No abstract

We studied the functional role of highly conserved VISIT-DG sequence residues αIle-346 and αIle-348 in the catalytic sites of Escherichia coli F1Fo ATP synthase. αIle-346 is in close proximity, 2.98 and 3.63 Å, to the two known phosphate binding residues αR376 and βR182; αIle-348 is situated within 3.66 Å from βR182. Single or double mutants of both αI346 and αI348 resulted in a variable loss of oxidative phosphorylation and ATPase activity. Azide, fluoroaluminate, and fluoroscandium caused insignificant to significant inhibition of mutants. Whereas the wild-type enzyme was completely inhibited by NBD-Cl (7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole), a variable extent of inhibition was observed for αI346 and αI348 mutants. MgPi protection against NBD-Cl induced inhibition of wild-type, αI346, and αI348 demonstrated that, although strongly conserved, αI346 and αI348 have no direct role in phosphate binding. Insertion of Arginine in the form of αI346R/βR182A, αI346R/αR376A, or αI348R/βR182A was able to compensate for the absence of known phosphate-binding Arginine residues βR182 and αR376. Results also suggest that αIle-346 and αIle-348 seem to have functional importance in upholding the phosphate-binding subdomain and transition state stabilization in the catalytic sites of E. coli ATP synthase. Keywords

BackgroundAntibiotic resistance is becoming a huge threat with devastating effects on public health and the global economy. Therefore, finding new alternatives to combat antibiotic resistant microbes is of paramount importance. Selective inhibition of bacterial ATP synthase provides a fascinating opportunity to deal with this ongoing problem. ATP synthase is vital for cellular energy production in almost all organisms from bacteria to man. Inhibition of ATP synthase can deprive cells of ATP resulting in cell death. Many peptides are known to have antimicrobial and antitumor properties. Earlier some peptide were shown to inhibit Escherichia coli ATP synthase establishing that the antimicrobial effects of peptides can be connected to the blocking of ATP generation in bacterial cells. Currently, our lab is exploring the connection between a variety of antimicrobial venom peptides and E. coli ATP synthase.MethodThe growth properties of wild‐type and mutant E. coli strains along with null strains were verified on fermentable and non‐fermentable carbon sources before harvesting cells in minimal media to isolate ATP synthase. Membrane bound F1Fo ATP synthase inhibitory studies and E. coli cell growth assays were performed in presence of varied concentrations of natural and modified venom peptides. A null control with deleted ATPase gene was also used in all the studies.ResultsOur preliminary results show that the natural and modified venom peptides affected degree of inhibition. It seems that inhibitory potency of venom peptides can be enhanced by addition of a c‐terminal NH2 groups. We also observed that venom peptides follow a differential pattern of inhibition between wild type and mutant ATP synthase. Our initial results also indicate that incremental additions of positive charges can augment the extent of inhibition. Our early results suggest a feasible connection between the antimicrobial properties of venom peptides and bacterial ATP synthase. We are finalizing our preliminary results and we are investigating the synergetic effects of venom peptides on ATP synthase.ConclusionsApparently, antimicrobial venom peptides inhibit E. coli ATP synthase and bind at the βDELSEED‐motif of ATP synthase. Our preliminary data suggests that the inhibitory potency of venom peptides can be augmented through structural modification of venom peptides. Also, selective inhibition of ATP synthase by venom peptides offers a valuable alternative to combat antibiotic resistant bacterial infection.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.