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Aims Streptococcus mutans is highly sensitive to inhibitors of proton-pumping F-type ATPase (F-ATPase) under acidic conditions. Herein, we investigated the role of S. mutans F-ATPase in acid tolerance using a bacterium expressing the F-ATPase β subunit at lower levels than the wild-type strain. Methods and results We generated a mutant S. mutans expressing the catalytic β subunit of F-ATPase at lower levels than the wild-type bacterium. The mutant cells exhibited a significantly slower growth rate at pH 5.30, whereas the rate was essentially the same as that of wild-type cells at pH 7.40. In addition, the colony-forming ability of the mutant was decreased at pH <4.30 but not at pH 7.40. Thus, the growth rate and survival of S. mutans expressing low levels of the β subunit were reduced under acidic conditions. Conclusions Together with our previous observations, this study indicates that F-ATPase is involved in the acid tolerance mechanism of S. mutans by secreting protons from the cytoplasm. [ABSTRACT FROM AUTHOR]

Ductins are a family of homologous and structurally similar membrane proteins with 2 or 4 trans-membrane alpha-helices. The active forms of the Ductins are membranous ring- or star-shaped oligomeric assemblies and they provide various pore, channel, gap-junction functions, assist in membrane fusion processes and also serve as the rotor c-ring domain of V-and F-ATPases. All functions of the Ductins have been reported to be sensitive to the presence of certain divalent metal cations (Me2+), most frequently Cu2+ or Ca2+ ions, for most of the better known members of the family, and the mechanism of this effect is not yet known. Given that we have earlier found a prominent Me2+ binding site in a well-characterised Ductin protein, we hypothesise that certain divalent cations can structurally modulate the various functions of Ductin assemblies via affecting their stability by reversible non-covalent binding to them. A fine control of the stability of the assembly ranging from separated monomers through a loosely/weakly to tightly/strongly assembled ring might render precise regulation of Ductin functions possible. The putative role of direct binding of Me2+ to the c-ring subunit of active ATP hydrolase in autophagy and the mechanism of Ca2+-dependent formation of the mitochondrial permeability transition pore are also discussed.

Ion-translocating ATPases and ATP synthases (F-, V-, A-type ATPases, and several P-type ATPases and ABC-transporters) catalyze ATP hydrolysis or ATP synthesis coupled with the ion transport across the membrane. F-, V-, and A-ATPases are protein nanomachines that combine transmembrane transport of protons or sodium ions with ATP synthesis/hydrolysis by means of a rotary mechanism. These enzymes are composed of two multisubunit subcomplexes that rotate relative to each other during catalysis. Rotary ATPases phosphorylate/dephosphorylate nucleotides directly, without the generation of phosphorylated protein intermediates. F-type ATPases are found in chloroplasts, mitochondria, most eubacteria, and in few archaea. V-type ATPases are eukaryotic enzymes present in a variety of cellular membranes, including the plasma membrane, vacuoles, late endosomes, and trans-Golgi cisternae. A-type ATPases are found in archaea and some eubacteria. F- and A-ATPases have two main functions: ATP synthesis powered by the proton motive force (pmf) or, in some prokaryotes, sodium-motive force (smf) and generation of the pmf or smf at the expense of ATP hydrolysis. In prokaryotes, both functions may be vitally important, depending on the environment and the presence of other enzymes capable of pmf or smf generation. In eukaryotes, the primary and the most crucial function of F-ATPases is ATP synthesis. Eukaryotic V-ATPases function exclusively as ATP-dependent proton pumps that generate pmf necessary for the transmembrane transport of ions and metabolites and are vitally important for pH regulation. This review describes the diversity of rotary ion-translocating ATPases from different organisms and compares the structural, functional, and regulatory features of these enzymes. [ABSTRACT FROM AUTHOR]

A method is proposed for analyzing fast (10 µs) single-molecule rotation trajectories in F1 adenosinetriphosphatase (F1-ATPase). This method is based on the distribution of jumps in the rotation angle that occur in the transitions during the steps between subsequent catalytic dwells. The method is complementary to the "stalling" technique devised by H. Noji et al. [Biophys. Rev. 9, 103-118, 2017], and can reveal multiple states not directly detectable as steps. A bimodal distribution of jumps is observed at certain angles, due to the system being in either of 2 states at the same rotation angle. In this method, a multistate theory is used that takes into account a viscoelastic fluctuation of the imaging probe. Using an established sequence of 3 specific states, a theoretical profile of angular jumps is predicted, without adjustable parameters, that agrees with experiment for most of the angular range. Agreement can be achieved at all angles by assuming a fourth state with an ~10 µs lifetime and a dwell angle about 40° after the adenosine 5'-triphosphate (ATP) binding dwell. The latter result suggests that the ATP binding in one β subunit and the adenosine 5'-diphosphate (ADP) release from another β subunit occur via a transient whose lifetime is ~10 µs and is about 6 orders of magnitude smaller than the lifetime for ADP release from a singly occupied F1-ATPase. An internal consistency test is given by comparing 2 independent ways of obtaining the relaxation time of the probe. They agree and are ~15 µs. [ABSTRACT FROM AUTHOR]

Streptococcus mutans , a bacterium mainly inhabiting the tooth surface, is a major pathogen of dental caries. The bacterium metabolizes sugars to produce acids, resulting in an acidic microenvironment in the dental plaque. Hence, S. mutans should possess a mechanism for surviving under acidic conditions. In the current study, we report the effects of inhibitors of Escherichia coli proton-pumping F-type ATPase (F-ATPase) on the activity of S. mutans enzyme, and the growth and survival of S. mutans under acidic conditions. Piceatannol, curcumin, and demethoxycurcumin strongly reduced the ATPase activity of S. mutans F-ATPase. Interestingly, these compounds inhibited the growth of S. mutans at pH 5.3 but not at pH 7.3. They also significantly reduced the colony-forming ability of S. mutans after incubation at pH 4.3, while showing essentially no effect at pH 7.3. These observations indicate that S. mutans is highly sensitive to F-ATPase inhibitors under acidic conditions and that F-ATPase plays an important role in acid tolerance of this bacterium. • Piceatannol, curcumin, and demethoxycurcumin inhibited S. mutans F-ATPase. • The compounds strongly reduced bacterial growth and survival under acidic conditions. • Data suggest that S. mutans F-ATPase plays an important role in acid tolerance. [ABSTRACT FROM AUTHOR]

Proton-translocating FOF1-ATP synthase (F-type ATPase, F-ATPase or FOF1) performs ATP synthesis/hydrolysis coupled to proton transport across the membrane in mitochondria, chloroplasts, and most eubacteria. The ATPase activity of the enzyme is suppressed in the absence of protonmotive force by several regulatory mechanisms. The most conserved of these mechanisms is noncompetitive inhibition of ATP hydrolysis by the MgADP complex (ADP-inhibition) which has been found in all the enzymes studied. When MgADP binds without phosphate in the catalytic site, the enzyme enters an inactive state, and MgADP gets locked in the catalytic site and does not exchange with the medium. The degree of ADP-inhibition varies in FOF1 enzymes from different organisms. In the Escherichia coli enzyme, ADP-inhibition is relatively weak and, in contrast to other organisms, is enhanced rather than suppressed by phosphate. In this study, we used sitedirected mutagenesis to investigate the role of amino acid residues β139, β158, β189, and β319 of E. coli FOF1-ATP synthase in the mechanism of ADP-inhibition and its modulation by the protonmotive force. The amino acid residues in these positions differ in the enzymes from beta- and gammaproteobacteria (including E. coli) and FOF1-ATP synthases from other eubacteria, mitochondria, and chloroplasts. The βN158L substitution produced no effect on the enzyme activity, while substitutions βF139Y, βF189L, and βV319T only slightly affected ATP (1 mM) hydrolysis. However, in a mixture of ATP and ADP, the activity of the mutants was less suppressed than that of the wild-type enzyme. In addition, mutations βF189L and βV319T weakened the ATPase activity inhibition by phosphate in the presence of ADP. We suggest that residues β139, β189, and β319 are involved in the mechanism of ADP-inhibition and its modulation by phosphate. [ABSTRACT FROM AUTHOR]

Hydrolysis of ATP by the mitochondrial F‐ATPase is inhibited by a protein called IF1. In the parasitic flagellate, Trypanosoma brucei, this protein, known as TbIF1, is expressed exclusively in the procyclic stage, where the F‐ATPase is synthesizing ATP. In the bloodstream stage, where TbIF1 is absent, the F‐ATPase hydrolyzes ATP made by glycolysis and compensates for the absence of a proton pumping respiratory chain by translocating protons into the intermembrane space, thereby maintaining the essential mitochondrial membrane potential. We have defined regions and amino acid residues of TbIF1 that are required for its inhibitory activity by analyzing the binding of several modified recombinant inhibitors to F1‐ATPase isolated from the procyclic stage of T. brucei. Kinetic measurements revealed that the C‐terminal portion of TbIF1 facilitates homodimerization, but it is not required for the inhibitory activity, similar to the bovine and yeast orthologs. However, in contrast to bovine IF1, the inhibitory capacity of the C‐terminally truncated TbIF1 diminishes with decreasing pH, similar to full length TbIF1. This effect does not involve the dimerization of active dimers to form inactive tetramers. Over a wide pH range, the full length and C‐terminally truncated TbIF1 form dimers and monomers, respectively. TbIF1 has no effect on bovine F1‐ATPase, and this difference in the mechanism of regulation of the F‐ATPase between the host and the parasite could be exploited in the design of drugs to combat human and animal African trypanosomiases. The essential ATPase activity of mitochondrial ATP synthase in the pathogenic stage of Trypanosoma brucei is inhibited by a protein inhibitor TbIF1. TbIF1 forms dimers, acts in a pH‐dependent manner, and does not inhibit a mammalian F1‐ATPase. The inhibition of F1‐ATPase by TbIF1 exhibits several parasite‐specific features. Differences between the parasite and host F1‐ATPase inhibition can be exploited in drug design. [ABSTRACT FROM AUTHOR]

Hydrolysis of ATP by the mitochondrial F‐ATPase is inhibited by a protein called IF1. In the parasitic flagellate, Trypanosoma brucei, this protein, known as TbIF1, is expressed exclusively in the procyclic stage, where the F‐ATPase is synthesizing ATP. In the bloodstream stage, where TbIF1 is absent, the F‐ATPase hydrolyzes ATP made by glycolysis and compensates for the absence of a proton pumping respiratory chain by translocating protons into the intermembrane space, thereby maintaining the essential mitochondrial membrane potential. We have defined regions and amino acid residues of TbIF1 that are required for its inhibitory activity by analyzing the binding of several modified recombinant inhibitors to F1‐ATPase isolated from the procyclic stage of T. brucei. Kinetic measurements revealed that the C‐terminal portion of TbIF1 facilitates homodimerization, but it is not required for the inhibitory activity, similar to the bovine and yeast orthologs. However, in contrast to bovine IF1, the inhibitory capacity of the C‐terminally truncated TbIF1 diminishes with decreasing pH, similar to full length TbIF1. This effect does not involve the dimerization of active dimers to form inactive tetramers. Over a wide pH range, the full length and C‐terminally truncated TbIF1 form dimers and monomers, respectively. TbIF1 has no effect on bovine F1‐ATPase, and this difference in the mechanism of regulation of the F‐ATPase between the host and the parasite could be exploited in the design of drugs to combat human and animal African trypanosomiases. The essential ATPase activity of mitochondrial ATP synthase in the pathogenic stage of Trypanosoma brucei is inhibited by a protein inhibitor TbIF1. TbIF1 forms dimers, acts in a pH‐dependent manner, and does not inhibit a mammalian F1‐ATPase. The inhibition of F1‐ATPase by TbIF1 exhibits several parasite‐specific features. Differences between the parasite and host F1‐ATPase inhibition can be exploited in drug design. [ABSTRACT FROM AUTHOR]

During brief 120° transitions between long catalytic dwells, single F _1 -ATPase molecules exhibit angular jumps that vary with rotation angles. Using the angular jump profile enables the detection of fast states in the mechano-chemical scheme of the enzyme, states that are difficult to capture from single-molecule trajectories due to the fluctuations of the imaging nanoprobe. In a previous work, a short-lived, three occupancy state was postulated from a multi-state, probabilistic theory to explain the mean angular jump profile. An assumption in the theory was that the ‘mixing’ of chemical states is negligible during jumps. In a mixing event, two subsequent angular positions recorded by the imaging apparatus belong to two different chemical states of the motor enzyme due to fast reactions within a recording frame. In this paper, we provide an enhanced method for the detection of fast states. On one hand, we show using Langevin simulations that state mixing leads to faster mean angular jump, shifting up the profile. Consequently, the improved method provides a correction to the angular position and lifetime of the postulated three-occupancy metastable state. On the other hand, we show that when F _1 -ATPase is subject to torques opposing rotation in hydrolysis direction, the torques shift down the dwell angles without affecting the angle-dependent reaction rates. The torques improve detection capability for the fast state by increasing dwell times which is made evident by the flattening of the mean angular jump profile within 40°–60° from the catalytic dwell. In the three-occupancy state release of ADP occurs in concert with the binding of ATP to a different site in the F _1 -ATPase. Similarly, in the full ATP synthase when torques are created by the proton gradient in the F _O region, the release of the product ATP is presumably accelerated by the binding of ADP to a different site in the F _1 domain.

H+-FOF1-ATP synthase (F-ATPase, F-type ATPase, FOF1 complex) catalyzes ATP synthesis from ADP and inorganic phosphate in eubacteria, mitochondria, chloroplasts, and some archaea. ATP synthesis is powered by the transmembrane proton transport driven by the proton motive force (PMF) generated by the respiratory or photosynthetic electron transport chains. When the PMF is decreased or absent, ATP synthase catalyzes the reverse reaction, working as an ATP-dependent proton pump. The ATPase activity of the enzyme is regulated by several mechanisms, of which the most conserved is the non-competitive inhibition by the MgADP complex (ADP-inhibition). When ADP binds to the catalytic site without phosphate, the enzyme may undergo conformational changes that lock bound ADP, resulting in enzyme inactivation. PMF can induce release of inhibitory ADP and reactivate ATP synthase; the threshold PMF value required for enzyme reactivation might exceed the PMF for ATP synthesis. Moreover, membrane energization increases the catalytic site affinity to phosphate, thereby reducing the probability of ADP binding without phosphate and preventing enzyme transition to the ADP-inhibited state. Besides phosphate, oxyanions (e.g., sulfite and bicarbonate), alcohols, lauryldimethylamine oxide, and a number of other detergents can weaken ADP-inhibition and increase ATPase activity of the enzyme. In this paper, we review the data on ADP-inhibition of ATP synthases from different organisms and discuss the in vivo role of this phenomenon and its relationship with other regulatory mechanisms, such as ATPase activity inhibition by subunit ε and nucleotide binding in the noncatalytic sites of the enzyme. It should be noted that in Escherichia coli enzyme, ADP-inhibition is relatively weak and rather enhanced than prevented by phosphate. [ABSTRACT FROM AUTHOR]

The theory of elastic group transfer for the binding and release rate constants for nucleotides in F1-ATPase as a function of the rotor angle is further extended in several respects. (i) A method is described for predicting the experimentally observed lifetime distribution of long binding events in the controlled rotation experiments by taking into account the hydrolysis and synthesis reactions occurring during these events. (ii) A method is also given for treating the long binding events in the experiments and obtaining the rate constants for the hydrolysis and synthesis reactions occurring during these events. (iii) The theory in the previous paper is given in a symmetric form, an extension that simplifies the application of the theory to experiments. It also includes a theory-based correction of the reported "on" and "off" rates by calculating the missed events. A near symmetry of the data about the angle of -40° and a "turnover" in the binding rate data vs. rotor angle for angles greater than -40° is also discussed. [ABSTRACT FROM AUTHOR]

The recent experimental, theoretical and computational advances in the field of F1-ATPase single-molecule microscopy are briefly surveyed. The role of theory is revealed in the statistical analysis, interpretation and prediction of single-molecule experimental trajectories, and in linking them with atomistic simulations. In particular, a theoretical model of elastically coupled molecular group transfer is reviewed and a detailed method for its application in stalling and controlled rotation experiments is provided. It is shown how the model can predict, using previous experiments, the rates of ligand binding/release processes (steps) and their exponential dependence on rotor angle in these experiments. The concept of Brønsted slopes is reviewed in the context of the single-molecule experiments, and the rate versus rotor angle relations are explained using the elastic model. These experimental data are treated in terms of the effect of thermodynamic driving forces on the rates assuming that the rotor shaft is elastically coupled to stator ring subunits in which the steps occur. In the application of the group transfer model on an extended angular range processes leading up to the transfer are discussed. Implications for large-scale atomistic simulation are suggested for the treatment of torque-generating steps. [ABSTRACT FROM AUTHOR]

The F-ATPase is the catalytic portion of the FF ATP synthase and acts as a rotary molecular motor when it hydrolyzes ATP. Two decades have passed since the single-molecule rotation assay of F-ATPase was established. Although several fundamental issues remain elusive, basic properties of F-type ATPases as motor proteins have been well characterized, and a large part of the reaction scheme has been revealed by the combination of extensive structural, biochemical, biophysical, and theoretical studies. This review is intended to provide a concise summary of the fundamental features of F-ATPases, by use of the well-described model F from the thermophilic Bacillus PS3 (TF). In the last part of this review, we focus on the robustness of the rotary catalysis of F-ATPase to provide a perspective on the re-designing of novel molecular machines. [ABSTRACT FROM AUTHOR]

Ductins are a family of homologous and structurally similar membrane proteins with 2 or 4 trans-membrane alpha-helices. The active forms of the Ductins are membranous ring- or star-shaped oligomeric assemblies and they provide various pore, channel, gap-junction functions, assist in membrane fusion processes and also serve as the rotor c-ring domain of V-and F-ATPases. All functions of the Ductins have been reported to be sensitive to the presence of certain divalent metal cations (Me 2+ ), most frequently Cu 2+ or Ca 2+ ions, for most of the better known members of the family, and the mechanism of this effect is not yet known. Given that we have earlier found a prominent Me 2+ binding site in a well-characterised Ductin protein, we hypothesise that certain divalent cations can structurally modulate the various functions of Ductin assemblies via affecting their stability by reversible non-covalent binding to them. A fine control of the stability of the assembly ranging from separated monomers through a loosely/weakly to tightly/strongly assembled ring might render precise regulation of Ductin functions possible. The putative role of direct binding of Me 2+ to the c-ring subunit of active ATP hydrolase in autophagy and the mechanism of Ca 2+ -dependent formation of the mitochondrial permeability transition pore are also discussed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Sebők-Nagy, Blastyák, Juhász and Páli.)

The structures of F-ATPases have been determined predominantly with mitochondrial enzymes, but hitherto no F-ATPase has been crystallized intact. A high-resolution model of the bovine enzyme built up from separate sub-structures determined by X-ray crystallography contains about 85% of the entire complex, but it lacks a crucial region that provides a transmembrane proton pathway involved in the generation of the rotary mechanism that drives the synthesis of ATP. Here the isolation, characterization and crystallization of an integral F-ATPase complex from the α-proteobacterium Paracoccus denitrificans are described. Unlike many eubacterial F-ATPases, which can both synthesize and hydrolyse ATP, the P. denitrificans enzyme can only carry out the synthetic reaction. The mechanism of inhibition of its ATP hydrolytic activity involves a ζ inhibitor protein, which binds to the catalytic F1-domain of the enzyme. The complex that has been crystallized, and the crystals themselves, contain the nine core proteins of the complete F-ATPase complex plus the ζ inhibitor protein. The formation of crystals depends upon the presence of bound bacterial cardiolipin and phospholipid molecules; when they were removed, the complex failed to crystallize. The experiments open the way to an atomic structure of an F-ATPase complex.

Having a sufficient supply of energy, usually in the form of ATP, is essential for all living organisms. In this study, however, we demonstrate that it can be beneficial to reduce ATP availability when the objective is microbial production. By introducing the ATP hydrolyzing F-ATPase into a Lactococcus lactis strain engineered into producing acetoin, we show that production titer and yield both can be increased. At high F-ATPase expression level, the acetoin production yield could be increased by 10 %; however, because of the negative effect that the F-ATPase had on biomass yield and growth, this increase was at the cost of volumetric productivity. By lowering the expression level of the F-ATPase, both the volumetric productivity and the final yield could be increased by 5 % compared to the reference strain not overexpressing the F-ATPase, and in batch fermentation, it was possible to convert 176 mM (32 g/L) of glucose into 146.5 mM (12.9 g/L) acetoin with a yield of 83 % of the theoretical maximum. To further demonstrate the potential of the cell factory developed, we complemented it with the lactose plasmid pLP712, which allowed for growth and acetoin production from a dairy waste stream, deproteinized whey. Using this cheap and renewable feedstock, efficient acetoin production with a titer of 157 mM (14 g/L) acetoin was accomplished. [ABSTRACT FROM AUTHOR]

The mitochondrial permeability transition is an inner mitochondrial membrane event involving the opening of the permeability transition pore concomitant with a sudden efflux of matrix solutes and breakdown of membrane potential. The mitochondrial F o F 1 ATP synthase has been proposed as the molecular identity of the permeability transition pore. The likeliness of potential pore-forming sites in the mitochondrial F o F 1 ATP synthase is discussed and a new model, the death finger model, is described. In this model, movement of a p-side density that connects the lipid-plug of the c-ring with the distal membrane bending F o domain allows reversible opening of the c-ring and structural cross-talk with OSCP and the catalytic (αβ) 3 hexamer. This article is part of a Special Issue entitled ‘EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2–6, 2016’, edited by Prof. Paolo Bernardi. [ABSTRACT FROM AUTHOR]

ATP synthases (F o F 1 ) are found ubiquitously in energy-transducing membranes of bacteria, mitochondria, and chloroplasts. These enzymes couple proton transport and ATP synthesis or hydrolysis through subunit rotation, which has been studied mainly by observing single molecules. In this review, we discuss the mechanism of rotational catalysis of ATP synthases, mainly that from Escherichia coli , emphasizing the high-speed and stochastic rotation including variable rates and an inhibited state. Single molecule studies combined with structural information of the bovine mitochondrial enzyme and mutational analysis have been informative as to an understanding of the catalytic site and the interaction between rotor and stator subunits. We discuss the similarity and difference in structure and inhibitory regulation of F 1 from bovine and E. coli . Unlike the crystal structure of bovine F 1 (α 3 β 3 γ), that of E. coli contains a ε subunit, which is a known inhibitor of bacterial and chloroplast F 1 ATPases. The carboxyl terminal domain of E. coli ε (εCTD) interacts with the catalytic and rotor subunits (β and γ, respectively), and then inhibits rotation. The effects of phytopolyphenols on F 1 -ATPase are also discussed: one of them, piceatannol, lowered the rotational speed by affecting rotor/stator interactions. [ABSTRACT FROM AUTHOR]

Among the many types of bioenergy-transducing machineries, F- and V-ATPases are unique bio- and nano-molecular rotary motors. The rotational catalysis of F¹-ATPase has been investigated in detail, and molecular mechanisms have been proposed based on the crystal structures of the complex and on extensive single-molecule rotational observations. Recently, we obtained crystal structures of bacterial V1-ATPase (A3B3 and A3B3DF complexes) in the presence and absence of nucleotides. Based on these new structures, we present a novel model for the rotational catalysis mechanism of V1- ATPase, which is different from that of F1-ATPases. [ABSTRACT FROM AUTHOR]

The structures of F-ATPases have predominantly been determined from mitochondrial enzymes, and those of the enzymes in eubacteria have been less studied. Paracoccus denitrificans is a member of the α-proteobacteria and is related to the extinct protomitochondrion that became engulfed by the ancestor of eukaryotic cells. The P. denitrificans F-ATPase is an example of a eubacterial F-ATPase that can carry out ATP synthesis only, whereas many others can catalyse both the synthesis and the hydrolysis of ATP. Inhibition of the ATP hydrolytic activity of the P. denitrificans F-ATPase involves the ζ inhibitor protein, an α-helical protein that binds to the catalytic F1 domain of the enzyme. This domain is a complex of three α-subunits and three β-subunits, and one copy of each of the γ-, δ- and ɛ-subunits. Attempts to crystallize the F1-ζ inhibitor complex yielded crystals of a subcomplex of the catalytic domain containing the α- and β-subunits only. Its structure was determined to 2.3 Å resolution and consists of a heterodimer of one α-subunit and one β-subunit. It has no bound nucleotides, and it corresponds to the `open' or `empty' catalytic interface found in other F-ATPases. The main significance of this structure is that it aids in the determination of the structure of the intact membrane-bound F-ATPase, which has been crystallized. [ABSTRACT FROM AUTHOR]

Curcumin, a dietary phytopolyphenol isolated from a perennial herb ( Curcuma longa ), is a well-known compound effective for bacterial infections and tumors, and also as an antioxidant. In this study, we report the inhibitory effects of curcumin and its analogs on the Escherichia coli ATP synthase F 1 sector. A structure–activity relationship study indicated the importance of 4′-hydroxy groups and a β-diketone moiety for the inhibition. The 3′-demethoxy analog (DMC) inhibited F 1 more strongly than curcumin did. Furthermore, these compounds inhibited E. coli growth through oxidative phosphorylation, consistent with their effects on ATPase activity. These results suggest that the two compounds affected bacterial growth through inhibition of ATP synthase. Derivatives including bis(arylmethylidene)acetones (C 5 curcuminoids) exhibited only weak activity toward ATPase and bacterial growth. [ABSTRACT FROM AUTHOR]

F- and V-ATPases are unique bio- and nano-molecular rotary motors among many types of bioenergy-transducing machineries. The rotational catalysis of F1 -ATPase has been investigated in detail, and the molecular mechanisms have been proposed on the basis of crystal structures of the complex and extensive single-molecule observation of the rotation. Recently, we have obtained crystal structures of bacterial V1-ATPase (A3B3 and A3B3DF complexes) with and without nucleotide. On the basis of these new structures, we present a novel model of the rotational catalytic mechanism for V1-ATPase, which is apparently different from those of F1-ATPases. [ABSTRACT FROM AUTHOR]

ATP synthases (FF-ATPases) of chloroplasts, mitochondria, and bacteria catalyze ATP synthesis or hydrolysis coupled with the transmembrane transfer of protons or sodium ions. Their activity is regulated through their reversible inactivation resulting from a decreased transmembrane potential difference. The inactivation is believed to conserve ATP previously synthesized under conditions of sufficient energy supply against unproductive hydrolysis. This review is focused on the mechanism of nucleotide-dependent regulation of the ATP synthase activity where the so-called noncatalytic nucleotide binding sites are involved. Properties of these sites varying upon free enzyme transition to its membrane-bound form, their dependence on membrane energization, and putative mechanisms of noncatalytic site-mediated regulation of the ATP synthase activity are discussed. [ABSTRACT FROM AUTHOR]

In this article, we discuss single molecule observation of rotational catalysis by E. coli ATP synthase (F-ATPase) using small gold beads. Studies involving a low viscous drag probe showed the stochastic properties of the enzyme in alternating catalytically active and inhibited states. The importance of subunit interaction between the rotor and the stator, and thermodynamics of the catalysis are also discussed. 'Single Molecule Enzymology' is a new trend for understanding enzyme mechanisms in biochemistry and physiology. © 2013 IUBMB Life, 65(3):247-254, 2013 [ABSTRACT FROM AUTHOR]

The rate of rotation of the rotor in the yeast vacuolar proton-ATPase (V-ATPase), relative to the stator or steady parts of the enzyme, is estimated in native vacuolar membrane vesicles from Saccharomyces cerevisiae under standardised conditions. Membrane vesicles are formed spontaneously after exposing purified yeast vacuoles to osmotic shock. The fraction of total ATPase activity originating from the V-ATPase is determined by using the potent and specific inhibitor of the enzyme, concanamycin A. Inorganic phosphate liberated from ATP in the vacuolar membrane vesicle system, during ten min of ATPase activity at 20 °C, is assayed spectrophotometrically for different concanamycin A concentrations. A fit of the quadratic binding equation, assuming a single concanamycin A binding site on a monomeric V-ATPase (our data are incompatible with models assuming multiple binding sites), to the inhibitor titration curve determines the concentration of the enzyme. Combining this with the known ATP/rotation stoichiometry of the V-ATPase and the assayed concentration of inorganic phosphate liberated by the V-ATPase, leads to an average rate of ~10 Hz for full 360° rotation (and a range of 6-32 Hz, considering the ± standard deviation of the enzyme concentration), which, from the time-dependence of the activity, extrapolates to ~14 Hz (8-48 Hz) at the beginning of the reaction. These are lower-limit estimates. To our knowledge, this is the first report of the rotation rate in a V-ATPase that is not subjected to genetic or chemical modification and is not fixed to a solid support; instead it is functioning in its native membrane environment. [ABSTRACT FROM AUTHOR]

Abstract: We focus on the rotational catalysis of Escherichia coli F-ATPase (ATP synthase, FOF1). Using a probe with low viscous drag, we found stochastic fluctuation of the rotation rates, a flat energy pathway, and contribution of an inhibited state to the overall behavior of the enzyme. Mutational analyses revealed the importance of the interactions among β and γ subunits and the β subunit catalytic domain. We also discuss the V-ATPase, which has different physiological roles from the F-ATPase, but is structurally and mechanistically similar. We review the rotation, diversity of subunits, and the regulatory mechanism of reversible subunit dissociation/assembly of Saccharomyces cerevisiae and mammalian complexes. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012). [Copyright &y& Elsevier]

The ATP synthase consists of two opposing rotary motors, F and F, coupled to each other. When the F motor is not coupled to the F motor, it can work in the direction hydrolyzing ATP, as a nanomotor called F-ATPase. It has been reported that the stiffness of the protein varies nonlinearly with increasing load. The nonlinearity has an important effect on the rotating rate of the F-ATPase. Here, considering the nonlinearity of the γ shaft stiffness for the F-ATPase, a nonlinear chemo-mechanical coupled dynamic model of F motor is proposed. Nonlinear vibration frequencies of the γ shaft and their changes along with the system parameters are investigated. The nonlinear stochastic response of the elastic γ shaft to thermal excitation is analyzed. The results show that the stiffness nonlinearity of the γ shaft causes an increase of the vibration frequency for the F motor, which increases the motor's rotation rate. When the concentration of ATP is relatively high and the load torque is small, the effects of the stiffness nonlinearity on the rotating rates of the F motor are obvious and should be considered. These results are useful for improving calculation of the rotating rate for the F motor and provide insight about the stochastic wave mechanics of F-ATPase. [ABSTRACT FROM AUTHOR]

Abstract: Nidus Vespae (honeycomb) is a kind of traditional Chinese medicine that has been demonstrated to inhibit the growth and acid-production of oral cariogenic bacteria. Subsequent studies showed that the chloroform/methanol (Chl/MeOH) chemical extraction of Nidus Vespae was the most effective inhibitor of growth and acidogenicity of Streptococcus mutans. In this study, we isolated the chemical compounds of the Nidus Vespae Chl/MeOH extraction, tested their antimicrobial activity against six cariogenic bacteria and further evaluated the acid inhibition properties, anti-F-ATPase activity and anti-LDH activity against S. mutans. The isolated flavonoids, quercetin and kaempferol, inhibited the growth of bacteria (S. mutans, Streptococcus sobrinus, Streptococcus sanguis, Actinomyces viscosus, Actinomyces naeslundii and Lactobacillus rhamnosus) with minimum inhibitory concentrations (MICs) ranging from 1 to 4mg/ml and minimum bactericidal concentrations (MBCs) from 4 to 16mg/ml. In addition, quercetin and kaempferol at sub-MIC levels significantly inhibited acidogenicity and acidurity of S. mutans cells. Treated with the test agents, the F-ATPase activity was reduced by 47.37% with 1mg/ml quercetin and by 49.66% with 0.5mg/ml kaempferol. The results showed that quercetin and kaempferol contained in Chl/MeOH extraction presented remarkably biological activity, suggesting that Nidus Vespae might be useful as a potential preventive and therapeutic agent in dental caries. [Copyright &y& Elsevier]

F-ATPase is a molecular motor in which the γ subunit rotates inside the αβ ring upon adenosine triphosphate (ATP) hydrolysis. Recent works on single-molecule manipulation of F-ATPase have shown that kinetic parameters such as the on-rate of ATP and the off-rate of adenosine diphosphate (ADP) strongly depend on the rotary angle of the γ subunit (Hirono-Hara et al. ; Iko et al. ). These findings provide important insight into how individual reaction steps release energy to power F and also have implications regarding ATP synthesis and how reaction steps are reversed upon reverse rotation. An important issue regarding the angular dependence of kinetic parameters is that the angular position of a magnetic bead rotation probe could be larger than the actual position of the γ subunit due to the torsional elasticity of the system. In the present study, we assessed the stiffness of two different portions of F from thermophilic Bacillus PS3: the internal part of the γ subunit embedded in the αβ ring, and the complex of the external part of the γ subunit and the αβ ring (and streptavidin and magnetic bead), by comparing rotational fluctuations before and after crosslinkage between the rotor and stator. The torsional stiffnesses of the internal and remaining parts were determined to be around 223 and 73 pNnm/radian, respectively. Based on these values, it was estimated that the actual angular position of the internal part of the γ subunit is one-fourth of the magnetic bead position upon stalling using an external magnetic field. The estimated elasticity also partially explains the accommodation of the intrinsic step size mismatch between F and F-ATPase. [ABSTRACT FROM AUTHOR]

Abstract: Two proton pumps, the F-ATPase (ATP synthase, FoF1) and the V-ATPase (endomembrane proton pump), have different physiological functions, but are similar in subunit structure and mechanism. They are composed of a membrane extrinsic (F1 or V1) and a membrane intrinsic (Fo or Vo) sector, and couple catalysis of ATP synthesis or hydrolysis to proton transport by a rotational mechanism. The mechanism of rotation has been extensively studied by kinetic, thermodynamic and physiological approaches. Techniques for observing subunit rotation have been developed. Observations of micron-length actin filaments, or polystyrene or gold beads attached to rotor subunits have been highly informative of the rotational behavior of ATP hydrolysis-driven rotation. Single molecule FRET experiments between fluorescent probes attached to rotor and stator subunits have been used effectively in monitoring proton motive force-driven rotation in the ATP synthesis reaction. By using small gold beads with diameters of 40–60nm, the E. coli F1 sector was found to rotate at surprisingly high speeds (>400rps). This experimental system was used to assess the kinetics and thermodynamics of mutant enzymes. The results revealed that the enzymatic reaction steps and the timing of the domain interactions among the β subunits, or between the β and γ subunits, are coordinated in a manner that lowers the activation energy for all steps and avoids deep energy wells through the rotationally-coupled steady-state reaction. In this review, we focus on the mechanism of steady-state F1-ATPase rotation, which maximizes the coupling efficiency between catalysis and rotation. [Copyright &y& Elsevier]

The 2 nanomotors of rotary ATP synthase, ionmotive FO and chemically active F1, are mechanically coupled by a central rotor and an eccentric bearing. Both motors rotate, with 3 steps in F1 and 10-15 in FO. Simulation by statistical mechanics has revealed that an elastic power transmission is required for a high rate of coupled turnover. Here, we investigate the distribution in the FOF1 structure of compliant and stiff domains. The compliance of certain domains was restricted by engineered disulfide bridges between rotor and stator, and the torsional stiffness (κ) of unrestricted domains was determined by analyzing their thermal rotary fluctuations. A fluorescent magnetic bead was attached to single molecules of F1 and a fluorescent actin filament to FOF1, respectively. They served to probe first the functional rotation and, after formation of the given disulfide bridge, the stochastic rotational motion. Most parts of the enzyme, in particular the central shaft in F1, and the long eccentric bearing were rather stiff (torsional stiffness κ > 750 pNnm). One domain of the rotor, namely where the globular portions of subunits γ and ϵ of F1 contact the c-ring of FO, was more compliant (K ≅ 68 pNnm). This elastic buffer smoothes the cooperation of the 2 stepping motors. It is located were needed, between the 2 sites where the power strokes in FO and F1 are generated and consumed. [ABSTRACT FROM AUTHOR]

Triclosan was found to be a potent inhibitor of the F(H+)-ATPase of the oral pathogen Streptococcus mutans and to increase proton permeabilities of intact cells. Moreover, it acted additively with weak-acid transmembrane proton carriers, such as fluoride or sorbate, to sensitize glycolysis to acid inhibition. Even at neutral pH, triclosan could inhibit glycolysis more directly as an irreversible inhibitor of the glycolytic enzymes pyruvate kinase, lactic dehydro genase, aldolase, and the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Cell glycolysis in suspensions or biofilms was inhibited in a pH-dependent manner by triclosan at a concentration of about 0.1 mmol/L at pH 7, approximately the lethal concentration for S. mutans cells in suspensions. Cells in intact biofilms were almost as sensitive to triclosan inhibition of glycolysis as were cells in suspensions but were more resistant to killing. Targets for irreversible inhibition of glycolysis included the PTS and cytoplasmic enzymes, specifically pyruvate kinase, lactic dehydrogenase, and to a lesser extent, aldolase. General conclusions are that triclosan is a multi-target inhibitor for mutans streptococci, which lack a triclosan-sensitive FabI enoyl-ACP reductase, and that inhibition of glycolysis in dental plaque biofilms, in which triclosan is retained after initial or repeated exposure, would reduce cariogenicity. [ABSTRACT FROM AUTHOR]

Caries-causing oral bacteria such as Streptococcus mutans are protected by the actions of F-ATPases against acid damage in dental plaque acidified by glycolytic acid production or ingestion of acids foods and beverages. Catabolites such as glucose and sucrose were found to enhance the protection of S. mutans and also other oral lactic-acid bacteria against acid killing at lethal pH values as low as 2.5. Protection involved glycolysis with the production of lactate and ATP, which is a substrate for F-ATPases. ATP could also be produced by starved cells apparently through synthase activity of the F-ATPase associated with acid decline. Fluoride and the organic weak-acid indomethacin acted to diminish this protection, as did F-ATPase inhibitors such as dicyclohexylcarbodi-imide. Protection against acid killing involving catabolism and synthase activity is likely to be important for plaque cariogenicity. [ABSTRACT FROM AUTHOR]

We discuss the most recent reports on two proton pumps, F-ATPase (ATP synthase) and V-ATPase (endomembrane proton pump). They are formed from similar extrinsic (F 1 or V 1 ) and intrinsic (Fo or Vo) membrane sectors, and couple chemistry and proton transport through subunit rotation for apparently different physiological roles. Emphasis is placed on the stochastic rotational catalysis of F-ATPase and isoforms of V-ATPase. iubmb Life , 58: 318–322, 2006 [ABSTRACT FROM AUTHOR]

Vma5p (subunit C) of the yeast V-ATPase was produced in Escherichia coli and purified to homogeneity. Analysis of secondary structure by circular dichroism spectroscopy showed that Vma5p comprises 64% α-helix and 17% β-sheet content. The molecular mass of this subunit, determined by gel filtration analysis and small angle X-ray scattering (SAXS), was approximately 51 ± 4 kDa, indicating a high hydration level of the protein in solution. The radius of gyration and the maximum size of Vma5p were determined to be 3.74 ± 0.03 and 12.5 ± 0.1 nm, respectively. Using two independent ab initio approaches, the first low-resolution shape of the protein was determined. Vma5p is an elongated boot-shaped particle consisting of two distinct domains. Co-reconstitution of Vma5p to V1 without C from Manduca sexta resulted in a V1–Vma5p hybrid complex and a 20% increase in ATPase hydrolysis activity. [Copyright &y& Elsevier]

The vacuolar H[SUP+]-ATPase (V-ATPase) is one of the most fundamental enzymes in nature. It functions in almost every eukaryotic cell and energizes a wide variety of organelles and membranes. V-ATPase has a structure and mechanism of action similar to F-ATPase and several of their subunits probably evolved from common ancestors. In eukaryotic cells, F-ATPase is confined to the semiautonomous organelles, chloroplasts and mitochondria, which contain their own genes that encode some of the FATPase subunits. In contrast to F-ATPases, whose primary function in eukaryotic cells is to form ATP at the expense of the protonmotive force (pmf), V-ATPases function exclusively as ATP-dependent proton pumps. The pmf generated by V-ATPases in organelles and membranes of eukaryotic cells is utilized as a driving force for numerous secondary transport processes. It was the survival of the yeast mutant without the active enzyme and yeast genetics that allowed the identification of genuine subunits of the V-ATPase. It also revealed special properties of individual subunits, factors that are involved in the enzyme's biogenesis and assembly, as well as the involvement of V-ATPase in the secretory pathway, endocytosis, and respiration. It may be the insect V-ATPase that unconventionally resides in the plasma membrane of their midgut, that will give the first structure resolution of this complex. [ABSTRACT FROM AUTHOR]