/farmacologia , DNA Girase/metabolismo , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/genética , Aptidão Genética , Humanos , Testes de Sensibilidade Microbiana , Fenótipo , Salmonella typhimurium , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genéticaRESUMO
The Arabidopsis thaliana genome contains four genes that were originally annotated as potentially encoding DNA gyrase: ATGYRA, ATGYRB1, ATGYRB2, and ATGYRB3. Although we subsequently showed that ATGYRB3 does not encode a gyrase subunit, the other three genes potentially encode subunits of a plant gyrase. We also showed evidence for the existence of supercoiling activity in A. thaliana and that the plant is sensitive to quinolone and aminocoumarin antibiotics, compounds that target DNA gyrase in bacteria. However, it was not possible at that time to show whether the A. thaliana genes encoded an active gyrase enzyme, nor whether that enzyme is indeed the target for the quinolone and aminocoumarin antibiotics. Here we show that an A. thaliana mutant resistant to the quinolone drug ciprofloxacin has a point mutation in ATGYRA. Moreover we show that, as in bacteria, the quinolone-sensitive (wild-type) allele is dominant to the resistant gene. Further we have heterologously expressed ATGYRA and ATGYRB2 in a baculovirus expression system and shown supercoiling activity of the partially purified enzyme. Expression/purification of the quinolone-resistant A. thaliana gyrase yields active enzyme that is resistant to ciprofloxacin. Taken together these experiments now show unequivocally that A. thaliana encodes an organelle-targeted DNA gyrase that is the target of the quinolone drug ciprofloxacin; this has important consequences for plant physiology and the development of herbicides.
Assuntos
Antibacterianos/farmacologia , Proteínas de Arabidopsis/antagonistas & inibidores , Arabidopsis/enzimologia , Cloroplastos/efeitos dos fármacos , Ciprofloxacina/farmacologia , DNA Girase/metabolismo , Inibidores da Topoisomerase II/farmacologia , Substituição de Aminoácidos , Animais , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cloroplastos/enzimologia , Cloroplastos/ultraestrutura , DNA Girase/química , DNA Girase/genética , DNA Girase/isolamento & purificação , Resistência a Medicamentos , Técnicas de Inativação de Genes , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Mitocôndrias/ultraestrutura , Forma das Organelas/efeitos dos fármacos , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/ultraestrutura , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/ultraestrutura , Mutação Puntual , Conformação Proteica , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/genética , Subunidades Proteicas/isolamento & purificação , Subunidades Proteicas/metabolismo , Interferência de RNA , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Células Sf9 , SpodopteraRESUMO
DNA topoisomerases control the topology of DNA. Type II topoisomerases exhibit topology simplification, whereby products of their reactions are simplified beyond that expected based on thermodynamic equilibrium. The molecular basis for this process is unknown, although DNA bending has been implicated. To investigate the role of bending in topology simplification, the DNA bend angles of four enzymes of different types (IIA and IIB) were measured using atomic force microscopy (AFM). The enzymes tested were Escherichia coli topo IV and yeast topo II (type IIA enzymes that exhibit topology simplification), and Methanosarcina mazei topo VI and Sulfolobus shibatae topo VI (type IIB enzymes, which do not). Bend angles were measured using the manual tangent method from topographical AFM images taken with a novel amplitude-modulated imaging mode: small amplitude small set-point (SASS), which optimises resolution for a given AFM tip size and minimises tip convolution with the sample. This gave improved accuracy and reliability and revealed that all 4 topoisomerases bend DNA by a similar amount: ~120° between the DNA entering and exiting the enzyme complex. These data indicate that DNA bending alone is insufficient to explain topology simplification and that the 'exit gate' may be an important determinant of this process.
Assuntos
DNA Topoisomerases Tipo II/metabolismo , DNA/química , DNA/metabolismo , Microscopia de Força Atômica , Peso Molecular , Conformação de Ácido Nucleico , Ligação ProteicaRESUMO
DNA topoisomerases are highly exploited targets for antimicrobial drugs. The spread of antibiotic resistance represents a significant threat to public health and necessitates the discovery of inhibitors that target topoisomerases in novel ways. However, the traditional assays for topoisomerase activity are not suitable for the high-throughput approaches necessary for drug discovery. In this study we validate a novel assay for screening topoisomerase inhibitors. A library of 960 compounds was screened against Escherichia coli DNA gyrase and archaeal Methanosarcina mazei DNA topoisomerase VI. Several novel inhibitors were identified for both enzymes, and subsequently characterised in vitro and in vivo. Inhibitors from the M. mazei topoisomerase VI screen were tested for their ability to inhibit Arabidopsis topoisomerase VI in planta. The data from this work present new options for antibiotic drug discovery and provide insight into the mechanism of topoisomerase VI.
Assuntos
Proteínas Arqueais/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores da Topoisomerase II , Inibidores da Topoisomerase/farmacologia , Antraquinonas/farmacologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/enzimologia , Arabidopsis/crescimento & desenvolvimento , DNA Topoisomerases Tipo II , Avaliação Pré-Clínica de Medicamentos/instrumentação , Escherichia coli/enzimologia , Hexilresorcinol/farmacologia , Methanosarcina/enzimologia , Mitoxantrona/farmacologia , Quinacrina/farmacologia , Sulfolobus/enzimologia , Suramina/farmacologiaRESUMO
Tuberculosis and other bacterial diseases represent a significant threat to human health. The DNA topoisomerases are excellent targets for chemotherapy, and DNA gyrase in particular is a well-validated target for antibacterial agents. Naphthoquinones (e.g. diospyrin and 7-methyljuglone) have been shown to have therapeutic potential, particularly against Mycobacterium tuberculosis. We have found that these compounds are inhibitors of the supercoiling reaction catalyzed by M. tuberculosis gyrase and other gyrases. Our evidence strongly suggests that the compounds bind to the N-terminal domain of GyrB, which contains the ATPase active site, but are not competitive inhibitors of the ATPase reaction. We propose that naphthoquinones bind to GyrB at a novel site close to the ATPase site. This novel mode of action could be exploited to develop new antibacterial agents.
Assuntos
DNA Girase/química , Naftoquinonas/química , Trifosfato de Adenosina/química , Anti-Infecciosos/farmacologia , Sítios de Ligação , Domínio Catalítico , DNA/genética , DNA Girase/metabolismo , Escherichia coli/metabolismo , Humanos , Concentração Inibidora 50 , Espectrometria de Massas/métodos , Modelos Químicos , Mycobacterium tuberculosis/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Staphylococcus aureus/metabolismo , Ressonância de Plasmônio de Superfície , Tuberculose/tratamento farmacológicoRESUMO
Bacterial toxin-antitoxin (TA) systems encode two proteins, a potent inhibitor of cell proliferation (toxin) and its specific antidote (antitoxin). Structural data has revealed striking similarities between the two model TA toxins CcdB, a DNA gyrase inhibitor encoded by the ccd system of plasmid F, and Kid, a site-specific endoribonuclease encoded by the parD system of plasmid R1. While a common structural fold seemed at odds with the two clearly different modes of action of these toxins, the possibility of functional crosstalk between the parD and ccd systems, which would further point to their common evolutionary origin, has not been documented. Here, we show that the cleavage of RNA and the inhibition of protein synthesis by the Kid toxin, two activities that are specifically counteracted by its cognate Kis antitoxin, are altered, but not inhibited, by the CcdA antitoxin. In addition, Kis was able to inhibit the stimulation of DNA gyrase-mediated cleavage of DNA by CcdB, albeit less efficiently than CcdA. We further show that physical interactions between the toxins and antitoxins of the different systems do occur and define the stoichiometry of the complexes formed. We found that CcdB did not degrade RNA nor did Kid have any reproducible effect on the tested DNA gyrase activities, suggesting that these toxins evolved to reach different, rather than common, cellular targets.
Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Proteínas de Escherichia coli/química , Proteínas de Bactérias/genética , Cloranfenicol/química , Clivagem do DNA , DNA Circular/química , Proteínas de Ligação a DNA/genética , Estabilidade Enzimática , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Evolução Molecular , Luciferases/biossíntese , Peso Molecular , Ligação Proteica , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas/química , Clivagem do RNA , RNA Bacteriano/químicaRESUMO
DNA gyrase is the only type II topoisomerase in Mycobacterium tuberculosis and needs to catalyse DNA supercoiling, relaxation and decatenation reactions in order to fulfil the functions normally carried out by gyrase and DNA topoisomerase IV in other bacteria. We have obtained evidence for the existence of a Ca(2+)-binding site in the GyrA subunit of M. tuberculosis gyrase. Ca(2+) cannot support topoisomerase reactions in the absence of Mg(2+), but partial removal of Ca(2+) from GyrA by dialysis against EGTA leads to a modest loss in relaxation activity that can be restored by adding back Ca(2+). More extensive removal of Ca(2+) by denaturation of GyrA and dialysis against EGTA results in an enzyme with greatly reduced enzyme activities. Mutation of the proposed Ca(2+)-binding residues also leads to loss of activity. We propose that Ca(2+) has a regulatory role in M. tuberculosis gyrase and suggest a model for the modulation of gyrase activity by Ca(2+) binding.
Assuntos
Cálcio/fisiologia , DNA Girase/química , Mycobacterium tuberculosis/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Cálcio/química , Biologia Computacional , Clivagem do DNA , DNA Girase/genética , DNA Girase/metabolismo , DNA Super-Helicoidal/metabolismo , Escherichia coli/enzimologia , Magnésio , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteólise , Alinhamento de SequênciaRESUMO
In a previous study, we presented the dimer structure of DNA gyrase B' domain (GyrB C-terminal domain) from Mycobacterium tuberculosis and proposed a 'sluice-like' model for T-segment transport. However, the role of the dimer structure is still not well understood. Cross-linking and analytical ultracentrifugation experiments showed that the dimer structure exists both in the B' protein and in the full-length GyrB in solution. The cross-linked dimer of GyrB bound GyrA very weakly, but bound dsDNA with a much higher affinity than that of the monomer state. Using cross-linking and far-western analyses, the dimer state of GyrB was found to be involved in the ternary GyrA-GyrB-DNA complex. The results of mutational studies reveal that the dimer structure represents a state before DNA cleavage. Additionally, these results suggest that the dimer might also be present between the cleavage and reunion steps during processive transport.
Assuntos
DNA Girase/química , DNA/química , Mycobacterium tuberculosis/enzimologia , Biocatálise , DNA/metabolismo , Clivagem do DNA , DNA Girase/metabolismo , Dimerização , Modelos Moleculares , Estrutura Terciária de Proteína , Subunidades Proteicas/metabolismoRESUMO
OBJECTIVES: Aminocoumarin antibiotics are potent inhibitors of bacterial DNA gyrase. We investigated the inhibitory and antibacterial activity of naturally occurring aminocoumarin antibiotics and six structural analogues (novclobiocins) against DNA gyrase and DNA topoisomerase IV from Escherichia coli and Staphylococcus aureus as well as the effect of potassium and sodium glutamate on the activity of these enzymes. METHODS: The inhibitory concentrations of the aminocoumarins were determined in gyrase supercoiling assays and topoisomerase IV decatenation assays. Both subunits of S. aureus topoisomerase IV were purified as His-Tag proteins in E. coli. The MIC was tested in vivo for the control organisms E. coli ATCC 25922 and S. aureus ATCC 29213. RESULTS: DNA gyrase is the primary target in vitro of all investigated aminocoumarins. With the exception of simocyclinone D8, all other aminocoumarins inhibited S. aureus gyrase on average 6-fold more effectively than E. coli gyrase. Potassium glutamate is essential for the activity of S. aureus gyrase and increases the sensitivity of E. coli gyrase to aminocoumarins ≥ 10-fold. The antibacterial activity of the tested compounds mirrored their relative activities against topoisomerases. CONCLUSIONS: The study provides insights about the substituents that are important for the inhibitory activity of aminocoumarins against the target enzymes, which will facilitate the rational design of improved antibiotics.
Assuntos
Aminocumarinas/farmacologia , Antibacterianos/farmacologia , DNA Topoisomerase IV/antagonistas & inibidores , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/enzimologia , Inibidores da Topoisomerase II , Inibidores da Topoisomerase/farmacologia , Aminocumarinas/química , Antibacterianos/química , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/isolamento & purificação , Clonagem Molecular , DNA Girase/genética , DNA Topoisomerase IV/genética , DNA Super-Helicoidal/química , Escherichia coli/genética , Glutamatos/farmacologia , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Glutamato de Sódio/farmacologia , Staphylococcus aureus/genética , Relação Estrutura-AtividadeRESUMO
It has long been known that type II topoisomerases require divalent metal ions in order to cleave DNA. Kinetic, mutagenesis and structural studies indicate that the eukaryotic enzymes utilize a novel variant of the canonical two-metal-ion mechanism to promote DNA scission. However, the role of metal ions in the cleavage reaction mediated by bacterial type II enzymes has been controversial. Therefore, to resolve this critical issue, this study characterized the DNA cleavage reaction of Escherichia coli topoisomerase IV. We utilized a series of divalent metal ions with varying thiophilicities in conjunction with oligonucleotides that replaced bridging and non-bridging oxygen atoms at (and near) the scissile bond with sulfur atoms. DNA scission was enhanced when thiophilic metal ions were used with substrates that contained bridging sulfur atoms. In addition, the metal-ion dependence of DNA cleavage was sigmoidal in nature, and rates and levels of DNA cleavage increased when metal ion mixtures were used in reactions. Based on these findings, we propose that topoisomerase IV cleaves DNA using a two-metal-ion mechanism in which one of the metal ions makes a critical interaction with the 3'-bridging atom of the scissile phosphate and facilitates DNA scission by the bacterial type II enzyme.
Assuntos
Clivagem do DNA , DNA Topoisomerase IV/química , DNA/química , Metais/química , Cátions Bivalentes/química , DNA/metabolismo , DNA Topoisomerase IV/metabolismo , Escherichia coli/enzimologia , Fosfatos/químicaRESUMO
The chromosomally encoded Qnr homolog protein from Enterococcus faecalis (EfsQnr), when expressed, confers to its host a decreased susceptibility to quinolones and consists mainly of tandem repeats, which is consistent with belonging to the pentapeptide repeat family of proteins (PRPs). EfsQnr was cloned with an N-terminal 6× His tag and purified to homogeneity. EfsQnr partially protected DNA gyrase from fluoroquinolone inhibition at concentrations as low as 20 nM. EfsQnr inhibited the ATP-dependent supercoiling activity of DNA gyrase with a 50% inhibitory concentration (IC(50)) of 1.2 µM, while no significant inhibition of ATP-independent relaxation activity was observed. EfsQnr was cytotoxic when overexpressed in Escherichia coli, resulting in the clumping of cells and a loss of viability. The X-ray crystal structure of EfsQnr was determined to 1.6-Å resolution. EfsQnr exhibits the right-handed quadrilateral beta-helical fold typical of PRPs, with features more analogous to MfpA (mycobacterium fluoroquinolone resistance pentapeptide) than to the PRPs commonly found in cyanobacteria.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/farmacologia , Enterococcus faecalis/metabolismo , Inibidores da Topoisomerase II , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Enterococcus faecalis/genética , Escherichia coli/citologia , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , Viabilidade Microbiana , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , Multimerização Proteica , Estrutura Secundária de Proteína , Homologia de Sequência de AminoácidosRESUMO
DNA gyrase is an essential bacterial enzyme required for the maintenance of chromosomal DNA topology. This enzyme is the target of several protein toxins encoded in toxin-antitoxin (TA) loci as well as of man-made antibiotics such as quinolones. The genome of Vibrio cholerae, the cause of cholera, contains three putative TA loci that exhibit modest similarity to the RK2 plasmid-borne parDE TA locus, which is thought to target gyrase although its mechanism of action is uncharacterized. Here we investigated the V. cholerae parDE2 locus. We found that this locus encodes a functional proteic TA pair that is active in Escherichia coli as well as V. cholerae. ParD2 co-purified with ParE2 and interacted with it directly. Unlike many other antitoxins, ParD2 could prevent but not reverse ParE2 toxicity. ParE2, like the unrelated F-encoded toxin CcdB and quinolones, targeted the GyrA subunit and stalled the DNA-gyrase cleavage complex. However, in contrast to other gyrase poisons, ParE2 toxicity required ATP, and it interfered with gyrase-dependent DNA supercoiling but not DNA relaxation. ParE2 did not bind GyrA fragments bound by CcdB and quinolones, and a set of strains resistant to a variety of known gyrase inhibitors all exhibited sensitivity to ParE2. Together, our findings suggest that ParE2 and presumably its many plasmid- and chromosome-encoded homologues inhibit gyrase in a different manner than previously described agents.
Assuntos
DNA Bacteriano/metabolismo , DNA Super-Helicoidal/metabolismo , Inibidores Enzimáticos/metabolismo , Proteínas de Escherichia coli/antagonistas & inibidores , Escherichia coli/enzimologia , Plasmídeos/metabolismo , Inibidores da Topoisomerase II , Vibrio cholerae/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Girase/genética , DNA Girase/metabolismo , DNA Bacteriano/genética , DNA Super-Helicoidal/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Loci Gênicos/fisiologia , Humanos , Plasmídeos/genética , Vibrio cholerae/genéticaRESUMO
Simocyclinones are bifunctional antibiotics that inhibit bacterial DNA gyrase by preventing DNA binding to the enzyme. We report the crystal structure of the complex formed between the N-terminal domain of the Escherichia coli gyrase A subunit and simocyclinone D8, revealing two binding pockets that separately accommodate the aminocoumarin and polyketide moieties of the antibiotic. These are close to, but distinct from, the quinolone-binding site, consistent with our observations that several mutations in this region confer resistance to both agents. Biochemical studies show that the individual moieties of simocyclinone D8 are comparatively weak inhibitors of gyrase relative to the parent compound, but their combination generates a more potent inhibitor. Our results should facilitate the design of drug molecules that target these unexploited binding pockets.