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Antimicrobial Agents and Chemotherapy, July 2001, p. 1994-2000, Vol. 45, No. 7
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.7.1994-2000.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Interaction between DNA Gyrase and Quinolones: Effects of Alanine Mutations at GyrA Subunit Residues Ser83 and Asp87

Faye M. Barnarddagger and Anthony Maxwell*

Department of Biochemistry, University of Leicester, Leicester LE1 7RH, United Kingdom

Received 16 January 2001/Returned for modification 8 March 2001/Accepted 16 April 2001

DNA gyrase is a target of quinolone antibacterial agents, but the molecular details of the quinolone-gyrase interaction are not clear. Quinolone resistance mutations frequently occur at residues Ser83 and Asp87 of the gyrase A subunit, suggesting that these residues are involved in drug binding. Single and double alanine substitutions were created at these positions (Ala83, Ala87, and Ala83 Ala87), and the mutant proteins were assessed for DNA supercoiling, DNA cleavage, and resistance to a number of quinolone drugs. The Ala83 mutant was fully active in supercoiling, whereas the Ala87 and the double mutant were 2.5- and 4- to 5-fold less active, respectively; this loss in activity may be partly due to an increased affinity of these mutant proteins for DNA. Supercoiling inhibition and cleavage assays revealed that the double mutant has a high level of resistance to certain quinolones while the mutants with single alanine substitutions show low-level resistance. Using a drug-binding assay we demonstrated that the double-mutant enzyme-DNA complex has a lower affinity for ciprofloxacin than the wild-type complex. Based on the pattern of resistance to a series of quinolones, an interaction between the C-8 group of the quinolone and the double-mutant gyrase in the region of residues 83 and 87 is proposed.

* Corresponding author. Present address: Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Colney, Norwich, NR4 7UH, United Kingdom. Phone: 01603 450771. Fax: 01603 450018. E-mail: tony.maxwell{at}bbsrc.ac.uk.

dagger Present address: Institute of Infections and Immunity, University Hospital, Queen's Medical Centre, Nottingham NG7 2UH, United Kingdom.

Antimicrobial Agents and Chemotherapy, July 2001, p. 1994-2000, Vol. 45, No. 7
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.7.1994-2000.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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