TY - JOUR
T1 - A surface plasmon resonance study of the intermolecular interaction between Escherichia coli topoisomerase i and pBAD/Thio supercoiled plasmid DNA
AU - Tiwari, Purushottam Babu
AU - Annamalai, Thirunavukkarasu
AU - Cheng, Bokun
AU - Narula, Gagandeep
AU - Wang, Xuewen
AU - Tse-Dinh, Yuk Ching
AU - He, Jin
AU - Darici, Yesim
N1 - Funding Information:
P. Tiwari would like to thank Florida International University (FIU) School of Integrated Science & Humanity, College Arts & Sciences for the research assistantship. This work was supported by NIH Grant R01GM054226 (Y.T.) and the start-up funds from FIU (J.H.).
PY - 2014/3/7
Y1 - 2014/3/7
N2 - 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 EctopoI-pBAD/Thio interactions was determined to be about 8 nM. We then studied the effect of Mg2+ on the catalysis of EctopoI-pBAD/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+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 Mg2+ and furthers the understanding of importance of the Mg2+ ion for bacterial topoisomerase I catalytic activity.
AB - 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 EctopoI-pBAD/Thio interactions was determined to be about 8 nM. We then studied the effect of Mg2+ on the catalysis of EctopoI-pBAD/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+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 Mg2+ and furthers the understanding of importance of the Mg2+ ion for bacterial topoisomerase I catalytic activity.
KW - Bacterial topoisomerase I
KW - Capture covalent method
KW - Equilibrium dissociation constant
KW - Supercoiled plasmid DNA
KW - Surface plasmon resonance
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U2 - 10.1016/j.bbrc.2014.02.015
DO - 10.1016/j.bbrc.2014.02.015
M3 - Article
C2 - 24530905
AN - SCOPUS:84896066378
SN - 0006-291X
VL - 445
SP - 445
EP - 450
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 2
ER -