ABSTRACT
Microcin B17 (MccB17) is a 3.1-kDa Escherichia coli antibiotic that contains thiazole and oxazole heterocycles in a peptide backbone. MccB17 inhibits its cellular target, DNA gyrase, by trapping the enzyme in a complex that is covalently bound to double-strand cleaved DNA, in a manner similar to the well-known quinolone drugs. The identification of gyrase as the target of MccB17 provides an opportunity to analyze the relationship between the structure of this unusual antibiotic and its activity. In this report, steady-state parameters are used to describe the induction of the cleavable complex by MccB17 analogs containing modified bisheterocyclic sites. The relative potency of these analogs corresponds to the capacity of the compounds to prevent growth of sensitive cells. In contrast to previously reported experiments, inhibition of DNA gyrase supercoiling activity by wild-type MccB17 also was observed. These results suggest that DNA gyrase is the main intracellular target of MccB17. This study probes the structure-function relationship of a new class of gyrase inhibitors and demonstrates that these techniques could be used to analyze compounds in the search for clinically useful antibiotics that block DNA gyrase.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacteriocins/pharmacology , Enzyme Inhibitors/pharmacology , Topoisomerase II Inhibitors , Anti-Bacterial Agents/metabolism , Bacteriocins/chemistry , Bacteriocins/metabolism , DNA Topoisomerases, Type II/metabolism , Enzyme Inhibitors/metabolismABSTRACT
The B subunit of DNA gyrase (GyrB) consists of a 43 kDa N-terminal domain, containing the site of ATP binding and hydrolysis, and a 47 kDa C-terminal domain that is thought to play a role in interactions with GyrA and DNA. In cells containing a deletion of topA (the gene encoding DNA topoisomerase I) a compensatory mutation is found in gyrB. This mutation (gyrB-225) results in a two amino acid insertion in the N-terminal domain of GyrB. We found that cells containing this mutation are more sensitive than wild-type cells to quinolone drugs with respect to bacteriostatic and lethal action. We have characterised the mutant GyrB protein in vitro and found it to have reduced DNA supercoiling, relaxation, ATPase, and cleavage activities. The mutant enzyme is up to threefold more sensitive to quinolones than wild-type. The mutation also increases the affinity of GyrB for GyrA and DNA, while the affinity of quinolone for the enzyme-DNA complex is unaffected. We propose that the loss in activity is due to misfolding of the GyrB-225 protein, providing an example in which misfolding of one protein, DNA gyrase, suppresses a deficiency of another, topoisomerase I. The increased quinolone sensitivity is proposed to be a consequence of an altered conformation of the protein that renders quinolones better able to disrupt, rather than generate, gyrase-drug-DNA complexes.
Subject(s)
Anti-Infective Agents/pharmacology , DNA Topoisomerases, Type II/metabolism , DNA Topoisomerases, Type I/deficiency , Escherichia coli/enzymology , Suppression, Genetic/genetics , Topoisomerase II Inhibitors , Adenosine Triphosphatases/antagonists & inhibitors , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Alleles , Amino Acid Substitution/genetics , Anti-Infective Agents/metabolism , Ciprofloxacin/metabolism , Ciprofloxacin/pharmacology , DNA Gyrase , DNA Topoisomerases, Type I/genetics , DNA Topoisomerases, Type II/chemistry , DNA Topoisomerases, Type II/genetics , DNA, Superhelical/chemistry , DNA, Superhelical/genetics , DNA, Superhelical/metabolism , Drug Tolerance , Escherichia coli/drug effects , Escherichia coli/genetics , Genetic Complementation Test , Kinetics , Models, Molecular , Nucleic Acid Conformation , Oxolinic Acid/metabolism , Oxolinic Acid/pharmacology , Plasmids/chemistry , Plasmids/genetics , Plasmids/metabolism , Protein Binding/drug effects , Protein Folding , Protein Structure, Tertiary , Surface Plasmon Resonance , ThermodynamicsABSTRACT
Microcin B17 is a 3.1-kDa bactericidal peptide; the putative target of this antibiotic is DNA gyrase. Microcin B17 has no detectable effect on gyrase-catalysed DNA supercoiling or relaxation activities in vitro and is unable to stabilise DNA cleavage in the absence of nucleotides. However, in the presence of ATP, or the non-hydrolysable analogue 5'-adenylyl beta,gamma-imidodiphosphate, microcin B17 stabilises a gyrase-dependent DNA cleavage complex in a manner reminiscent of quinolones, Ca(2+), or the bacterial toxin CcdB. The pattern of DNA cleavage produced by gyrase in the presence of microcin B17 is different from that produced by quinolones and more closely resembles Ca(2+)-mediated cleavage. Several gyrase mutants, including well-known quinolone-resistant mutants, are cross resistant to microcin-induced DNA cleavage. We suggest that microcin exerts its effects through a mechanism that has similarities to those of both the bacterial toxin CcdB and the quinolone antibacterial agents.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacteriocins/pharmacology , Peptides , Topoisomerase II Inhibitors , Adenosine Triphosphate/metabolism , Adenylyl Imidodiphosphate/metabolism , Anti-Bacterial Agents/chemistry , Anti-Infective Agents/pharmacology , Bacterial Proteins/pharmacology , Bacterial Toxins/pharmacology , Bacteriocins/chemistry , Calcium/pharmacology , Ciprofloxacin/chemistry , Ciprofloxacin/pharmacology , Coumarins/pharmacology , Cytotoxins/chemistry , Cytotoxins/pharmacology , DNA Gyrase , DNA Replication/drug effects , DNA Topoisomerases, Type II/chemistry , DNA Topoisomerases, Type II/genetics , DNA Topoisomerases, Type II/metabolism , DNA, Superhelical/chemistry , DNA, Superhelical/genetics , DNA, Superhelical/metabolism , DNA-Directed DNA Polymerase/metabolism , Escherichia coli/drug effects , Escherichia coli/enzymology , Escherichia coli/genetics , Kinetics , Models, Molecular , Mutation/genetics , Nucleic Acid Synthesis Inhibitors , Protein Conformation , Quinolones/pharmacology , Substrate Specificity , Yeasts/enzymologyABSTRACT
DNA gyrase supercoils DNA in bacteria. The fact that it is essential in all bacteria and absent from eukaryotes makes it an ideal drug target. We discuss the action of coumarin and quinolone drugs on gyrase. In the case of coumarins, the drugs are known to be competitive inhibitors of the gyrase ATPase reaction. From a combination of structural and biochemical studies, the molecular details of the gyrase-coumarin complex are well established. In the case of quinolones, the drugs are thought to act by stabilising a cleavage complex between gyrase and DNA that arrests polymerases in vivo. The exact nature of the gyrase-quinolone-DNA complex is not known; we propose a model for this complex based on structural and biochemical data.
