Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:5.99.1.2 (topoisomerase)
9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Fluoroquinolones are potent antibacterial agents being used clinically against multidrug-resistant tuberculosis. Treatment failure is thought to arise from acquisition of fluoroquinolone resistance by Mycobacterium tuberculosis. A collection of 13 resistant clinical isolates of M. tuberculosis was examined for ciprofloxacin sensitivity relative to controls exhibiting the same IS6110 DNA type. Specific alleles were associated with distinct levels of drug susceptibility for 11 isolates that contained nucleotide changes expected to alter the amino acid sequence of the A subunit of DNA gyrase. Five different gyrA (ciprofloxacin resistance) alleles were present among 7 isolates having the W DNA subtype. These isolates, which are representative of an outbreak strain, constitute a panel of organisms that can be used to evaluate contributions of gyrase and DNA topoisomerase IV to resistance.
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PMID:Fluoroquinolone resistance associated with specific gyrase mutations in clinical isolates of multidrug-resistant Mycobacterium tuberculosis. 889 23

The complete gene encoding Topoisomerase 1 (Topo I) from Mycobacterium tuberculosis (MTb), Erdman strain, has been isolated and sequenced. The coding region of this gene is 2700 nt encoding a polypeptide of 900 amino acids with a calculated molecular mass of 99353 Da. The amino-acid sequence identity compared to E. coli and Synechococcus Topo I is 22 and 30%, respectively. The gene was expressed in E. coli BL21(DE3) and purified to near homogeneity. Recombinant MTb Topo I is enzymatically active, relaxing negatively supercoiled DNA in a magnesium-dependent, ATP-independent reaction. Spermidine, a typical inhibitor of prokaryotic type I DNA topoisomerase, inhibits the activity. Unlike the more well-characterized E. coli Topo I, MTb Topo I does not contain a zinc-finger DNA-binding motif in the C-terminal domain of the protein.
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PMID:Cloning, expression, purification and characterization of DNA topoisomerase I of Mycobacterium tuberculosis. 892 93

A type I topoisomerase has been purified to homogeneity from Mycobacterium smegmatis. It is the largest single subunit enzyme of this class having molecular mass of 110 kDa. The enzyme is Mg2+ dependent and can relax negatively supercoiled DNA, catenate, and knot single-stranded DNA, thus having typical properties of type I topoisomerases. Furthermore, the enzyme makes single-stranded nicks and the 5'-phosphoryl end of the nicked DNA gets covalently linked with a tyrosine residue of the enzyme. However, M. smegmatis enzyme shows some distinctive features from the prototype Escherichia coli topoisomerase I. The enzyme is relatively stable at higher temperatures and not inhibited by spermidine. It apparently does not contain any bound Zn2+ and on modification of cysteine residues retains the activity, suggesting the absence of the zinc-finger motif in DNA binding. Partially purified Mycobacterium tuberculosis topoisomerase I exhibits very similar properties with respect to size, stability, and reaction characteristics. Sequence comparison of topoisomerase I from E. coli and M. tuberculosis shows the absence of zinc-finger motifs in mycobacterial enzyme. Using a two-substrate assay system, we demonstrate that the enzyme acts processively at low ionic strength and switches over to distributive mode at high Mg2+ concentration. Significantly, the enzyme activity is stimulated by single strand DNA-binding protein. There is a potential to exploit the characteristics of the enzyme to develop it as a molecular target against mycobacterial infections.
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PMID:DNA topoisomerase I from Mycobacterium smegmatis. An enzyme with distinct features. 959 41

Mycobacterium smegmatis topoisomerase I has several distinctive features. The absence of the zinc finger motif found in other prokaryotic type I topoisomerases and the ability of the enzyme to recognise single-stranded and duplex DNA are unique characteristics of the enzyme. We have mapped the strong topoisomerase sites of the enzyme on genomic DNA sequences from Mycobacterium tuberculosis and M.smegmatis. The enzyme does not nick DNA in random fashion and DNA cleavage occurred at a few specific sites. Mapping of these sites revealed conservation of a pentanucleotide motif CG/TCT/T at the cleavage site (/ represents the cleavage site). The enzyme binds and cleaves consensus oligo-nucleotides having this sequence motif. The protein exhibits a very high preference for C or a G residue at the +2 position with respect to the cleavage site. Based on earlier and the present studies we propose that the enzyme functions in vivo mainly at these specific sites to carry out topological reactions.
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PMID:Determination of the recognition sequence of Mycobacterium smegmatis topoisomerase I on mycobacterial genomic sequences. 1073 3

DNA gyrase is an essential type II topoisomerase found in bacteria. We have previously characterized DNA gyrase from Mycobacterium tuberculosis and Mycobacterium smegmatis. In this study, several monoclonal antibodies were generated against the gyrase A subunit (GyrA) of M. smegmatis. Three, MsGyrA:C3, MsGyrA:H11 and MsGyrA:E9, were further analyzed for their interaction with the enzyme. The monoclonal antibodies showed high degree of cross-reactivity with both fast-growing and slow-growing mycobacteria. In contrast, none recognized Escherichia coli GyrA. All the three monoclonal antibodies were of IgG1 isotype falling into two distinct types with respect to epitope recognition and interaction with the enzyme. MsGyrA:C3 and MsGyrA:H11 IgG, and their respective Fab fragments, inhibited the DNA supercoiling activity catalyzed by mycobacterial DNA gyrase. The epitope for the neutralizing monoclonal antibodies appeared to involve the region towards the N-terminus (residues 351-415) of the enzyme in a conformation-dependent manner. These monoclonal antibodies would serve as valuable tools for structure-function analysis and immunocytological studies of mycobacterial DNA gyrase. In addition, they would be useful for designing peptide inhibitors against DNA gyrase.
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PMID:Monoclonal antibodies to mycobacterial DNA gyrase A inhibit DNA supercoiling activity. 1127 26

DNA gyrase is an essential topoisomerase found in all bacteria. It is encoded by gyrB and gyrA genes. These genes are organized differently in different bacteria. Direct comparison of Mycobacterium tuberculosis and Mycobacterium smegmatis genomes reveals presence of an additional gyrB in M. smegmatis flanked by novel genes. Analysis of the amino acid sequence of GyrB from different organisms suggests that the orphan GyrB in M. smegmatis may have an important cellular role.
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PMID:An orphan gyrB in the Mycobacterium smegmatis genome uncovered by comparative genomics. 1271 39

Genome studies suggest that DNA gyrase is the sole type II topoisomerase and likely the unique target of quinolones in Mycobacterium tuberculosis. Despite the emerging importance of quinolones in the treatment of mycobacterial disease, the slow growth and high pathogenicity of M. tuberculosis have precluded direct purification of its gyrase and detailed analysis of quinolone action. To address these issues, we separately overexpressed the M. tuberculosis DNA gyrase GyrA and GyrB subunits as His-tagged proteins in Escherichia coli from pET plasmids carrying gyrA and gyrB genes. The soluble 97-kDa GyrA and 72-kDa GyrB subunits were purified by nickel chelate chromatography and shown to reconstitute an ATP-dependent DNA supercoiling activity. The drug concentration that inhibited DNA supercoiling by 50% (IC(50)) was measured for 22 different quinolones, and values ranged from 2 to 3 microg/ml (sparfloxacin, sitafloxacin, clinafloxacin, and gatifloxacin) to >1,000 microg/ml (pipemidic acid and nalidixic acid). By comparison, MICs measured against M. tuberculosis ranged from 0.12 microg/ml (for gatifloxacin) to 128 microg/ml (both pipemidic acid and nalidixic acid) and correlated well with the gyrase IC(50)s (R(2) = 0.9). Quinolones promoted gyrase-mediated cleavage of plasmid pBR322 DNA due to stabilization of the cleavage complex, which is thought to be the lethal lesion. Surprisingly, the measured concentrations of drug inducing 50% plasmid linearization correlated less well with the MICs (R(2) = 0.7). These findings suggest that the DNA supercoiling inhibition assay may be a useful screening test in identifying quinolones with promising activity against M. tuberculosis. The quinolone structure-activity relationship demonstrated here shows that C-8, the C-7 ring, the C-6 fluorine, and the N-1 cyclopropyl substituents are desirable structural features in targeting M. tuberculosis gyrase.
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PMID:Mycobacterium tuberculosis DNA gyrase: interaction with quinolones and correlation with antimycobacterial drug activity. 1504 30

The antimicrobial agents used in the treatment of mycobacterial infections have remained largely unchanged for several decades. Primary treatment of tuberculosis relies on four drugs, isoniazid, a rifamycin, pyrazinamide, and ethambutol (or streptomycin), and generally results in >95% cure in uncomplicated tuberculosis infection. Drug resistance greatly complicates treatment of this disease. Treatment of tuberculosis caused by multiply drug-resistant strains with "second-line" drugs remains complex, and is generally tailored to the individual patient and strain. Several of the fluoroquinolones have shown promise as second line drugs for treatment of active disease and, in combination with clarithromycin or azithromycin, ethambutol, and other agents, for treatment of Mycobacterium avium complex infection. While large clinical trials are not possible with second line drugs, clinical treatment data are available and suggest that the quinolones have various degrees of promise in treatment of these infections. Bacterial type II DNA topoisomerases, DNA gyrase and topoisomerase IV, are the targets of quinolones, and provide the genetic basis for quinolone activity in mycobacteria. Mutations in these enzymes results in resistance, and characterization of resistant mutants allows correlation of genotype with susceptibility phenotype. Structure-activity relationship studies have provided further insight into optimal use of quinolones in mycobacterial infections. Care should be taken in treating pneumonia with fluoroquinolones if there is a degree of suspicion of tuberculosis, since quinolone monotherapy may rapidly select for quinolone resistance, thereby removing that class of antibiotic from the small range of treatment options.
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PMID:Fluoroquinolones as chemotherapeutics against mycobacterial infections. 1554 10

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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PMID:A monoclonal antibody that inhibits mycobacterial DNA gyrase by a novel mechanism. 1593 Jan 58

Genome deciphering revealed that Mycobacterium tuberculosis encodes a single type II topoisomerase contrary to common bacteria harboring two type II topoisomerases (DNA gyrase and topoisomerase IV). Functions of the M. tuberculosis type II topoisomerase were explored after cloning and expressing the subunits encoding genes in Escherichia coli. M. tuberculosis type II topoisomerase supercoiled relaxed pBR322 with a specific activity close to that of DNA gyrases of common bacteria whereas it exhibited DNA relaxation and formation of cleavable complexes with activities significantly higher than other DNA gyrases. Intermolecular passage activity evaluated by the decatenation of kinetoplast DNA was 25-fold lower than that of the topoisomerase IV from Streptococcus pneumoniae, but was markedly higher than that of the E. coli gyrase. Overall, the type II topoisomerase of M. tuberculosis exhibits classical polyvalent activities of DNA gyrase for supercoiling but enhanced relaxation, cleavage, and decatenation activities.
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PMID:First functional characterization of a singly expressed bacterial type II topoisomerase: the enzyme from Mycobacterium tuberculosis. 1687 25


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