Monoclonal antibodies to mycobacterial DNA gyrase A inhibit DNA supercoiling activity

DNA gyrase is an essential type II topoisomerase found in bacteria. We have previously characterized DNA gyrase from Mycobacterium tuberculosis and My cobacterium smegmatis. In this study, several monoclonal antibodies were ge nerated against the gyrase A subunit (GyrA) of M. smegmatis. Three, MsGyrA: C3, MsGyrA:H11 and MsGyrA:E9, were further analyzed for their interaction w ith the enzyme. The monoclonal antibodies showed high degree of crossreacti vity with both fast-growing and slow-growing mycobacteria. In contrast, non e recognized Escherichia coli GyrA. All the three monoclonal antibodies wer e of IgG(1) isotype falling into two distinct types with respect to epitope recognition and interaction with the enzyme. MsGyrA:C3 and MsGyrA:H11 IgG, and their respective Fab fragments, inhibited the DNA supercoiling activit y catalyzed by mycobacterial DNA gyrase. The epitope for the neutralizing m onoclonal antibodies appeared to involve the region towards the N-terminus (residues 351-415) of the enzyme in a conformation-dependent manner. These monoclonal antibodies would serve as valuable tools for structure-function analysis and immunocytological studies of mycobacterial DNA gyrase. In addi tion, they would be useful for designing peptide inhibitors against DNA gyr ase.