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

• Main Authors: Hotra, Adam, Suter, Manuel, Biuković, Goran, Priya, Ragunathan, Kundu, Subhashri, Dick, Thomas, Grüber, Gerhard
• Other Authors: School of Biological Sciences
• Format: Article
• Language: English
• Published: 2016
• Subjects: Bioenergetics; FATP synthase
• Online Access: https://hdl.handle.net/10356/83414; http://hdl.handle.net/10220/41395
• Institution: Nanyang Technological University

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 α3:β3 headpiece to drive the γ subunit, which couples ATP cleavage with proton pumping in the c ring of FO via the bottom of the γ subunit. ATPase-driven H+ pumping is latent in mycobacteria. The presence of a unique 14 amino acid residue loop of the mycobacterial γ 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 γ loop by deleting the respective loop-encoding sequence (γ166–179) in the genome of M. smegmatis. ATP hydrolysis-driven H+ pumping was observed in IMVs containing the Δγ166–179 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 Δγ166–179 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.