CRYSTAL STRUCTURE OF THE DNA GYRASE B PROTEIN FROM B. stearothermophilus. F. T. F. Tsai1, H. S. Subramanya1, J. A, Brannigan2, A J. Wilkinson2,T. Skarzynski3, O. M. P. Singh3, A. J. Wonacott3, and D. B. Wigley1, 1Laboratory of Molecular Biophysics, Rex Richards Building, Oxford University, Oxford OX1 3QU, UK, 2Dept. of Chemistry, York University, Heslington, York YO1 5DD, UK, 3Department of Biomolecular Structure, Glaxo Wellcome Research and Development Ltd. Medicine Research Centre, Stevenage SG1 2NY, UK
Topoisomerases are DNA-binding proteins that are found in all living organisms. They catalyse the interconversion of different topological forms of DNA by breaking, passing and resealing duplex DNA, and thereby alter the DNA superhelicity in the cell; a process which is essential in DNA replication.
Bacterial DNA gyrase is a type II DNA topoisomerase which uniquely catalyses the negative supercoiling of closed circular DNA in vitro utilising the free energy released by ATP hydrolysis. The protein from B. stearothermophilus is a heterotetrameric enzyme of 334kDa molecular weight, that consist of two pairs of subunits A (GyrA, 97kDa) and B (GyrB,70kDa). Enzymatically, the larger GyrA subunit is responsible for the DNA breakage and religation activity, while the smaller GyrB protein is associated with the ATP binding and hydrolysis activity.
The recent structural information obtained of eukaryotic and prokaryotic type II topoisomerase fragments suggested a functional mechanism for type II topoisomerases. However, it is still unclear why gyrases, in contrast to eukaryotic type II topoisomerases, are able to catalyse the negative supercoiling of closed circular DNA.
The intact GyrB protein from B. stearothermophilus has been purified by standard chromatographic techniques to homogeneity and has been crystallised by dialysis in the presence ADPNP. The crystals belong to the cubic space group I23, with unit cell dimensions a = 249 Å and one dimer in the asymmetric unit (Vm = 4.7Å3Da-1). The structure has been solved to 4.2Å resolution using molecular replacement and isomorphous replacement methods. The collection of high resolution data are currently underway.