title: A surface plasmon resonance study of the intermolecular interaction between Escherichia coli topoisomerase I and pBAD/Thio supercoiled plasmid DNA.
abstract: 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 Mg(2+). 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 EctopoI-pBAD/Thio interactions was determined to be about 8 nM. We then studied the effect of Mg(2+) on the catalysis of EctopoI-pBAD/Thio reaction. A slightly higher equilibrium dissociation constant (~15 nM) was obtained for Mg(2+) coordinated EctopoI (Mg(2+)EctopoI)-pBAD/Thio interactions. In addition, we observed a larger dissociation rate constant (kd) for Mg(2+)EctopoI-pBAD/Thio interactions (~0.043 s(-1)), compared to EctopoI-pBAD/Thio interactions (~0.017 s(-1)). These results suggest that enzyme turnover during plasmid DNA relaxation is enhanced due to the presence of Mg(2+) and furthers the understanding of importance of the Mg(2+) ion for bacterial topoisomerase I catalytic activity.
authors: Department of Physics, Florida International University, Miami, FL 33199, United States.
authors: Purushottam Babu
authors: PB
authors: Tiwari
authors: Department of Chemistry and Biochemistry, Florida International University, Miami, FL 33199, United States.
authors: Thirunavukkarasu
authors: T
authors: Annamalai
authors: Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY 10595, United States.
authors: Bokun
authors: B
authors: Cheng
authors: Department of Chemistry and Biochemistry, Florida International University, Miami, FL 33199, United States.
authors: Gagandeep
authors: G
authors: Narula
authors: Department of Physics, Florida International University, Miami, FL 33199, United States.
authors: Xuewen
authors: X
authors: Wang
authors: Department of Chemistry and Biochemistry, Florida International University, Miami, FL 33199, United States. Electronic address: ytsedinh@fiu.edu.
authors: Yuk-Ching
authors: YC
authors: Tse-Dinh
authors: Department of Physics, Florida International University, Miami, FL 33199, United States. Electronic address: jinhe@fiu.edu.
authors: Jin
authors: J
authors: He
authors: Department of Physics, Florida International University, Miami, FL 33199, United States. Electronic address: dariciy@fiu.edu.
authors: Yesim
authors: Y
authors: Darici
mesh_terms: D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D008274:Magnesium / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism*; D020349:Surface Plasmon Resonance
chemical_list: D004278:DNA, Superhelical; D004264:DNA Topoisomerases, Type I; D008274:Magnesium
keywords: Bacterial topoisomerase I; Capture covalent method; Equilibrium dissociation constant; Supercoiled plasmid DNA; Surface plasmon resonance