To date, the bacterial DNA topoisomerases are one of the major target biomolecules for the discovery of new antibacterial drugs. DNA topoisomerase regulates the topological state of DNA, which is very important for replication, transcription and recombination. The relaxation of negatively supercoiled DNA is catalyzed by bacterial DNA topoisomerase I (topoI) and this reaction requires Mg2+. In this report, we first quantitatively studied the intermolecular interactions between Escherichia coli topoisomerase I (EctopoI) and pBAD/Thio supercoiled plasmid DNA using surface plasmon resonance (SPR) technique. The equilibrium dissociation constant (Kd) for EctopoIpBAD/Thio interactions was determined to be about 8 nM. We then studied the effect of Mg2+ on the catalysis of EctopoIpBAD/Thio reaction. A slightly higher equilibrium dissociation constant (~15 nM) was obtained for Mg2+ coordinated EctopoI (Mg2+EctopoI)pBAD/Thio interactions. In addition, we observed a larger dissociation rate constant (kd) for Mg2+EctopoIpBAD/Thio interactions (~0.043 s-1), compared to EctopoIpBAD/Thio interactions (~0.017 s-1). These results suggest that enzyme turnover during plasmid DNA relaxation is enhanced due to the presence of Mg2+ and furthers the understanding of importance of the Mg2+ ion for bacterial topoisomerase I catalytic activity.
Tiwari, Purushottam Babu, Thirunavukkarasu Annamalai, Bokun Cheng, Gagandeep Narula, Xuewen Wang, Yuk-Ching Tse-Dinh, Jin He, Yesim Darici
Biochemical and biophysical research communications