scholarly journals Intergenic suppression of the γM23K uncoupling mutation in F0F1 ATP synthase by βGlu-381 substitutions: the role of the β380DELSEED386 segment in energy coupling

1998 ◽  
Vol 330 (2) ◽  
pp. 707-712 ◽  
Author(s):  
J. Christian KETCHUM ◽  
Marwan K. AL-SHAWI ◽  
K. Robert NAKAMOTO
Keyword(s):  
Hydrogen Bond ◽  
Transition State ◽  
Thermodynamic Parameters ◽  
Atp Synthase ◽  
Energy Coupling ◽  
Crystallographic Structure ◽  
Β Subunit ◽  
Wild Type ◽  
F0f1 Atp Synthase

We previously demonstrated that the Escherichia coli F0F1-ATP synthase mutation, γM23K, caused increased energy of interaction between γ- and β-subunits which was correlated to inefficient coupling between catalysis and transport [Al-Shawi, Ketchum and Nakamoto (1997) J. Biol. Chem. 272, 2300-2306]. Based on these results and the X-ray crystallographic structure of bovine F1-ATPase [Abrahams, Leslie, Lutter and Walker (1994) Nature (London) 370, 621-628] γM23K is believed to form an ionized hydrogen bond with βGlu-381 in the conserved β380DELSEED386 segment. In this report, we further test the role of γ-β-subunit interactions by introducing a series of substitutions for βGlu-381 and γArg-242, the residue which forms a hydrogen bond with βGlu-381 in the wild-type enzyme. βE381A, D, and Q were able to restore efficient coupling when co-expressed with γM23K. All three mutations reversed the increased transition state thermodynamic parameters for steady state ATP hydrolysis caused by γM23K. βE381K by itself caused inefficient coupling, but opposite from the effect of γM23K, the transition state thermodynamic parameters were lower than wild-type. These results suggest that the βE381K mutation perturbs the γ-β-subunit interaction and the local conformation of the β380DELSEED386 segment in a specific way that disrupts the communication of coupling information between transport and catalysis. βE381A, L, K, and R, and γR242L and E mutations perturbed enzyme assembly and stability to varying degrees. These results provide functional evidence that the β380DELSEED386 segment and its interactions with the γ-subunit are involved in the mechanism of coupling.

Molecular Features of Energy Coupling in the F0F1 ATP Synthase

Physiology ◽  
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.

scholarly journals Role of βAsn-243 in the Phosphate-binding Subdomain of Catalytic Sites ofEscherichia coliF1-ATPase

2004 ◽  
Vol 279 (44) ◽  
pp. 46057-46064 ◽  
Author(s):  
Zulfiqar Ahmad ◽  
Alan E. Senior
Keyword(s):  
Atpase Activity ◽  
Atp Synthase ◽  
Catalytic Mechanism ◽  
Wild Type ◽  
Phosphate Binding ◽  
Γ Subunit ◽  
Catalytic Sites ◽  
The Face ◽  
Extensive Involvement

In the catalytic mechanism of ATP synthase, phosphate (Pi) binding and release steps are believed to be correlated to γ-subunit rotation, and Pibinding is proposed to be prerequisite for binding ADP in the face of high cellular [ATP]/[ADP] ratios. In x-ray structures, residue βAsn-243 appears centrally located in the Pi-binding subdomain of catalytic sites. Here we studied the role of βAsn-243 inEscherichia coliATP synthase by mutagenesis to Ala and Asp. Mutation βN243A caused 30-fold impairment of F1-ATPase activity; 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole inhibited this activity less potently than in wild type and Piprotected from inhibition. ADP-fluoroaluminate was more inhibitory than in wild-type, but ADP-fluoroscandium was less inhibitory. βN243D F1-ATPase activity was impaired by 1300-fold and was not inhibited by ADP-fluoroaluminate or ADP-fluoroscandium. 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole activated βN243D F1-ATPase, and Pidid not affect activation. We conclude that residue βAsn-243 is not involved in Pibinding directly but is necessary for correct organization of the transition state complex through extensive involvement in hydrogen bonding to neighboring residues. It is also probably involved in orientation of the “attacking water” and of an associated second water.

scholarly journals A dominant trifluoperazine resistance gene from Saccharomyces cerevisiae has homology with F0F1 ATP synthase and confers calcium-sensitive growth.

1988 ◽  
Vol 8 (8) ◽  
pp. 3094-3103 ◽  
Author(s):  
C K Shih ◽  
R Wagner ◽  
S Feinstein ◽  
C Kanik-Ennulat ◽  
N Neff
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Atp Synthase ◽  
Resistance Mutation ◽  
Amino Acid Sequences ◽  
Wild Type ◽  
Reading Frame ◽  
F0f1 Atp Synthase ◽  
General Inhibitor

The antipsychotic drug trifluoperazine has been long considered a calmodulin inhibitor from in vitro studies but may function in vivo as a more general inhibitor by disturbing ion fluxes and altering the membrane potential. Resistance to trifluoperazine can arise in Saccharomyces cerevisiae cells by alterations in at least three distinct genetic loci. One locus, defined by a spontaneous dominant trifluoperazine resistance mutation (TFP1-408), was isolated and sequenced. The sequence of the TFP1-408 gene revealed a large open reading frame coding for a large protein of 1,031 amino acids with predicted hydrophobic transmembrane domains. A search of existing amino acid sequences revealed a significant homology with F0F1 ATP synthase. Mutant TFP1-408 cells did not grow efficiently in the presence of 50 mM CaCl2, whereas wild-type cells did. Wild-type cells became resistant to trifluoperazine in the presence of 50 mM CaCl2 or 50 mM MgCl2. Mutant cells showed a higher rate of calcium transport relative to wild-type cells. These data suggest that the TFP1 gene product codes for a transmembrane ATPase-like enzyme possibly involved in Ca2+ transport or in generating a transmembrane ion gradient between two cellular compartments.

Probing conformations of the β subunit of F0F1-ATP synthase in catalysis

2006 ◽  
Vol 342 (3) ◽  
pp. 800-807 ◽  
Author(s):  
Tomoko Masaike ◽  
Toshiharu Suzuki ◽  
Satoshi P. Tsunoda ◽  
Hiroki Konno ◽  
Masasuke Yoshida
Keyword(s):  
Atp Synthase ◽  
Β Subunit ◽  
F0f1 Atp Synthase

scholarly journals Insights into the Physiology and Metabolism of a Mycobacterial Cell in an Energy-Compromised State

2019 ◽  
Vol 201 (19) ◽  
Author(s):  
Varsha Patil ◽  
Vikas Jain
Keyword(s):  
Oxidative Phosphorylation ◽  
Atp Synthase ◽  
Drug Targets ◽  
Energy State ◽  
Β Subunit ◽  
Wild Type ◽  
Content Type ◽  
Substrate Level Phosphorylation ◽  
Substrate Level ◽  
Physiological Differences

ABSTRACT Mycobacterium tuberculosis, a bacterium that causes tuberculosis, poses a serious threat, especially due to the emergence of drug-resistant strains. M. tuberculosis and other mycobacterial species, such as M. smegmatis, are known to generate an inadequate amount of energy by substrate-level phosphorylation and mandatorily require oxidative phosphorylation (OXPHOS) for their growth and metabolism. Hence, antibacterial drugs, such as bedaquiline, targeting the multisubunit ATP synthase complex, which is required for OXPHOS, have been developed with the aim of eliminating pathogenic mycobacteria. Here, we explored the influence of suboptimal OXPHOS on the physiology and metabolism of M. smegmatis. M. smegmatis harbors two identical copies of atpD, which codes for the β subunit of ATP synthase. We show that upon deletion of one copy of atpD (M. smegmatis ΔatpD), M. smegmatis synthesizes smaller amounts of ATP and enters into an energy-compromised state. The mutant displays remarkable phenotypic and physiological differences from the wild type, such as respiratory slowdown, reduced biofilm formation, lesser amounts of cell envelope polar lipids, and increased antibiotic sensitivity compared to the wild type. Additionally, M. smegmatis ΔatpD overexpresses genes belonging to the dormancy operon, the β-oxidation pathway, and the glyoxylate shunt, suggesting that the mutant adapts to a low energy state by switching to alternative pathways to produce energy. Interestingly, M. smegmatis ΔatpD shows significant phenotypic, metabolic, and physiological similarities with bedaquiline-treated wild-type M. smegmatis. We believe that the identification and characterization of key metabolic pathways functioning during an energy-compromised state will enhance our understanding of bacterial adaptation and survival and will open newer avenues in the form of drug targets that may be used in the treatment of mycobacterial infections. IMPORTANCE M. smegmatis generates an inadequate amount of energy by substrate-level phosphorylation and mandatorily requires oxidative phosphorylation (OXPHOS) for its growth and metabolism. Here, we explored the influence of suboptimal OXPHOS on M. smegmatis physiology and metabolism. M. smegmatis harbors two identical copies of the atpD gene, which codes for the ATP synthase β subunit. Here, we carried out the deletion of only one copy of atpD in M. smegmatis to understand the bacterial survival response in an energy-deprived state. M. smegmatis ΔatpD shows remarkable phenotypic, metabolic, and physiological differences from the wild type. Our study thus establishes M. smegmatis ΔatpD as an energy-compromised mycobacterial strain, highlights the importance of ATP synthase in mycobacterial physiology, and further paves the way for the identification of novel antimycobacterial drug targets.

scholarly journals The Role of Adenine Nucleotide Translocase in the Mitochondrial Permeability Transition

Cells ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 2686
Author(s):  
Nickolay Brustovetsky
Keyword(s):  
Atp Synthase ◽  
Mitochondrial Membrane ◽  
Mitochondrial Permeability Transition ◽  
Adenine Nucleotide ◽  
Permeability Transition ◽  
Adenine Nucleotide Translocase ◽  
Inner Mitochondrial Membrane ◽  
Mitochondrial Permeability ◽  
F0f1 Atp Synthase

The mitochondrial permeability transition, a Ca2+-induced significant increase in permeability of the inner mitochondrial membrane, plays an important role in various pathologies. The mitochondrial permeability transition is caused by induction of the permeability transition pore (PTP). Despite significant effort, the molecular composition of the PTP is not completely clear and remains an area of hot debate. The Ca2+-modified adenine nucleotide translocase (ANT) and F0F1 ATP synthase are the major contenders for the role of pore in the PTP. This paper briefly overviews experimental results focusing on the role of ANT in the mitochondrial permeability transition and proposes that multiple molecular entities might be responsible for the conductance pathway of the PTP. Consequently, the term PTP cannot be applied to a single specific protein such as ANT or a protein complex such as F0F1 ATP synthase, but rather should comprise a variety of potential contributors to increased permeability of the inner mitochondrial membrane.

A dominant trifluoperazine resistance gene from Saccharomyces cerevisiae has homology with F0F1 ATP synthase and confers calcium-sensitive growth

1988 ◽  
Vol 8 (8) ◽  
pp. 3094-3103 ◽  
Author(s):  
C K Shih ◽  
R Wagner ◽  
S Feinstein ◽  
C Kanik-Ennulat ◽  
N Neff
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Atp Synthase ◽  
Resistance Mutation ◽  
Amino Acid Sequences ◽  
Wild Type ◽  
Reading Frame ◽  
F0f1 Atp Synthase ◽  
General Inhibitor

The antipsychotic drug trifluoperazine has been long considered a calmodulin inhibitor from in vitro studies but may function in vivo as a more general inhibitor by disturbing ion fluxes and altering the membrane potential. Resistance to trifluoperazine can arise in Saccharomyces cerevisiae cells by alterations in at least three distinct genetic loci. One locus, defined by a spontaneous dominant trifluoperazine resistance mutation (TFP1-408), was isolated and sequenced. The sequence of the TFP1-408 gene revealed a large open reading frame coding for a large protein of 1,031 amino acids with predicted hydrophobic transmembrane domains. A search of existing amino acid sequences revealed a significant homology with F0F1 ATP synthase. Mutant TFP1-408 cells did not grow efficiently in the presence of 50 mM CaCl2, whereas wild-type cells did. Wild-type cells became resistant to trifluoperazine in the presence of 50 mM CaCl2 or 50 mM MgCl2. Mutant cells showed a higher rate of calcium transport relative to wild-type cells. These data suggest that the TFP1 gene product codes for a transmembrane ATPase-like enzyme possibly involved in Ca2+ transport or in generating a transmembrane ion gradient between two cellular compartments.

Essential Role of Tyrosine 229 of the Oxaloacetate Decarboxylase β-Subunit in the Energy Coupling Mechanism of the Na+Pump†

Biochemistry ◽  
2000 ◽  
Vol 39 (15) ◽  
pp. 4320-4326 ◽  
Author(s):  
Petra Jockel ◽  
Markus Schmid ◽  
Thomas Choinowski ◽  
Peter Dimroth
Keyword(s):  
Essential Role ◽  
Energy Coupling ◽  
Coupling Mechanism ◽  
Β Subunit ◽  
Oxaloacetate Decarboxylase ◽  
Na Pump
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