Regulation of ATP hydrolysis by the ε subunit, ζ subunit and Mg-ADP in the ATP synthase of Paracoccus denitrificans

In oxidative phosphorylation, ATP synthases interconvert two forms of free energy: they are driven by the proton-motive force across an energy-transducing membrane to synthesize ATP and displace the ADP/ATP ratio from equilibrium. For thermodynamically efficient energy conversion they must be reversible catalysts. However, in many species ATP synthases are unidirectional catalysts (their rates of ATP hydrolysis are negligible), and in others mechanisms have evolved to regulate or minimize hydrolysis. Unidirectional catalysis by Paracoccus denitrificans ATP synthase has been attributed to its unique ζ subunit, which is structurally analogous to the mammalian inhibitor protein IF 1 . Here, we used homologous recombination to delete the ζ subunit from the P. denitrificans genome, and compared ATP synthesis and hydrolysis by the wild-type and knockout enzymes in inverted membrane vesicles and the F 1 -ATPase subcomplex. ATP synthesis was not affected by loss of the ζ subunit, and the rate of ATP hydrolysis increased by less than twofold, remaining negligible in comparison with the rates of the Escherichia coli and mammalian enzymes. Therefore, deleting the P. denitrificans ζ subunit is not sufficient to activate ATP hydrolysis. We close by considering our conclusions in the light of reversible catalysis and regulation in ATP synthase enzymes.

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Faculty Opinions recommendation of Deleting the IF1-like ζ subunit from Paracoccus denitrificans ATP synthase is not sufficient to activate ATP hydrolysis.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.732573222.793542250 ◽

2018 ◽

Author(s):

Stephen Spiro

Keyword(s):

Atp Synthase ◽

Atp Hydrolysis ◽

Paracoccus Denitrificans

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Aerobic Growth of Escherichia coli Is Reduced, and ATP Synthesis Is Selectively Inhibited when Five C-terminal Residues Are Deleted from the ϵ Subunit of ATP Synthase

Journal of Biological Chemistry ◽

10.1074/jbc.m115.665059 ◽

2015 ◽

Vol 290 (34) ◽

pp. 21032-21041 ◽

Cited By ~ 20

Author(s):

Naman B. Shah ◽

Thomas M. Duncan

Keyword(s):

Escherichia Coli ◽

Atp Synthase ◽

Atp Hydrolysis ◽

Atp Synthesis ◽

Intrinsic Rate ◽

Synthesis Rate ◽

Final Segment ◽

Rotary Catalysis

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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Purification and Biochemical Characterization of the F1Fo-ATP Synthase from Thermoalkaliphilic Bacillus sp. Strain TA2.A1

Journal of Bacteriology ◽

10.1128/jb.185.15.4442-4449.2003 ◽

2003 ◽

Vol 185 (15) ◽

pp. 4442-4449 ◽

Cited By ~ 45

Author(s):

Gregory M. Cook ◽

Stefanie Keis ◽

Hugh W. Morgan ◽

Christoph von Ballmoos ◽

Ulrich Matthey ◽

...

Keyword(s):

Atp Synthase ◽

Biochemical Characterization ◽

Atp Hydrolysis ◽

Sodium Dodecyl ◽

Electrical Potential ◽

Atp Synthesis ◽

Intrinsic Property ◽

Protein Sequencing ◽

Hydrolysis Activity

ABSTRACT We describe here purification and biochemical characterization of the F1Fo-ATP synthase from the thermoalkaliphilic organism Bacillus sp. strain TA2.A1. The purified enzyme produced the typical subunit pattern of an F1Fo-ATP synthase on a sodium dodecyl sulfate-polyacrylamide gel, with F1 subunits α, β, γ, δ, and ε and Fo subunits a, b, and c. The subunits were identified by N-terminal protein sequencing and mass spectroscopy. A notable feature of the ATP synthase from strain TA2.A1 was its specific blockage in ATP hydrolysis activity. ATPase activity was unmasked by using the detergent lauryldimethylamine oxide (LDAO), which activated ATP hydrolysis >15-fold. This activation was the same for either the F1Fo holoenzyme or the isolated F1 moiety, and therefore latent ATP hydrolysis activity is an intrinsic property of F1. After reconstitution into proteoliposomes, the enzyme catalyzed ATP synthesis driven by an artificially induced transmembrane electrical potential (Δψ). A transmembrane proton gradient or sodium ion gradient in the absence of Δψ was not sufficient to drive ATP synthesis. ATP synthesis was eliminated by the electrogenic protonophore carbonyl cyanide m-chlorophenylhydrazone, while the electroneutral Na+/H+ antiporter monensin had no effect. Neither ATP synthesis nor ATP hydrolysis was stimulated by Na+ ions, suggesting that protons are the coupling ions of the ATP synthase from strain TA2.A1, as documented previously for mesophilic alkaliphilic Bacillus species. The ATP synthase was specifically modified at its c subunits by N,N′-dicyclohexylcarbodiimide, and this modification inhibited ATP synthesis.

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Structure of a bacterial ATP synthase

10.1101/463968 ◽

2018 ◽

Cited By ~ 1

Author(s):

Hui Guo ◽

Toshiharu Suzuki ◽

John L. Rubinstein

Keyword(s):

Atp Synthase ◽

Biochemical Analysis ◽

Atp Hydrolysis ◽

Genetic Manipulation ◽

Proton Translocation ◽

Atp Synthesis ◽

Mitochondrial Complex ◽

Rotational States ◽

Membrane Region ◽

Atp Synthases

AbstractATP synthases produce ATP from ADP and inorganic phosphate with energy from a transmembrane proton motive force. Bacterial ATP synthases have been studied extensively because they are the simplest form of the enzyme and because of the relative ease of genetic manipulation of these complexes. We expressed theBacillusPS3 ATP synthase inEschericia coli, purified it, and imaged it by cryo-EM, allowing us to build atomic models of the complex in three rotational states. The position of subunitεshows how it is able to inhibit ATP hydrolysis while allowing ATP synthesis. The architecture of the membrane region shows how the simple bacterial ATP synthase is able to perform the same core functions as the equivalent, but more complicated, mitochondrial complex. The structures reveal the path of transmembrane proton translocation and provide a model for understanding decades of biochemical analysis interrogating the roles of specific residues in the enzyme.

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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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Structure of the ATP synthase from Mycobacterium smegmatis provides targets for treating tuberculosis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2111899118 ◽

2021 ◽

Vol 118 (47) ◽

pp. e2111899118

Author(s):

Martin G. Montgomery ◽

Jessica Petri ◽

Tobias E. Spikes ◽

John E. Walker

Keyword(s):

Atp Synthase ◽

Mycobacterium Smegmatis ◽

Atp Hydrolysis ◽

Terminal Region ◽

Catalytic Cycle ◽

Α Subunit ◽

Antitubercular Drugs ◽

Safe Mechanism ◽

Fail Safe ◽

Α Subunits

The structure has been determined by electron cryomicroscopy of the adenosine triphosphate (ATP) synthase from Mycobacterium smegmatis. This analysis confirms features in a prior description of the structure of the enzyme, but it also describes other highly significant attributes not recognized before that are crucial for understanding the mechanism and regulation of the mycobacterial enzyme. First, we resolved not only the three main states in the catalytic cycle described before but also eight substates that portray structural and mechanistic changes occurring during a 360° catalytic cycle. Second, a mechanism of auto-inhibition of ATP hydrolysis involves not only the engagement of the C-terminal region of an α-subunit in a loop in the γ-subunit, as proposed before, but also a “fail-safe” mechanism involving the b′-subunit in the peripheral stalk that enhances engagement. A third unreported characteristic is that the fused bδ-subunit contains a duplicated domain in its N-terminal region where the two copies of the domain participate in similar modes of attachment of the two of three N-terminal regions of the α-subunits. The auto-inhibitory plus the associated “fail-safe” mechanisms and the modes of attachment of the α-subunits provide targets for development of innovative antitubercular drugs. The structure also provides support for an observation made in the bovine ATP synthase that the transmembrane proton-motive force that provides the energy to drive the rotary mechanism is delivered directly and tangentially to the rotor via a Grotthuss water chain in a polar L-shaped tunnel.

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F1F0-ATP synthase from bovine heart mitochondria: development of the purification of a monodisperse oligomycin-sensitive ATPase

Biochemical Journal ◽

10.1042/bj2950799 ◽

1993 ◽

Vol 295 (3) ◽

pp. 799-806 ◽

Cited By ~ 30

Author(s):

R Lutter ◽

M Saraste ◽

H S van Walraven ◽

M J Runswick ◽

M Finel ◽

...

Keyword(s):

Atp Synthase ◽

Atp Hydrolysis ◽

Gel Filtration ◽

Primary Objective ◽

Membrane Domain ◽

Mitochondrial Membranes ◽

F1 Atpase ◽

Ammonium Sulphate Precipitation ◽

F1f0 Atp Synthase ◽

Hydrolysis Activity

A new procedure for the isolation of ATP synthase from bovine mitochondria has been developed, with the primary objective of producing enzyme suitable for crystallization trials. Proteins were extracted from mitochondrial membranes with dodecyl-beta-D-maltoside, and the ATP synthase was purified from the extract in the presence of the same detergent by a combination of ion-exchange and gel-filtration chromatography and ammonium sulphate precipitation. This simple and rapid procedure yields 20-30 mg of highly pure and monodisperse enzyme, evidently consisting of 14 different subunits, amongst them, in apparently stoichiometric amounts with the established subunits, subunit e, a recently discovered subunit of unknown function. The enzyme preparation has an oligomycin-sensitive ATP hydrolysis activity, and so the F1 domain is functionally associated with the membrane domain, F0. In contrast with the N-termini of some of the subunits of bovine mitochondrial F1-ATPase, those of the F1F0-ATP synthase are not degraded by proteolysis during the isolation procedure. This preparation therefore satisfies prerequisites for crystallization trials.

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Uni-Site ATP Hydrolysis Catalyzed by the ATP-Synthase from Chloroplasts

Current Research in Photosynthesis ◽

10.1007/978-94-009-0511-5_445 ◽

1990 ◽

pp. 1943-1946

Author(s):

A. Labahn ◽

P. Gräber

Keyword(s):

Atp Synthase ◽

Atp Hydrolysis

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The structure of the catalytic domain of the ATP synthase fromMycobacterium smegmatisis a target for developing antitubercular drugs

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1817615116 ◽

2019 ◽

Vol 116 (10) ◽

pp. 4206-4211 ◽

Cited By ~ 19

Author(s):

Alice Tianbu Zhang ◽

Martin G. Montgomery ◽

Andrew G. W. Leslie ◽

Gregory M. Cook ◽

John E. Walker

Keyword(s):

Atp Synthase ◽

Mycobacterium Smegmatis ◽

Catalytic Domain ◽

Atp Hydrolysis ◽

Atp Synthesis ◽

Geobacillus Stearothermophilus ◽

Antitubercular Drugs ◽

Atp Binding Site ◽

E Coli ◽

Catalytic Cycles

The crystal structure of the F1-catalytic domain of the adenosine triphosphate (ATP) synthase has been determined fromMycobacterium smegmatiswhich hydrolyzes ATP very poorly. The structure of the α3β3-component of the catalytic domain is similar to those in active F1-ATPases inEscherichia coliandGeobacillus stearothermophilus. However, its ε-subunit differs from those in these two active bacterial F1-ATPases as an ATP molecule is not bound to the two α-helices forming its C-terminal domain, probably because they are shorter than those in active enzymes and they lack an amino acid that contributes to the ATP binding site in active enzymes. InE. coliandG. stearothermophilus, the α-helices adopt an “up” state where the α-helices enter the α3β3-domain and prevent the rotor from turning. The mycobacterial F1-ATPase is most similar to the F1-ATPase fromCaldalkalibacillus thermarum, which also hydrolyzes ATP poorly. The βE-subunits in both enzymes are in the usual “open” conformation but appear to be occupied uniquely by the combination of an adenosine 5′-diphosphate molecule with no magnesium ion plus phosphate. This occupation is consistent with the finding that their rotors have been arrested at the same point in their rotary catalytic cycles. These bound hydrolytic products are probably the basis of the inhibition of ATP hydrolysis. It can be envisaged that specific as yet unidentified small molecules might bind to the F1domain inMycobacterium tuberculosis, prevent ATP synthesis, and inhibit the growth of the pathogen.

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