Mycobacterial DNA gyrase: enzyme purification and characterization of supercoiling activity

Abstract

Putative structural genes encoding Mycobacterium bovis BCG gyrase A and gyrase B subunits were expressed in Escherichia coli under the control of a regulated promoter. Upon induction, high levels of proteins of M(r) 92,000 and 75,000 were generated. Purification and reconstitution of these proteins yielded an enzyme with bacterial DNA gyrase activity. DNA supercoiling activity of the mycobacterial enzyme required ATP, Mg2+, and spermidine. Like other bacterial DNA gyrases, the supercoiling activity of the mycobacterial enzyme was inhibited by low concentration of the classical gyrase B subunit inhibitors novobiocin and coumermycin. Older gyrase A subunit inhibitors, nalidixic and oxolinic acid, had no effect on the supercoiling activity at 400 to 800 micrograms/ml. However, in vitro assays to show the inhibition of supercoiling activity and stimulation of cleavable complex formation demonstrated that ciprofloxacin is a potent inhibitor of mycobacterial DNA gyrase. The availability of highly purified mycobacterial DNA gyrase could aid in future investigations of quinolone derivatives targeting Mycobacterium specifically.