For many years, DNA gyrase was thought to be responsible both for unlinking replicated daughter chromosomes and for controlling negative superhelical tension in bacterial DNA. However, in 1990 a homolog of gyrase, topoisomerase IV, that had a potent decatenating activity was discovered. It is now clear that topoisomerase IV, rather than gyrase, is responsible for decatenation of interlinked chromosomes. Moreover, topoisomerase IV is a target of the 4-quinolones, antibacterial agents that had previously been thought to target only gyrase. The key event in quinolone action is reversible trapping of gyrase-DNA and topoisomerase IV-DNA complexes. Complex formation with gyrase is followed by a rapid, reversible inhibition of DNA synthesis, cessation of growth, and induction of the SOS response. At higher drug concentrations, cell death occurs as double-strand DNA breaks are released from trapped gyrase and/or topoisomerase IV complexes. Repair of quinolone-induced DNA damage occurs largely via recombination pathways. In many gram-negative bacteria, resistance to moderate levels of quinolone arises from mutation of the gyrase A protein and resistance to high levels of quinolone arises from mutation of a second gyrase and/or topoisomerase IV site. For some gram-positive bacteria, the situation is reversed: primary resistance occurs through changes in topoisomerase IV while gyrase changes give additional resistance. Gyrase is also trapped on DNA by lethal gene products of certain large, low-copy-number plasmids. Thus, quinolone-topoisomerase biology is providing a model for understanding aspects of host-parasite interactions and providing ways to investigate manipulation of the bacterial chromosome by topoisomerases.

DNA gyrase, topoisomerase IV, and the 4-quinolones.

Thirteen spontaneous quinolone-resistant gyrB mutants of Escherichia coli KL16, including two that were examined previously, were divided into two types according to their quinolone resistance patterns. Type 1 mutants were resistant to all the quinolones tested, while type 2 mutants were resistant to acidic quinolones and were hypersusceptible to amphoteric quinolones. Nucleotide sequence analysis disclosed that all nine type 1 mutants had a point mutation from aspartic acid to asparagine at amino acid 426 and that all four type 2 mutants had a point mutation from lysine to glutamic acid at amino acid 447.

Quinolone resistance-determining region in the DNA gyrase gyrA gene of Escherichia coli.

INTRODUCTION In 1971, Wang (1) discovered the first DNA topoisomerase in Escherichia coli. The enzyme (E. coli DNA topoisomerase I, or w protein) catalyzed relaxation of negatively supercoiled DNA in the absence of any energy cofactor (1). Wang proposed that this enzyme also catalyzed transient nicking of the DNA double helix and possessed both DNase and ligase activity in one polypeptide (1). The lack of any energy cofactor for the reaction also led Wang to the proposal that the enzyme may form a high-energy covalent bond between itself and the transiently broken DNA phosphodiester bond (1). Both of these predictions have turned out to be correct (2). Since the discovery of E. coli topoisomerase I, investigators have isolated many other DNA topoisomerases from both prokaryotes and eukaryotes. In 1972, Champoux & Dulbecco isolated an enzyme with activity similar to that of E. coli topoisomerase I from mouse embryo cells (3). In 1976, Gellert and his colleagues identified an enzyme activity opposing E. coli DNA topoisomerase I (4). They demonstrated that this enzyme (E. coli DNA topoisomerase II, or gyrase) catalyzed the conversion of relaxed DNA into negatively supercoiled DNA in a reaction requiring ATP hydrolysis (4). These two opposing activities are important for maintaining the super­ helical state of the chromosomal DNA during various DNA transactions (5-8). In 1979, Liu et al (9) isolated an enzyme from bacteriophage

DNA Topoisomerases: Essential Enzymes and Lethal Targets

3.1. Chelation 564 3.2. Acid−Base Character 564 3.3. Photochemistry 565 4. In Vitro Antimicrobial Spectra 566 5. Structure−Activity Relationships 568 5.1. N-1 Ethyl Family 568 5.2. N-1 Cyclopropyl Family 568 5.3. N-1 to C-8 Bridged (Tricyclic) Family 568 5.4. N-1 Aryl Family 569 5.5. Positions C-2, C-3, and C-4 570 5.6. C-4a Substituted Analogues 571 5.7. C-5 Substituents 571 5.8. C-6 Substituents 571 5.9. C-7 Substituents 571 5.9.1. Piperazinyl and Related Moieties 572 5.9.2. Pyrrolidinyl and Related Moieties 572 5.9.3. Cyclobutylaminyl and Related Moieties 572 5.9.4. Bicycloaminyl Moieties 572 5.9.5. Carbon-Linked Substituents 572 5.10. Substituents at C-8 573 5.11. Resume of Structure−Activity Relationships of Quinolones 573

Bacterial topoisomerase inhibitors: quinolone and pyridone antibacterial agents.

DNA gyrase is an essential bacterial enzyme that catalyzes the ATP-dependent negative super-coiling of double-stranded closed-circular DNA. Gyrase belongs to a class of enzymes known as topoisomerases that are involved in the control of topological transitions of DNA. The mechanism by which gyrase is able to influence the topological state of DNA molecules is of inherent interest from an enzymological standpoint. In addition, much attention has been focused on DNA gyrase as the intracellular target of a number of antibacterial agents as a paradigm for other DNA topoisomerases. In this review we summarize the current knowledge concerning DNA gyrase by addressing a wide range of aspects of the study of this enzyme.

DNA Gyrase: Structure and Function

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.

Padam N. Sharma

Papers

Mechanism of inhibition of DNA gyrase by quinolone antibacterials: a cooperative drug--DNA binding model.