Hot-spot consensus of fluoroquinolone-mediated DNA cleavage by Gram-negative and Gram-positive type II DNA topoisomerases

Bacterial DNA gyrase and topoisomerase IV are selective targets of fluoroquinolones. Topoisomerase IV versus gyrase and Gram-positive versus Gram-negative behavior was studied based on the different recognition of DNA sequences by topoisomerase–quinolone complexes. A careful statistical analysis of preferred bases was performed on a large number (>400) of cleavage sites. We found discrete preferred sequences that were similar when using different enzymes (i.e. gyrase and topoisomerase IV) from the same bacterial source, but in part diverse when employing enzymes from different origins (i.e. Escherichia coli and Streptococcus pneumoniae). Subsequent analysis on the wild-type and mutated consensus sequences showed that: (i) Gn/Cn-rich sequences at and around the cleavage site are hot spots for quinolone-mediated strand breaks, especially for E. coli topoisomerases: we elucidated positions required for quinolone and enzyme recognition; (ii) for S. pneumoniae enzymes only, A and T at positions −2 and +6 are discriminating cleavage determinants; (iii) symmetry of the target sequence is a key trait to promote cleavage and (iv) the consensus sequence adopts a heteronomous A/B conformation, which may trigger DNA processing by the enzyme–drug complex

A Crystal Structure of the Bifunctional Antibiotic Simocyclinone D8, Bound to DNA Gyrase

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

Use of divalent metal ions in the DNA cleavage reaction of topoisomerase IV

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

DNA gyrase requires DNA for effective two-site coordination of divalent metal ions: further insight into the mechanism of enzyme action

The catalytic properties of DNA gyrase, an A2B2 complex, are modulated by the presence of divalent metal ions. Using circular dichroism, protein melting experiments and enzyme activity assays, we investigated the correlation between the A2B2 conformation, the nature of the metal ion cofactor and the enzyme activity in the presence and absence of DNA substrate. At room temperature, DNA gyrase structure is not appreciably affected by Ca2+ or Mg2+ but is modified by Mn2+. In addition, metal ions strongly affect the enzyme\u2019s thermal transitions, rendering the A2B2 structure more flexible. Using the B subunit, we were able to identify two distinct complexes with manganese ions. The first one exhibits a 1:1 stoichiometry and is not affected by the presence of DNA. The second complex is associated with a large protein structural modification that can be remarkably modulated by addition of the DNA substrate. This behavior is conserved in the reconstituted protein. Studies with two GyrB mutants indicate that Mn2+ interference with the TOPRIM region modulates gyrase supercoiling activity. In particular, considering the need for two divalent metal ions for an efficient catalytic cleavage of the phosphodiester bond, our data suggest that residue D500 participates in the first complexation event (DNA-independent), whereas residue D498 is involved mainly in the second process. In conclusion, a combination of the ion features (ionic size, electronegativity, coordination sphere) operating at the level of the catalytic region and of the ion-driven modifications in overall enzyme structure and flexibility contribute to the mechanism of gyrase activity. An effectual role for DNA recruiting the second catalytic metal ion is envisaged

Mapping Simocyclinone D8 Interaction with DNA Gyrase: Evidence for a New Binding Site on GyrB▿ †

Simocyclinone D8, a coumarin derivative isolated from Streptomyces antibioticus Tü 6040, represents an interesting new antiproliferative agent. It was originally suggested that this drug recognizes the GyrA subunit and interferes with the gyrase catalytic cycle by preventing its binding to DNA. To further characterize the mode of action of this antibiotic, we investigated its binding to the reconstituted DNA gyrase (A2B2) as well as to its GyrA and GyrB subunits and the individual domains of these proteins, by performing protein melting and proteolytic digestion studies as well as inhibition assays. Two binding sites were identified, one (anticipated) in the N-terminal domain of GyrA (GyrA59) and the other (unexpected) at the C-terminal domain of GyrB (GyrB47). Stabilization of the A subunit appears to be considerably more effective than stabilization of the B subunit. Our data suggest that these two distinct sites could cooperate in the reconstituted enzyme

Hot-spot consensus of fluoroquinolone-mediated DNA cleavage by Gram-negative and Gram-positive type II DNA topoisomerases

ABSTRACT Bacterial DNA gyrase and topoisomerase IV are selective targets of fluoroquinolones. Topoisomerase IV versus gyrase and Gram-positive versus Gram-negative behavior was studied based on the different recognition of DNA sequences by topoisomerase-quinolone complexes. A careful statistical analysis of preferred bases was performed on a large number (>400) of cleavage sites. We found discrete preferred sequences that were similar when using different enzymes (i.e. gyrase and topoisomerase IV) from the same bacterial source, but in part diverse when employing enzymes from different origins (i.e. Escherichia coli and Streptococcus pneumoniae). Subsequent analysis on the wild-type and mutated consensus sequences showed that: (i) Gn/Cn-rich sequences at and around the cleavage site are hot spots for quinolone-mediated strand breaks, especially for E. coli topoisomerases: we elucidated positions required for quinolone and enzyme recognition; (ii) for S. pneumoniae enzymes only, A and T at positions À2 and +6 are discriminating cleavage determinants; (iii) symmetry of the target sequence is a key trait to promote cleavage and (iv) the consensus sequence adopts a heteronomous A/B conformation, which may trigger DNA processing by the enzyme-drug complex