Deletion of a unique loop in the mycobacterial F-ATP synthase subunit sheds light on its inhibitory role in ATP hydrolysis-driven H+ pumping

The F1FO-ATP synthase is one of the enzymes that is essential to meet the energy requirement of both the proliferating aerobic and hypoxic dormant stages of the life cycle of mycobacteria. Most F-ATP synthases consume ATP in the (3):(3) headpiece to drive the subunit, which couples ATP cleavage with proton pumping in the c ring of F-O via the bottom of the subunit. ATPase-driven H+ pumping is latent in mycobacteria. The presence of a unique 14 amino acid residue loop of the mycobacterial subunit has been described and aligned in close vicinity to the c-ring loop Priya R et al. (2013) J Bioenerg Biomembr 45, 121-129 Here, we used inverted membrane vesicles (IMVs) of fast-growing Mycobacterium smegmatis and a variety of covalent and non-covalent inhibitors to characterize the ATP hydrolysis activity of the F-ATP synthase inside IMVs. These vesicles formed a platform to investigate the function of the unique mycobaterial loop by deleting the respective loop-encoding sequence ((166-179)) in the genome of M. smegmatis. ATP hydrolysis-driven H+ pumping was observed in IMVs containing the (166-179) mutant protein but not for IMVs containing the wild-type F-ATP synthase. In addition, when compared to the wild-type enzyme, IMVs containing the (166-179) mutant protein showed increased ATP cleavage and lower levels of ATP synthesis, demonstrating that the loop affects ATPase activity, ATPase-driven H+ pumping and ATP synthesis. These results further indicate that the loop may affect coupling of ATP hydrolysis and synthesis in a different mode.