title: 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.
abstract: The F1 FO -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.
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.|School of Physical and Mathematical Sciences, Nanyang Technological University, Singapore, Singapore.|Nanyang Institute of Technology in Health and Medicine, Interdisciplinary Graduate School, Nanyang Technological University, Singapore, Singapore.
authors.forename: Adam
authors.initials: A
authors.lastname: Hotra
authors.affiliation: Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
authors.forename: Manuel
authors.initials: M
authors.lastname: Suter
authors.affiliation: Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
authors.forename: Goran
authors.initials: G
authors.lastname: Biuković
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.
authors.forename: Priya
authors.initials: P
authors.lastname: Ragunathan
authors.affiliation: Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
authors.forename: Subhashri
authors.initials: S
authors.lastname: Kundu
authors.affiliation: Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
authors.forename: Thomas
authors.initials: T
authors.lastname: Dick
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Grüber
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D006859:Hydrogen / Q000378:metabolism; D006868:Hydrolysis; D020102:Mycobacterium smegmatis / Q000201:enzymology / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D006859:Hydrogen; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases
keywords: F-ATP synthase; Mycobacterium; bioenergetics; proton motive force; tuberculosis