Overexpression, purification and photoaffinity labeling with a 3H-analogue of norfloxacin, of the GyrA and GyrB subunits of the DNA gyrase.
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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) YesInhibitorDetails Pefloxacin DNA gyrase subunit A Protein Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd) YesInhibitorDetails