十字花科黑腐病菌 GyrA 蛋白 C 端 domain 之晶體結構研究

Abstract

Most eubacteria possess two homologous type IIA DNA topoisomerases, gyrase and topoisomerase IV (TopoIV).Both enzymes are heterotetramers assembled from two distinct polypeptides: GyrB and GyrA in the case of gyrase and ParE and ParC for TopoIV. GyrB/ParE contains an ATPase region and a toprim domain； GyrA/ParC harbors DNA cleavage domain and C-terminal domain（CTD）. While these two bacterial enzymes share a high degree of similarity, they appear to have distinct cellular functions. Gyrase is responsible for introducing negative supercoil into closed circular bacterial genomes, while TopoIV is the decatenating enzyme required for the segregation of inter-linked daughter chromosomes. Previous studies suggested that the homologous CTDs of GyrA subunit and ParC subunit may define the functional differences between gyrase and TopoIV. By comparing the crystal structure of GyrA-CTD from Borrelia burgdorferi （Bb GyrA-CTD）and Escherichia coli （Ec GyrA-CTD）,it was found that, though both homologous domains adopt β-pinwheel fold, there are obvious structural differences. Bb GyrA-CTD has smooth and closed appearances. But the Ec GyrA-CTD has a broken spiral shape that is more similar to the ParC-CTD of Bacillus stearothermophilus （Bs GyrA-CTD）.Interestingly, whereas Bb GyrA-CTD is expressed both independently and as a fusion with the N-terminal domain of GyrA, GyrA-CTDs of other bacteria have not been show to be expressed independently. Therefore, it has been proposed that the unique structure of Bb GyrA-CTD may have other functional implications. In contrast, the shape of Ec GyrA-CTD is likely to be more general. Whether the shape of GyrA-CTD is closed may not be the key determinant of the gyrase supercoiling activity. However, the structure of Ec GyrA-CTD does not include the highly conserved GyrA-box sequence in the N-terminal of GyrA-CTD. In addition, GyrA-box still may involve in the closure of GyrA-CTD； biochemical studies have shown that either deletion or substitutions of the positively charged residues in the GyrA-box abolishes the ability of gyrase to introduce negative supercoils. Thus it seems that the overall shape of bacterial GyrA-CTD and the functional significance of GyrA-box remain to be characterized. To gain additional insights into these questions, the goal of this experiment is to determine the GyrA-CTD structure of Xanthomonas campestris by X-ray crystallography.