PPEF: A bisbenzimdazole potent antimicrobial agent interacts at acidic triad of catalytic domain of E. coli topoisomerase IA

• Published in: Biochimica et biophysica acta. General subjects Vol. 1863; no. 10; pp. 1524 - 1535
• Main Authors: Singh, Raja, Pandey, Stuti, Sur, Souvik, Tandon, Vibha
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.10.2019
• Subjects: Acidic triad; Anti-Bacterial Agents - pharmacology; Bisbenzimidazole - metabolism; Bisbenzimidazole - pharmacology; Catalytic Domain - drug effects; Cleavage and Religation; Cloning, Molecular; DNA Topoisomerases, Type I - drug effects; DNA Topoisomerases, Type I - genetics; DNA Topoisomerases, Type I - metabolism; EcTopo 67; Escherichia coli - enzymology; Mutagenesis, Site-Directed; Poison inhibitor; PPEF; Thermodynamics; EcTopo 67; Poison inhibitor; Acidic triad; PPEF; Cleavage and Religation
• ISSN: 0304-4165; 1872-8006
• DOI: 10.1016/j.bbagen.2019.05.015

Topoisomerase is a well known target to develop effective antibacterial agents. In pursuance of searching novel antibacterial agents, we have established a novel bisbenzimidazole (PPEF) as potent E. coli topoisomerase IA poison inhibitor. In order to gain insights into the mechanism of action of PPEF and understanding protein-ligand interactions, we have produced wild type EcTopo 67 N-terminal domain (catalytic domain) and its six mutant proteins at acidic triad (D111, D113, E115). The DDE motif is replaced by alanine (A) to create three single mutants: D111A, D113A, E115A and three double mutants: D111A-D113A, D113A-E115A and D111A-E115A. Calorimetric study of PPEF with single mutants showed 10 fold lower affinity than that of wild type EcTopo 67 (7.32 × 106 M−1for wild type, 0.89 × 106 M−1for D111A) and 100 fold lower binding with double mutant D113A-E115A (0.02 × 106 M−1) was observed. The mutated proteins showed different CD signature as compared to wild type protein. CD and fluorescence titrations were done to study the interaction between EcTopo 67 and ligands. Molecular docking study validated that PPEF has decreased binding affinity towards mutated enzymes as compared to wild type. The overall study reveals that PPEF binds to D113 and E115 of acidic triad of EcTopo 67. Point mutations decrease binding affinity of PPEF towards DDE motif of topoisomerase. This study concludes PPEF as poison inhibitor of E. coli Topoisomerase IA, which binds to acidic triad of topoisomerase IA, responsible for its function. PPEF can be considered as therapeutic agent against bacteria. [Display omitted] •PPEF act as poison Inhibitor by stabilizing the cleavage religation complex.•Mutants of the acidic triad of EcTopo 67 by single mutation as well as double mutation cause the loss of religation activity•PPEF bind to the acid triad of EcTopo 67 and mutation will decrease the bind affinity of PPEF