Effects Of Interaction Between GyrB Key Region Mutants And GyrA Of DNA Gyrase From Mycobacterium Tube-rculosis On Its Holoenzymical Function

DNA gyrase is a unique type Ⅱ topoisomerase in M.tuberculosis, and thus it is the sole topoisomerase target for quinolones in M. tuberculosis. This enzyme works against DNA topological problems generated from transcription, replication and repair events, and thus is essential for cell survival and has been exploited as important drug targets for anticancer and antibacterial agents, such as fluoroquinolone, coumerin natural products novobiocin, pentapeptide repeat proteins MfpA Mt and QnrB4et al.. However, since the standard anti-TB drugs has been used for decades, resistance to these grugs has emerged and Multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) has also been reported worldwide. This signals the need for discovery of medicines effective against the variants.DNA gyrase is a tetrameric A2B2protein, separated GyrA or GyrB subunit does not show its enzymatic activity except the recombinant enzyme. In order to develop effective drugs for TB treatment, we need more specific information of the drug targets-DNA. Currently, research of the interaction region, structure, sites of GyrA and GyrB subunit are not so clearly. To determine the interaction area of GyrB and GyrA, a series of GyrB mutants has been constructed.Firstly, wild-type GyrA, GyrB, GyrB mutants (GyrB1-518, GyrB1-531, GyrB△531-550, GyrB△548-564, GyrB-CTD, GyrB-NTD) have been expressed, purified and concentrated.Secondly, the reaction conditions and main components of the relaxation and cleavage activity of DNA gyrase under the laboratory conditions were designed, the relaxation and cleavage reaction were37℃,30min and37℃,20min, respectively. Spermidine, DTT, tRNA were essential components, whereas EDTA can be omitted.Finally, the results further confirmed that the C-terminus of GyrB subunit is the the key area to interact with GyrA. In the relaxation and cleavage activity,531-550aa and548-564aa are necessary. Thay both located in the Toprim domain,531-550aa acts as an α-helix, and548-564aa is composed by an distorted α-helix and β-sheet. What’s more, Toprim domain is the essential area in the interaction of GyrA and GyrB subunits. Model functional sites "DXD" was located in the531-550aa, thus531-550aa shows both effects on enzyme activity and structure, whereas548-564aa makes more performance on structural support and construction.