In contrast with most bacteria which possess two type II topoisomerases (topoisomerase IV and DNA gyrase), Mycobacterium tuberculosis possesses only one, DNA gyrase, which is functionally a hybrid enzyme. Functional differences between the two type IIA topoisomerases are thought to be specified by a CTD (C-terminal DNA-binding domain), which controls DNA recognition. To explore the molecular mechanism responsible for the hybrid functions of the M. tuberculosis DNA gyrase, we conducted a series of sequence analyses and structural and biochemical experiments with the isolated GyrA CTD and the holoenzyme. Although the CTD displayed a global structure similar to that of bona fide GyrA and ParC paralogues, it harbours a second key motif similar in all respects to that of the conserved GyrA-box sequence motif. Biochemical assays showed that the GyrA-box is responsible for DNA supercoiling, whereas the second GyrA-box-l (GyrA-box-like motif) is responsible for the enhanced decatenation activity, suggesting that the mechanistic originality of M. tuberculosis DNA gyrase depends largely on the particular DNA path around the CTD allowed for by the presence of GyrA-box-l. The results of the present study also provide, through phylogenetic exploration of the entire Corynebacterineae suborder, a new and broader insight into the functional diversity of bacterial type IIA topoisomerases.