Anthony Maxwell

DNA gyrase: structure and function.

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.

Crystal structure of the breakage-reunion domain of DNA gyrase.

DNA gyrase is a type II DNA topoisomerase from bacteria that introduces supercoils into DNA,. 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 Å 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.

DNA gyrase as a drug target

DNA gyrase is a remarkable enzyme, catalysing the seemingly complex reaction of DNA supercoiling. As gyrase is essential in prokaryotes, it is a good target for antibacterial agents. These agents have diverse chemical structures and interact with gyrase in a variety of ways.

The nature of inhibition of DNA gyrase by the coumarins and the cyclothialidines revealed by X-ray crystallography.

This study describes the first crystal structures of a complex between a DNA topoisomerase and a drug. We present the structures of a 24 kDa N‐terminal fragment of the Escherichia coli DNA gyrase B protein in complexes with two different inhibitors of the ATPase activity of DNA gyrase, namely the coumarin antibiotic, novobiocin, and GR122222X, a member of the cyclothialidine family. These structures are compared with the crystal structure of the complex with an ATP analogue, adenylyl‐beta‐gamma‐imidodiphosphate (ADPNP). The likely mechanism, by which mutant gyrase B proteins become resistant to inhibition by novobiocin are discussed in light of these comparisons. The three ligands are quite dissimilar in chemical structure and bind to the protein in very different ways, but their binding is competitive because of a small degree of overlap of their binding sites. These crystal structures consequently describe a chemically well characterized ligand binding surface and provide useful information to assist in the design of novel ligands.

Mode of Action

Over recent years there has been an enormous explosion of interest in the quinolone drugs in general and the fluoroquinolones in particular. This is manifested by the production of this volume and other works on this subject (e.g. Fernandes 1989; Crumplin 1990; Siporin et al. 1990; Hooper and Wolfson 1993a). Details of the structures of a wide range of quinolones will be found elsewhere in this book, but the structures of the principal quinolones mentioned in this chapter are shown in Fig. 1. In this chapter, we discuss the mode of action of quinolones, i.e. how they are thought to act on their intracellular target, DNA gyrase. Other recent reviews which cover this topic include those by Drlica et al. (1990, 1995), Hooper and Wolfson (1991, 1993b), Reece and Maxwell (1991a), Maxwell (1992) and Palumbo et al. (1993). It is worth noting at this point the existence of a second target, DNA topoisomerase IV, which is discussed below. Before describing the effects of quinolones on bacteria and on gyrase, we will discuss the basic properties of DNA gyrase.

Exploiting bacterial DNA gyrase as a drug target: current state and perspectives

DNA gyrase is a type II topoisomerase that can introduce negative supercoils into DNA at the expense of ATP hydrolysis. It is essential in all bacteria but absent from higher eukaryotes, making it an attractive target for antibacterials. The fluoroquinolones are examples of very successful gyrase-targeted drugs, but the rise in bacterial resistance to these agents means that we not only need to seek new compounds, but also new modes of inhibition of this enzyme. We review known gyrase-specific drugs and toxins and assess the prospects for developing new antibacterials targeted to this enzyme.

The interaction between coumarin drugs and DNA gyrase

The coumarin group of antibiotics have as their target the bacterial enzyme DNA gyrase. The drugs bind to the B subunit of gyrase and inhibit DNA supercoiling by blocking the ATPase activity. Recent data show that the binding site for the drugs lies within the N‐terminal part of the B protein, and individual amino acids involved in coumarin interaction are being identified. The mode of inhibition of the gyrase ATPase reaction by coumarins is unlikely to be simple competitive inhibition, and the drugs may act by stabilizing a conformation of the enzyme with low affinity for ATP.

A single point mutation in the DNA gyrase A protein greatly reduces binding of fluoroquinolones to the gyrase-DNA complex.

Binding of the quinolone drug norfloxacin to gyrase and DNA has been investigated. We have detected binding to gyrase-DNA complex but find no significant binding to either gyrase or DNA alone. Enzyme containing gyrase A protein with the mutation Ser-83 to Trp (conferring quinolone resistance) showed greatly reduced drug binding.

The ATP-Binding Site of Type II Topoisomerases as a Target for Antibacterial Drugs

DNA topoisomerases are essential enzymes in all cell types and have been found to be valuable drug targets both for antibacterial and anti-cancer chemotherapy. Type II topoisomerases possess a binding site for ATP, which can be exploited as a target for chemo-therapeutic agents. High-resolution structures of protein fragments containing this site complexed with antibiotics or an ATP analogue have provided vital information for the understanding of the action of existing drugs and for the potential development of novel anti-bacterial agents. In this article we have reviewed the structure and function of the ATPase domain of DNA gyrase (bacterial topoisomerase II), particularly highlighting novel information that has been revealed by structural studies. We discuss the efficacy and mode of action of existing drugs and consider the prospects for the development of novel agents.

Interaction between DNA Gyrase and Quinolones: Effects of Alanine Mutations at GyrA Subunit Residues Ser83 and Asp87

ABSTRACT DNA gyrase is a target of quinolone antibacterial agents, but the molecular details of the quinolone-gyrase interaction are not clear. Quinolone resistance mutations frequently occur at residues Ser83 and Asp87 of the gyrase A subunit, suggesting that these residues are involved in drug binding. Single and double alanine substitutions were created at these positions (Ala83, Ala87, and Ala83Ala87), and the mutant proteins were assessed for DNA supercoiling, DNA cleavage, and resistance to a number of quinolone drugs. The Ala83 mutant was fully active in supercoiling, whereas the Ala87 and the double mutant were 2.5- and 4- to 5-fold less active, respectively; this loss in activity may be partly due to an increased affinity of these mutant proteins for DNA. Supercoiling inhibition and cleavage assays revealed that the double mutant has a high level of resistance to certain quinolones while the mutants with single alanine substitutions show low-level resistance. Using a drug-binding assay we demonstrated that the double-mutant enzyme-DNA complex has a lower affinity for ciprofloxacin than the wild-type complex. Based on the pattern of resistance to a series of quinolones, an interaction between the C-8 group of the quinolone and the double-mutant gyrase in the region of residues 83 and 87 is proposed.