Overexpression, purification and photoaffinity labeling with a 3H-analogue of norfloxacin, of the GyrA and GyrB subunits of the DNA gyrase.

Article Details

Citation

Copy to clipboard

Hombrouck C, Capmau ML, Moreau N

Overexpression, purification and photoaffinity labeling with a 3H-analogue of norfloxacin, of the GyrA and GyrB subunits of the DNA gyrase.

Cell Mol Biol (Noisy-le-grand). 1999 May;45(3):347-52.

PubMed ID

10386791 [ View in PubMed

]

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.

DrugBank Data that Cites this Article

Drug Targets

Drug	Target	Kind	Organism	Pharmacological Action	Actions
Norfloxacin	DNA gyrase subunit A	Protein	Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd)	Yes	Inhibitor	Details
Pefloxacin	DNA gyrase subunit A	Protein	Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd)	Yes	Inhibitor	Details