gyrB-225, a mutation of DNA gyrase that compensates for topoisomerase I deficiency: investigation of its low activity and quinolone hypersensitivity.
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.
Heddle
J G
JG
Department of Biochemistry, University of Leicester, Leicester, LE1 7RH, UK.
Lu
T
T
Zhao
X
X
Drlica
K
K
Maxwell
A
A
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000483:Alleles; D019943:Amino Acid Substitution / Q000235:genetics; D000890:Anti-Infective Agents / Q000378:metabolism / Q000494:pharmacology*; D002939:Ciprofloxacin / Q000378:metabolism / Q000494:pharmacology; D027081:DNA Gyrase; D004264:DNA Topoisomerases, Type I / Q000172:deficiency* / Q000235:genetics; D004250:DNA Topoisomerases, Type II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004278:DNA, Superhelical / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004361:Drug Tolerance; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D005816:Genetic Complementation Test; D007700:Kinetics; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D010093:Oxolinic Acid / Q000378:metabolism / Q000494:pharmacology; D010957:Plasmids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013489:Suppression, Genetic / Q000235:genetics*; D020349:Surface Plasmon Resonance; D013816:Thermodynamics; D059005:Topoisomerase II Inhibitors
D000890:Anti-Infective Agents; D004278:DNA, Superhelical; D059005:Topoisomerase II Inhibitors; D002939:Ciprofloxacin; D000251:Adenosine Triphosphatases; D004264:DNA Topoisomerases, Type I; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II; D010093:Oxolinic Acid