The DNA Gyrase-Quinolone Complex: ATP HYDROLYSIS AND THE MECHANISM OF DNA CLEAVAGE *
28 Aug 1998-Journal of Biological Chemistry (American Society for Biochemistry and Molecular Biology)-Vol. 273, Iss: 35, pp 22615-22626
TL;DR: It is demonstrated that quinolone binding and drug-induced DNA cleavage are separate processes constituting two sequential steps in the mechanism of action of quinOLones on DNA gyrase, which is capable of ATP hydrolysis through an alternative pathway involving two different conformations of the enzyme.
About: This article is published in Journal of Biological Chemistry.The article was published on 1998-08-28 and is currently open access. It has received 111 citations till now. The article focuses on the topics: DNA gyrase & DNA supercoil.
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TL;DR: The multilayered effects of drug–target interactions, including the essential cellular processes that are inhibited by bactericidal antibiotics and the associated cellular response mechanisms that contribute to killing are discussed.
Abstract: Antibiotic drug-target interactions, and their respective direct effects, are generally well characterized. By contrast, the bacterial responses to antibiotic drug treatments that contribute to cell death are not as well understood and have proven to be complex as they involve many genetic and biochemical pathways. In this Review, we discuss the multilayered effects of drug-target interactions, including the essential cellular processes that are inhibited by bactericidal antibiotics and the associated cellular response mechanisms that contribute to killing. We also discuss new insights into these mechanisms that have been revealed through the study of biological networks, and describe how these insights, together with related developments in synthetic biology, could be exploited to create new antibacterial therapies.
1,796 citations
Cites background from "The DNA Gyrase-Quinolone Complex: A..."
...that do not prevent quinolone binding, and studies that have shown that strand breakage can occur in the presence of quinolone...
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TL;DR: The quinolones are a group of synthetic antibacterial agents structurally related to nalidixic acid as mentioned in this paper, and the absorption of these agents is lower when they are consumed simultaneously with magnesium or aluminium antacids.
513 citations
TL;DR: In the present minireview, cell death is considered through a two-part “poison” hypothesis in which the quinolones form reversible drug-topoisomerase-DNA complexes that subsequently lead to several types of irreversible (lethal) damage.
Abstract: The fluoroquinolones are broad-spectrum antibacterial agents that are becoming increasingly popular as bacterial resistance erodes the effectiveness of other agents (fluoroquinolone sales accounted for 18% of the antibacterial market in 2006) (41). One of the attractive features of the quinolones is their ability to kill bacteria rapidly, an ability that differs widely among the various derivatives. For example, quinolones differ in rate and extent of killing, in the need for aerobic metabolism to kill cells, and in the effect of protein synthesis inhibitors on quinolone lethality. Understanding the mechanisms underlying these differences could lead to new ways for identifying the most bactericidal quinolone derivatives.
Before describing the types of damage caused by the quinolones, it is useful to define lethal activity. Operationally, it is the ability of drug treatment to reduce the number of viable cells, usually measured as CFU on drug-free agar after treatment. This assay is distinct from measurements that detect inhibition of growth (e.g., MIC), since with the latter bacteria are exposed to drug throughout the measurement. The distinction between killing and blocking growth is important because it allows susceptibility determinations to be related to particular biological processes. For example, inhibition of growth is typically reversed by the removal of drug, while cell death is not. Thus, biochemical events associated with blocking growth should be readily reversible, while those responsible for cell death should be difficult to reverse. Reversibility can be used to distinguish among quinolone derivatives and assign functions to particular aspects of drug structure. Moreover, protective functions, such as repair and stress responses, can be distinguished by whether their absence affects inhibition of growth, killing, or both.
The intracellular targets of the quinolones are two DNA topoisomerases: gyrase and topoisomerase IV. Gyrase tends to be the primary target in gram-negative bacteria, while topoisomerase IV is preferentially inhibited by most quinolones in gram-positive organisms (28). Both enzymes use a double-strand DNA passage mechanism, and it is likely that quinolone biochemistry is similar for both. However, physiological differences between the enzymes exist, some of which may bear on quinolone lethality.
In the present minireview we consider cell death through a two-part “poison” hypothesis in which the quinolones form reversible drug-topoisomerase-DNA complexes that subsequently lead to several types of irreversible (lethal) damage. Other consequences of quinolone treatment, such as depletion of gyrase and topoisomerase IV activity, are probably less immediate (42). To provide a framework for considering quinolone lethality, we begin by briefly describing the drug-topoisomerase-DNA complexes. Readers interested in a more comprehensive discussion of quinolones are referred to a previously published work (28).
470 citations
TL;DR: It is shown that superoxide‐mediated oxidation of iron–sulfur clusters promotes a breakdown of iron regulatory dynamics and drives the generation of highly destructive hydroxyl radicals via the Fenton reaction, and that blockage of hydroxy radical formation increases the survival of gyrase‐poisoned cells.
Abstract: Modulation of bacterial chromosomal supercoiling is a function of DNA gyrase-catalyzed strand breakage and rejoining. This reaction is exploited by both antibiotic and proteic gyrase inhibitors, which trap the gyrase molecule at the DNA cleavage stage. Owing to this interaction, doublestranded DNA breaks are introduced and replication machinery is arrested at blocked replication forks. This immediately results in bacteriostasis and ultimately induces cell death. Here we demonstrate, through a series of phenotypic and gene expression analyses, that superoxide and hydroxyl radical oxidative species are generated following gyrase poisoning and play an important role in cell killing by gyrase inhibitors. We show that superoxide-mediated oxidation of iron–sulfur clusters promotes a breakdown of iron regulatory dynamics; in turn, iron misregulation drives the generation of highly destructive hydroxyl radicals via the Fenton reaction. Importantly, our data reveal that blockage of hydroxyl radical formation increases the survival of gyrase-poisoned cells. Together, this series of biochemical reactions appears to compose a maladaptive response, that serves to amplify the primary effect of gyrase inhibition by oxidatively damaging DNA, proteins and lipids. Molecular Systems Biology 13 March 2007; doi:10.1038/msb4100135 Subject Categories: cellular metabolism; microbiology & pathogenesis
423 citations
TL;DR: When fluoroquinolones bind to gyrase or topoisomerase IV in the presence of DNA, they alter protein conformation and are trapped in ternary complexes with drug and cleaved DNA.
310 citations
References
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TL;DR: The results indicate that the binding of any non-interacting ligand covering more than one lattice residue results in non- linear (convex downward) Scatchard plots, and the introduction of positive ligand-ligand co-operativity antagonizes this non-linearity, and eventually leads to plots of the opposite curvature.
2,787 citations
TL;DR: The nalA locus is responsible for a second component needed for DNA gyrase activity in addition to the component determined by the previously described locus for resistance to novobiocin and coumermycin (cou), which appears to be involved in the nicking-closing activity required in the supercoiling reaction.
Abstract: ATP-dependent DNA supercoiling catalyzed by Escherichia coli DNA gyrase was inhibited by oxolinic acid, a compound similar to but more potent than nalidixic acid and a known inhibitor of DNA replication in E. coli. The supercoiling activity of DNA gyrase purified from nalidixic acid-resistant mutant (nalAR) bacteria was resistant to oxolinic acid. Thus, the nalA locus is responsible for a second component needed for DNA gyrase activity in addition to the component determined by the previously described locus for resistance to novobiocin and coumermycin (cou). Supercoiling of λ DNA in E. coli cells was likewise inhibited by oxolinic acid, but was resistant in the nalAR mutant. The inhibition by oxolinic acid of colicin E1 plasmid DNA synthesis in a cell-free system was largely relieved by adding resistant DNA gyrase.
In the absence of ATP, DNA gyrase preparations relaxed supercoiled DNA; this activity was also inhibited by oxolinic acid, but not by novobiocin. It appears that the oxolinic acid-sensitive component of DNA gyrase is involved in the nicking-closing activity required in the supercoiling reaction. In the presence of oxolinic acid, DNA gyrase forms a complex with DNA, which can be activated by later treatment with sodium dodecyl sulfate and a protease to produce double-strand breaks in the DNA. This process has some similarities to the known properties of relaxation complexes.
777 citations
TL;DR: The crystal structure of the breakage-reunion domain of DNA gyrase at 2.8 A resolution was presented in this paper, where it was shown that the two structures represent two principal conformations that participate in the enzymatic pathway.
Abstract: DNA gyrase is a type II DNA topoisomerase from bacteria that introduces supercoils into DNA1,2. It catalyses the breakage of a DNA duplex (the G segment), the passage of another segment (the T segment) through the break, and then the reunification of the break. This activity involves the opening and closing of a series of molecular ‘gates’ which is coupled to ATP hydrolysis. Here we present the crystal structure of the ‘breakage–reunion’ domain of the gyrase at 2.8 A resolution. Comparison of the structure of this 59K (relative molecular mass, 59,000) domain with that of a 92K fragment of yeast topoisomerase II (ref. 3) reveals a very different quaternary organization, and we propose that the two structures represent two principal conformations that participate in the enzymatic pathway. The gyrase structure reveals a new dimer contact with a grooved concave surface for binding the G segment and a cluster of conserved charged residues surrounding the active-site tyrosines. It also shows how breakage of the G segment can occur and, together with the topoisomerase II structure, suggests a pathway by which the T segment can be released through the second gate of the enzyme. Mutations that confer resistance to the quinolone antibacterial agents cluster at the new dimer interface, indicating how these drugs might interact with the gyrase–DNA complex.
441 citations
TL;DR: A cooperative quinolone-DNA binding model for the inhibition of DNA gyrase and the unique self-association phenomenon (from which the cooperativity is derived) of the drug molecules to fit the binding pocket with a high degree of flexibility is proposed.
Abstract: We have proposed a cooperative quinolone-DNA binding model for the inhibition of DNA gyrase. The essential feature of the model is that bound gyrase induces a specific quinolone binding site in the relaxed DNA substrate in the presence of ATP. The binding affinity and specificity are derived from two unique and equally important functional features: the specific conformation of the proposed single-stranded DNA pocket induced by the enzyme and the unique self-association phenomenon (from which the cooperativity is derived) of the drug molecules to fit the binding pocket with a high degree of flexibility. Supporting evidence for and implications of this model are provided.
412 citations
TL;DR: It is postulate that ATP and App[NH]p are allosteric effectors of a conformational change of gyrase that leads to one round of supercoiling and that cyclic conformational changes accompanying alteration in nucleotide affinity also seem to be a common feature of energy transduction in other diverse processes including muscle contraction, protein synthesis, and oxidative phosphorylation.
Abstract: Escherichia coli DNA gyrase catalyzes negative supercoiling of closed duplex DNA at the expense of ATP. Two additional activities of the enzyme that have illuminated the energy coupling component of the supercoiling reaction are the DNA-dependent hydrolysis of ATP to ADP and Pi and the alteration by ATP of the DNA site specificity of the gyrase cleavage reaction. This cleavage of both DNA strands results from treatment with sodium dodecyl sulfate of the stable gyrase-DNA complex that is trapped by the inhibitor oxolinic acid. Either ATP or a nonhydrolyzable analogue, adenyl-5′-yl-imidodiphosphate (App[NH]p), shifts the primary cleavage site on ColE1 DNA. The prevention by novobiocin and coumermycin A1 of this cleavage rearrangement places the site of action of the antibiotics at a reaction step prior to ATP hydrolysis. The step blocked is the binding of ATP because coumermycin A1 and novobiocin interact competitively with ATP in the ATPase and supercoiling assays; the Ki values are more than four orders of magnitude less than the Km for ATP. This simple mechanism accounts for all effects of the drugs on DNA gyrase. Studies with App[NH]p, another potent competitive inhibitor of reactions catalyzed by gyrase, show that cleavage of a high energy bond is not required for driving DNA into the higher energy supercoiled form. With substrate levels of gyrase, App[NH]p induces supercoiling that is proportional to the amount of enzyme; a -0.3 superhelical turn was introduced per gyrase protomer A. We postulate that ATP and App[NH]p are allosteric effectors of a conformational change of gyrase that leads to one round of supercoiling. Nucleotide dissociation favored by hydrolysis of ATP returns gyrase to its original conformation and thereby permits enzyme turnover. Such cyclic conformational changes accompanying alteration in nucleotide affinity also seem to be a common feature of energy transduction in other diverse processes including muscle contraction, protein synthesis, and oxidative phosphorylation.
371 citations