Turnover Number of Escherichia coli F0F1 ATP Synthase for ATP Synthesis in Membrane Vesicles

F-type ATP synthases are rotary nanomotor enzymes involved in cellular energy metabolism in eukaryotes and eubacteria. The ATP synthase from Gram-positive and -negative model bacteria can be autoinhibited by the C-terminal domain of its ϵ subunit (ϵCTD), but the importance of ϵ inhibition in vivo is unclear. Functional rotation is thought to be blocked by insertion of the latter half of the ϵCTD into the central cavity of the catalytic complex (F1). In the inhibited state of the Escherichia coli enzyme, the final segment of ϵCTD is deeply buried but has few specific interactions with other subunits. This region of the ϵCTD is variable or absent in other bacteria that exhibit strong ϵ-inhibition in vitro. Here, genetically deleting the last five residues of the ϵCTD (ϵΔ5) caused a greater defect in respiratory growth than did the complete absence of the ϵCTD. Isolated membranes with ϵΔ5 generated proton-motive force by respiration as effectively as with wild-type ϵ but showed a nearly 3-fold decrease in ATP synthesis rate. In contrast, the ϵΔ5 truncation did not change the intrinsic rate of ATP hydrolysis with membranes. Further, the ϵΔ5 subunit retained high affinity for isolated F1 but reduced the maximal inhibition of F1-ATPase by ϵ from >90% to ∼20%. The results suggest that the ϵCTD has distinct regulatory interactions with F1 when rotary catalysis operates in opposite directions for the hydrolysis or synthesis of ATP.

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Targeting Mycobacterial F-ATP Synthase C-Terminal α Subunit Interaction Motif on Rotary Subunit γ

Antibiotics ◽

10.3390/antibiotics10121456 ◽

2021 ◽

Vol 10 (12) ◽

pp. 1456

Author(s):

Amaravadhi Harikishore ◽

Chui-Fann Wong ◽

Priya Ragunathan ◽

Dennis Litty ◽

Volker Müller ◽

...

Keyword(s):

Atp Synthase ◽

Atp Hydrolysis ◽

Atp Synthesis ◽

Membrane Vesicles ◽

Α Subunit ◽

Rotational States ◽

C Terminus ◽

Inverted Membrane Vesicles ◽

Hydrolysis Of ◽

Micromolar Range

Mycobacteria regulate their energy (ATP) levels to sustain their survival even in stringent living conditions. Recent studies have shown that mycobacteria not only slow down their respiratory rate but also block ATP hydrolysis of the F-ATP synthase (α3:β3:γ:δ:ε:a:b:b’:c9) to maintain ATP homeostasis in situations not amenable for growth. The mycobacteria-specific α C-terminus (α533-545) has unraveled to be the major regulative of latent ATP hydrolysis. Its deletion stimulates ATPase activity while reducing ATP synthesis. In one of the six rotational states of F-ATP synthase, α533-545 has been visualized to dock deep into subunit γ, thereby blocking rotation of γ within the engine. The functional role(s) of this C-terminus in the other rotational states are not clarified yet and are being still pursued in structural studies. Based on the interaction pattern of the docked α533-545 region with subunit γ, we attempted to study the druggability of the α533-545 motif. In this direction, our computational work has led to the development of an eight-featured α533-545 peptide pharmacophore, followed by database screening, molecular docking, and pose selection, resulting in eleven hit molecules. ATP synthesis inhibition assays using recombinant ATP synthase as well as mycobacterial inverted membrane vesicles show that one of the hits, AlMF1, inhibited the mycobacterial F-ATP synthase in a micromolar range. The successful targeting of the α533-545-γ interaction motif demonstrates the potential to develop inhibitors targeting the α site to interrupt rotary coupling with ATP synthesis.

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Escherichia coli proton-translocating F0F1-ATP synthase and its association with solute secondary transporters and/or enzymes of anaerobic oxidation–reduction under fermentation

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2004.02.005 ◽

2004 ◽

Vol 315 (4) ◽

pp. 1051-1057 ◽

Cited By ~ 59

Author(s):

Armen Trchounian

Keyword(s):

Escherichia Coli ◽

Atp Synthase ◽

Anaerobic Oxidation ◽

F0f1 Atp Synthase ◽

Oxidation Reduction

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Molecular Features of Energy Coupling in the F0F1 ATP Synthase

Physiology ◽

10.1152/physiologyonline.1999.14.1.40 ◽

1999 ◽

Vol 14 (1) ◽

pp. 40-46

Author(s):

Robert K. Nakamoto

Keyword(s):

Atp Synthase ◽

Catalytic Domain ◽

Atp Synthesis ◽

Energy Coupling ◽

F0f1 Atp Synthase ◽

Molecular Features ◽

Rotary Mechanism ◽

Transport Domain

H+ translocation is coupled to ATP synthesis in the F0F1 ATP synthase via a rotary mechanism. Catalytic turnover, site-site cooperativity, and H+ transport obligatorily involve rotation of a set of subunits. The transport domain in the membranous F0 and the catalytic domain in the F1 are mechanisms designed for generating torque.

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