Study On The Key Region Of Interaction Between GyrB And GyrA Of DNA Gyrase From Mycobacterium Tuberculosis H₃₇ R_V

DNA gyrase is a basic enzyme in prokaryotes and is essential for DNA transcription, replication, and recombination. DNA gyrase is potential medicine targets of antibacterial and anticancer. Mycobacterium tuberculosis DNA gyrase play a important role in tuberculosis treatment. Research of DNA gyrase catalytic mechanism and medicine targes have beeing a hot point.The protein from Mtb is a homodimer whereas the bacterial enzymes are heterotetramers as B2A2, the two subunits of gyrase are GyrB and GyrA. But the ineraction mechanism between GyrB and GyrA of DNA gyrase from Mycobacterium tuberculosis still need to be explored. We have proved that the Toprim domain in GyrB is a role ineraction region, According to this outcomes, if the key interaction points or regions between GyrA and GyrB were known, we can effectively design medicine targets.In order to maker sure of the key interactional domain of GyrB with GyrA, the study constructed several truncated mutants which was based on the structure of Mtb GyrB: gyrB1-518, gyrB1-531, gyrBl-550, gyrBΔ531-550, gyrBΔ548-564. Purified proteins after ligasing these fragments to expression vector pET28a and pGADT7, We used some technologies to research the interaction between GyrA and truncated mutants, the outcomes of yeast two hybrid system, GST pull down and SPR assay suggested as follow: First, the CTD of GyrB is essential for the interaction of GyrB and GyrA, because the interactive activity lost when the CTD of GyrB was deleted; Second, the a helix (531-550aa)in Toprim domain is the key interaction region between GyrA and GyrB, it may play a role in GyrB'constructure; Third, the short peptide 548-550 aa may brace the basicα/βstructure of Toprim domain, its activities should coordinate with some other proteins.