AAC Accepts, published online ahead of print on 26 October 2009

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Antimicrob. Agents Chemother. doi:10.1128/AAC.00972-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Mapping simocyclinone D8 interaction to DNA gyrase: evidence for a new binding site on GyrB

C. Sissi, E. Vazquez, A. Chemello, L. A. Mitchenall, A. Maxwell, and M. Palumbo

Department of Pharmaceutical Sciences, University of Padova, Via Marzolo, 5- 35131 Padova, Italy; Department of Biological Chemistry, John Innes Centre, Colney, Norwich, NR4 7UH, UK

Simocyclinone D8, a coumarin derivative isolated from Streptomyces antibioticus Tü 6040, represents an interesting new antiproliferative agent. This drug has been originally suggested to recognize the GyrA subunit and interfere with the gyrase catalytic cycle by preventing its binding to DNA. To further characterise the mode of action of this antibiotic, we investigated its binding to the reconstituted DNA gyrase (A₂B₂) as well as to its subunits GyrA and GyrB, and individual domains of these proteins, by performing protein melting and proteolytic digestion studies as well as inhibition assays. Two binding sites were identified, one (anticipated) in the N-terminal domain of GyrA (GyrA59), the other (unexpected) at the C-terminal domain of GyrB (GyrB47). Stabilisation of the A subunit appears to be considerably more effective than stabilisation of the B subunit. Our data suggest that these two distinct sites could cooperate in the reconstituted enzyme.