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

Abstract

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.

Crystal engineering: deletion mutagenesis of the 24 kDa fragment of the DNA gyrase B subunit from Staphylococcus aureus

“…Analysis of the X-ray structure of the SaGyrB(1±234)± cyclothialidine binary complex as well as comparison with the E. coli p24±novobiocin complex revealed two regions for which there was no electron density. In the E. coli complex, electron density is lacking for the N-terminal 13 amino acids and the loop comprising residues 99±118 is disordered (Lewis et al, 1996). In the NgyrB±ADPNP complex, this glycine-rich loop forms a lid over the ATP-binding site, but in the p24± novobiocin complex it is not involved in ligand interactions.…”

“…coumermycin and novobiocin) act by inhibiting ATP hydrolysis by the B subunit of DNA gyrase. The X-ray structure of the 43 kDa N-terminal fragment±AMPNP complex (NGyrB±AMPNP) and the 24 kDa N-terminal fragment±novobiocin complex of the gyrase B subunit from Escherichia coli have been described (Lewis et al, 1996;Wigley et al, 1991). The 24 kDa fragment of DNA gyrase has no inherent activity of its own; it has been shown to bind a novobiocin±Sepharose af®nity column with similar avidity to GyrB (Gilbert & Maxwell, 1994).…”

Mass spectrometric characterization of a three-enzyme tandem reaction for assembly and modification of the novobiocin skeleton

“…Previous structural analysis and kinetic studies of the aminocoumarin antibiotics have revealed critical interactions between the carbamoylated noviosyl ring and the ATP binding site of the gyrase B subunit (6)(7)(8). The strategy in the design of improved coumarin-based antibiotics is to maintain the noviose decorations necessary for binding while introducing additional functionalities to improve the inhibitory properties of the analog or modulate solubility.…”

“…This family of antibiotics exerts its antibacterial activity via the inhibition of the type II DNA topoisomerase DNA gyrase (2)(3)(4)(5). X-ray crystallographic analysis of a 24-kDa N-terminal fragment of DNA gyrase with bound novobiocin (6)(7)(8) or clorobiocin (9) reveals that the antibiotics bind competitively at the ATP site and that the aminocoumarin bicyclic ring is the scaffold for presenting the decorated L-noviosyl sugar moiety to the target enzyme.…”

Methyltransferase genes in Streptomyces rishiriensis: new coumermycin derivatives from gene-inactivation experiments b bThe GenBank accession number for the sequence reported in this paper is AF235050.

“…crystallographic examinations demonstrated that the aminocoumarin moiety and the substituted deoxysugar moiety are essential for the binding of these compounds to the B subunit of gyrase (Ali et al, 1993 ;Lewis et al, 1996 ;Maxwell, 1993 ;Tsai et al, 1997). The affinity of coumermycin A " for intact gyrase is extremely high : 50 % inhibition of gyrase is reportedly achieved by coumermycin A " in a concentration of only 0n004 µM, compared to 0n1 µM for novobiocin, 1n8 µM for norfloxacin and 110 µM for nalidixic acid (Peng & Marians, 1993).…”