DNA cleavage activities of Staphylococcus aureus gyrase and topoisomerase IV stimulated by quinolones and 2-pyridones

We have cloned Staphylococcus aureus DNA gyrase and topoisomerase TV and ex pressed them in Escherichia coli as polyhistidine-tagged proteins to facili tate purification and eliminate contamination by host enzymes. The enzyme p reparations had specific activities similar to previously reported values. Potassium glutamate (K-Glu) stimulated the drug-induced DNA cleavage activi ty and was optimal between 100 and 200 mM for gyrase and peaked at 100 mM f or topoisomerase TV. Higher concentrations of K-Glu inhibited the cleavage activities of both enzymes. Using a common buffer system containing 100 mM K-Glu, we tested the enzyme-mediated DNA cleavage activities of both gyrase and topoisomerase IV with oxolinic acid, norfloxacin, ciprofloxacin, trova floxacin, clinafloxacin, and the 2-pyridone ABT-719, As expected, all drugs tested demonstrated greater potency against topoisomerase IV than against gyrase, In addition, cleavage activity was found to correlate well with ant ibacterial activity.