Overexpression, purification and photoaffinity labeling with a 3H-analogue of norfloxacin, of the GyrA and GyrB subunits of the DNA gyrase.
In spite of much work on DNA gyrase and quinolones for many years, our knowledge of the molecular basis of quinolone-gyrase action is still incomplete. We designed a photoaffinity labeling reagent for the quinolone target, and synthesized a norfloxacin analogue with an azide function which, under UV irradiation, becomes covalently linked to its target. For that, a large amount of purified gyrase was needed. Both subunits were purified using exclusion and affinity chromatography. A plasmid was used that allowed the overproduction of GyrA as a fusion-protein with six histidine residues at its carboxy-terminal domain. GyrA-(His)6 was purified after chromatography on a nickel-containing column, and native GyrB after chromatography on immobilized novobiocin. Reconstituted DNA gyrase (A2B2) had supercoiling activity. Photoaffinity labeling showed covalent binding of the 3H-photoaffinity analogue of norfloxacin to the gyrase-DNA complex, and mainly to the GyrA. The specific binding site remains to be explored.
Hombrouck
C
C
Laboratoire de Recherche Moléculaire sur les Antibiotiques, Université Paris 6, France.
Capmau
M L
ML
Moreau
N
N
D003001:Cloning, Molecular; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics; D007553:Isotope Labeling; D009643:Norfloxacin / Q000737:chemistry*; D019920:Photoaffinity Labels / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014316:Tritium
D019920:Photoaffinity Labels; D011994:Recombinant Proteins; D014316:Tritium; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II; D009643:Norfloxacin