title: Overexpression, purification and photoaffinity labeling with a 3H-analogue of norfloxacin, of the GyrA and GyrB subunits of the DNA gyrase.
abstract: 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.
authors.affiliation: Laboratoire de Recherche Moléculaire sur les Antibiotiques, Université Paris 6, France.
authors.forename: C
authors.initials: C
authors.lastname: Hombrouck
authors.forename: M L
authors.initials: ML
authors.lastname: Capmau
authors.forename: N
authors.initials: N
authors.lastname: Moreau
mesh_terms: 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
chemical_list: D019920:Photoaffinity Labels; D011994:Recombinant Proteins; D014316:Tritium; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II; D009643:Norfloxacin