Bacterial ATP Synthase Research Articles

Abstract 2144 Determinants of N-side proton transport in bacterial ATP synthase

Abstract 2144 Determinants of N-side proton transport in bacterial ATP synthase

Mode of Antibacterial Action of Tomatidine C3-Diastereoisomers.

Tomatidine (TO) is a natural narrow-spectrum antibiotic acting on the Staphylococcus aureus small colony variant (SCV) with a minimal inhibitory concentration (MIC) of 0.06 µg/mL while it shows no activity against prototypical strains (MIC > 128 µg/mL). To expand the spectrum of activity of TO, the 3β-hydroxyl group was substituted with an ethane-1,2-diamine, resulting in two diastereoisomers, TM-02 (C3-β) and TM-03 (C3-α). These molecules are equally potent against prototypical S. aureus and E. coli strains (MIC 8 and 32 µg/mL, respectively), whereas TM-02 is more potent against SCV (MIC 0.5 µg/mL) and hyperpermeable E. coli strains (MIC 1 µg/mL). The differences in their modes of action were investigated. We used membrane vesicles to confirm the inhibition of the bacterial ATP synthase, the documented target of TO, and measured effects on bacterial cell membranes. Both molecules inhibited E. coli ATP synthase, with Ki values of 1.1 µM and 3.5 µM for TM-02 and TM-03, respectively, and the bactericidal effect of TM-02 was linked to ATP synthase inhibition. Furthermore, TM-02 had no major effect on the membrane fluidity and gradually reduced membrane potential. In contrast, TM-03 caused structural damages to membranes and completely disrupted the membrane potential (>90%). We were successful in broadening the spectrum of activity of TO. C3-β-diastereoisomers may have more specific antibacterial action than C3-α.

From garden to lab: C-3 chemical modifications of tomatidine unveil broad-spectrum ATP synthase inhibitors to combat bacterial resistance

Antibiotic resistance is escalating alarmingly worldwide. Bacterial resistance mechanisms are surfacing and proliferating across the globe, jeopardizing our capacity to manage prevalent infectious illnesses. Without drastic measures, we risk entering a post-antibiotic era, where even trivial infections and injuries can cause death again. In this context, we have developed a new class of antibiotics based on tomatidine (TO), a natural product derived from tomato plants, with a novel mode of action by targeting bacterial ATP synthases. The first generation of compounds proved highly specific for small-colony variants (SCVs) of Staphylococcus aureus. However, optimization of this scaffold through extensive structure-activity relationship studies has enabled us to broaden its effectiveness to include both Gram-positive and Gram-negative bacteria. Notably, the results showed that specific C3-modification of TO could improve ATP synthase inhibition and also bypass the outer membrane barrier of Gram-negative bacteria to gain substantial growth inhibition including against multi-resistant strains.

Shikonin and Alkannin inhibit ATP synthase and impede the cell growth in Escherichia coli

Naturally occurring naphthoquinones, shikonin and alkannin, are important ingredients of traditional Chinese medicine Zicao. These constituents are reported to have many therapeutic uses, such as wound healing; scar treatment; and anti-inflammation, anti-acne, anti-ulcer, anti-HIV, anticancer, and antibacterial properties. The primary objective of this investigation was to explore the effect of shikonin and alkannin on Escherichia coli ATP synthase and its cell growth. Shikonin caused complete (100 %) inhibition, and alkannin caused partial (79 %) inhibition of wild-type E. coli ATP synthase. Both caused partial (4 %–27 %) inhibition of ATP synthase with genetically modified phytochemical binding site. The growth inhibition of strains expressing normal, deficient, and mutant ATP synthase by shikonin and alkannin, corroborated the inhibition observed in isolated normal wild-type and mutant ATP synthase. Trivial inhibition of mutant enzymes indicated αR283D, αE284R, βV265Q, and γT273A are essential for formation of the phytochemical binding site where shikonin and alkannin bind. Further, shikonin was a potent inhibitor of ATP synthase than alkannin. The antimicrobial properties of shikonin and alkannin were tied to the binding at phytochemical site of microbial ATP synthase. Selective targeting of bacterial ATP synthase by shikonin and alkannin may be an advantageous alternative to address the antibiotic resistance issue.

Mechanism of ATP hydrolysis dependent rotation of bacterial ATP synthase

F1 domain of ATP synthase is a rotary ATPase complex in which rotation of central γ-subunit proceeds in 120° steps against a surrounding α3β3 fueled by ATP hydrolysis. How the ATP hydrolysis reactions occurring in three catalytic αβ dimers are coupled to mechanical rotation is a key outstanding question. Here we describe catalytic intermediates of the F1 domain in FoF1 synthase from Bacillus PS3 sp. during ATP mediated rotation captured using cryo-EM. The structures reveal that three catalytic events and the first 80° rotation occur simultaneously in F1 domain when nucleotides are bound at all the three catalytic αβ dimers. The remaining 40° rotation of the complete 120° step is driven by completion of ATP hydrolysis at αDβD, and proceeds through three sub-steps (83°, 91°, 101°, and 120°) with three associated conformational intermediates. All sub-steps except for one between 91° and 101° associated with phosphate release, occur independently of the chemical cycle, suggesting that the 40° rotation is largely driven by release of intramolecular strain accumulated by the 80° rotation. Together with our previous results, these findings provide the molecular basis of ATP driven rotation of ATP synthases.

Inhibitors of ATP Synthase as New Antibacterial Candidates.

ATP, the power of all cellular functions, is constantly used and produced by cells. The enzyme called ATP synthase is the energy factory in all cells, which produces ATP by adding inorganic phosphate (Pi) to ADP. It is found in the inner, thylakoid and plasma membranes of mitochondria, chloroplasts and bacteria, respectively. Bacterial ATP synthases have been the subject of multiple studies for decades, since they can be genetically manipulated. With the emergence of antibiotic resistance, many combinations of antibiotics with other compounds that enhance the effect of these antibiotics have been proposed as approaches to limit the spread of antibiotic-resistant bacteria. ATP synthase inhibitors, such as resveratrol, venturicidin A, bedaquiline, tomatidine, piceatannol, oligomycin A and N,N-dicyclohexylcarbodiimide were the starting point of these combinations. However, each of these inhibitors target ATP synthase differently, and their co-administration with antibiotics increases the susceptibility of pathogenic bacteria. After a brief description of the structure and function of ATP synthase, we aim in this review to highlight therapeutic applications of the major bacterial ATP synthase inhibitors, including animal's venoms, and to emphasize their importance in decreasing the activity of this enzyme and subsequently eradicating resistant bacteria as ATP synthase is their source of energy.

Synthesis and Evaluation of Pseudomonas aeruginosa ATP Synthase Inhibitors.

New antibiotics with unique biological targets are desperately needed to combat the growing number of resistant bacterial pathogens. ATP synthase, a critical protein found in all life, has recently become a target of interest for antibiotic development due to the success of the anti-tuberculosis drug bedaquiline, and while many groups have worked on developing drugs to target bacterial ATP synthase, few have been successful at inhibiting Pseudomonas aeruginosa (PA) ATP synthase specifically. PA is one of the leading causes of resistant nosocomial infections across the world and is extremely challenging to treat due to its various antibiotic resistance mechanisms for most commonly used antibiotics. Herein, we detail the synthesis and evaluation of a series of C1/C2 quinoline analogues for their ability to inhibit PA ATP synthase and act as antibiotics against wild-type PA. From this survey, we found six compounds capable of inhibiting PA ATP synthase in vitro showing that bulky/hydrophobic C1/C2 substitutions are preferred. The strongest inhibitor showed an IC50 of 10 μg/mL and decreased activity of PA ATP synthase to 24% relative to the control. While none of the compounds were able to inhibit wild-type PA in cell culture, two showed improved inhibition of PA growth when permeability of the outer membrane was increased or efflux was knocked out, thus demonstrating that these compounds could be further developed into efficacious antibiotics.

Inhibition of Escherichia coliATP synthase by dietary pomegranate phenolics

BackgroundA wide range of natural and synthetic compounds bind and inhibit ATP synthase. Dietary polyphenols found in fruits, vegetables, and spices such as tea, olives, grapes, cantaloupes, garlic, and pomegranate possess antimicrobial activities. Dietary pomegranate polyphenols (DPPs) have been shown to exhibit anticancer, antioxidant, and antimicrobial properties. In the current study we are exploring the connection between antimicrobial properties of DPPs and the inhibition of microbial ATP synthase. ATP synthase is the main source of ATP in almost all organisms from bacteria to man. Selective inhibition of bacterial ATP synthase could help in combating microbial infections.MethodsWild type E. coli pBWU13.4 is grown on minimal media to late log phase. Cells are harvested, French Pressed and centrifuged to isolate and purify membrane bound F1FO ATP synthase. Our null control is E. coli pUC118 with deleted ATPase gene. DPPs and its analogs induced inhibition of wild‐type and mutant membrane‐bound ATP synthase is performed at room temperature. Wild‐type, mutant, and null cell growth assays on limiting glucose are evaluated at OD595 using AccuSkan Plate reader over a period of 24 hours.ResultsVariable oxidative phosphorylation and ATPase activity pattern was observed between wild type phytochemical binding site mutant strains. Western blot showed purity and integrity of wild type and mutant enzymes. DPPs and its analogs caused variable inhibition of ATP synthase. The wild‐type enzyme was fully inhibited while mutant enzyme showed variable resistance in presence of DPPs. Variation in the growth pattern was in agreement with the inhibitory profiles of isolated enzymes.ConclusionsThe inhibitory profiles of isolated E. coli membrane‐bound ATP synthase shows that DPPs are potent inhibitors of ATP synthase. Inhibition of E. coli cell growth was also related to DPPs caused ATP synthase inhibition suggesting that ATP synthase is a valuable molecular drug target to combat microbial infection.

Synthesis and structure-activity relationships for a new class of tetrahydronaphthalene amide inhibitors of Mycobacterium tuberculosis

Drug resistant tuberculsosis (TB) is global health crisis that demands novel treatment strategies. Bacterial ATP synthase inhibitors such as bedaquiline and next-generation analogues (such as TBAJ-876) have shown promising efficacy in patient populations and preclinical studies, respectively, suggesting that selective targeting of this enzyme presents a validated therapeutic strategy for the treatment of TB. In this work, we report tetrahydronaphthalene amides (THNAs) as a new class of ATP synthase inhibitors that are effective in preventing the growth of Mycobacterium tuberculosis (M.tb) in culture. Design, synthesis and comprehensive structure-activity relationship studies for approximately 80 THNA analogues are described, with a small selection of compounds exhibiting potent (in some cases MIC90 <1 μg/mL) in vitro M.tb growth inhibition taken forward to pharmacokinetic and off-target profiling studies. Ultimately, we show that some of these THNAs possess reduced lipophilic properties, decreased hERG liability, faster mouse/human liver microsomal clearance rates and shorter plasma half-lives compared with bedaquiline, potentially addressing of the main concerns of persistence and phospholipidosis associated with bedaquiline.

Modeling the interaction of thiozonide and atphase M.tuberculosis by the method of molecular docking

The mechanism of interaction of a new anti-tuberculosis drug thiosonide with a probable target of M. tuberculosis was studied. According to the data obtained, the most likely target with a thiozonide binding pocket is the subunit of bacterial ATP synthase, an enzyme that plays a key role in the processes of energy exchange in the cell, coupling the reaction of ATP synthesis/hydrolysis with transmembrane proton transfer. Original models of the three subunits of this enzyme (alpha, delta, and epsilon) were built. To simulate docking in silico, the AutoDock program version 4.2 was used, which is included in the MGL Tools version 1.5.6. It was shown that all three subunits have clusters with the minimum binding energies for the thiosinide molecule, and the epsilon subunit has two clusters with equal probabilities of being a thiosinide binding site.

Exploring proteins in living cells

<p indent="0mm">Proteins have been studied for over <sc>200 years,</sc> but much of that work has been performed <italic>in vitro</italic>. This article briefly summarized the current status of protein exploration in living cells, and then presented some of my own representative findings in this field. First, I summarized the major methodologies hitherto developed for studying proteins <italic>in vivo</italic>, including visualizing a target protein by fusing it with the green fluorescent protein; investigating the tertiary structure of the target protein via in-cell nuclear magnetic resonance spectroscopy; and exploring the behaviors of a target protein by genetically labeling it with different types of unnatural amino acids. Next, I described how my laboratory applied and modified an <italic>in vivo</italic> methodology in which the genetic introduction of a photoactivatable, unnatural amino acid at a selected residue position in a target protein then mediates crosslinking of that protein with other proteins when the cells are irradiated by UV light. By improving that method, my laboratory contributed the following: (1) The creation of a bacterial strain with a genome that has a gene that encodes the orthogonal aminoacyl-tRNA synthetase and one that encodes the tRNA that are both needed to incorporate an unnatural amino acid (i.e., Bpa) into any target protein. (2) The development of a system in which the unnatural amino acid is randomly introduced at each of the target protein’s amino acid residue positions and, after detecting a meaningful interaction, DNA sequencing subsequently identifies that unnatural amino acid’s exact position. (3) The incorporation of the unnatural amino acid into a target protein by directly modifying the endogenous target gene in the genomic DNA so that the variant protein is produced spatiotemporally identical to that of the wild-type protein. (4) The development of a high-throughput polyacrylamide gel electrophoresis system in which 384 protein samples can be analyzed in a single gel. Finally, I described a few representative results from my lab by using <italic>in vivo</italic> protein photocrosslinking analysis: (1) The serendipitous discovery of a reversible subcellular structure (regrowth-delay body) that forms when a highly selected group of important proteins assembles only in non-dividing bacterial cells, and then dissolves when the cells resume growth. Their presence thus marks dormant persister bacterial cells that are known to tolerate antibiotics. (2) A revelation of a “gear shifting” mechanism that modulates bacterial ATP synthase activity via adaptation of its ε-subunit to the “inserted” or “non-inserted” conformation as the cell responds to a variety of favorable to unfavorable cellular conditions. (3) The identification of a supramolecular protein complex that is composed of proteins present in the cytoplasm, inner membrane, periplasm and outer membrane of Gram-negative bacteria, and that mediates the biogenesis of a group of outer membrane proteins. (4) The unveiling of a unique way that the acid stress chaperone HdeA acts. It exhibits biological activity while its tertiary structure becomes largely disordered, and then it protects other chaperones under acidic conditions. (5) A clarification that the long-designated bifunctional protein DegP acts primarily as a proteinase with little chaperone activity. (6) The discovery that small heat shock proteins are activated in a temperature-responsive, cascade manner that allows them to significantly increase their biological activity at high temperatures. I then conclude with a list of unresolved issues regarding protein exploration in living cells.

Heat treatment of thioredoxin fusions increases the purity of α-helical transmembrane protein constructs.

Membrane proteins play key roles in cellular signaling and transport, represent the majority of drug targets, and are implicated in many diseases. Their relevance renders them important subjects for structural, biophysical, and functional investigations. However, obtaining membrane proteins in high purities is often challenging with conventional purification steps alone. To address this issue, we present here an approach to increase the purity of α‐helical transmembrane proteins. Our approach exploits the Thioredoxin (Trx) tag system, which is able to confer some of its favorable properties, such as high solubility and thermostability, to its fusion partners. Using Trx fusions of transmembrane helical hairpin constructs derived from the human cystic fibrosis transmembrane conductance regulator (CFTR) and a bacterial ATP synthase, we establish conditions for the successful implementation of the selective heat treatment procedure to increase sample purity. We further examine systematically its efficacy with respect to different incubation times and temperatures using quantitative gel electrophoresis. We find that minute‐timescale heat treatment of Trx‐tagged fusion constructs with temperatures ranging from 50 to 90°C increases the purity of the membrane protein samples from ~60 to 98% even after affinity purification. We show that this single‐step approach is even applicable in cases where regular selective heat purification from crude extracts, as reported for Trx fusions to soluble proteins, fails. Overall, our approach is easy to integrate into existing purification strategies and provides a facile route for increasing the purity of membrane protein constructs after purification by standard chromatography approaches.

Inhibition of Escherichia coli ATP synthase by dietary ginger phenolics

For centuries, dietary ginger has been known for its antioxidant, anticancer, and antibacterial properties. In the current study, we examined the link between antibacterial properties of 7 dietary ginger phenolics (DGPs)—gingerenone A, 6-gingerol, 8-gingerol, 10-gingerol, paradol, 6-shogaol, and zingerone—and inhibition of bacterial ATP synthase. DGPs caused complete (100%) inhibition of wild-type Escherichia coli membrane-bound F1Fo ATP synthase, but partial and variable (0%–87%) inhibition of phytochemical binding site mutant enzymes αR283D, αE284R, βV265Q, and γT273A. The mutant enzyme ATPase activity was 16-fold to 100-fold lower than that of the wild-type enzyme. The growth of wild-type, null, and mutant strains in the presence of the 7 DGPs were abrogated to variable degrees on limiting glucose and succinate media. DGPs-caused variable inhibitory profiles of wild-type and mutant ATP synthase confirm that residues of α-, β-, and γ-subunits are involved in the formation of phytochemical binding site. The variable degree of growth in the presence of DGPs also indicates the possibility of molecular targets other than ATP synthase. Our results establish that antibacterial properties of DGPs can be linked to the binding and inhibition of bacterial ATP synthase. Therefore, bacterial ATP synthase is a valuable molecular target for DGPs.

Dietary Ginger Phenolics Potent Inhibition of E. coli ATP Synthase

The broad long-term goal of our lab is to find and characterize potent selective inhibitors of ATP synthase. ATP synthase is the main source of cellular energy in all organisms. Selective inhibition of bacterial ATP synthase may be utilized to fight the drug-resistant microbial infections. Many phytochemicals, including phenolics, are known to bind at the phytochemical binding site of ATP synthase. In the current study, we are interested in the effect of dietary ginger phenolics (DGPs) on bacterial ATP synthase. DGPs exhibit antimicrobial and anticancer properties similar to many other categories of phytochemicals. Previously our lab has documented that a variety of dietary phytochemicals have selective inhibitory effects on ATP synthase. This leads to the experimental question of this project: What is the effect of DGP on E. coli cell growth and ATP synthase activity? This question will be further addressed in the following specific aims. Specific Aim 1: To determine the effects of DGP on E. coli membrane bound F1F0 ATP synthase activity. Wild type and mutant E. coli enzymes will be isolated and examined for activity of the enzyme. Assays of E. coli strains will be conducted in the presence of DGPs. DGP-induced inhibitory profiles will yield a dose response curve measured at OD595. ATP synthase activity should decrease as the concentration is increased for each mutant strain, as well as in the presence of the ginger phenolics. Specific Aim 2: To determine the effects of DGP on growth in wild type and mutant E. coli strains. Wild type, null, and mutant E. coli strains will be grown in presence and absence of DGP. Growth will be measured on fermentable limiting glucose and non-fermentable succinate media. Abrogation of growth in presence of DGP will be compared to isolated E. coli ATP synthase growth on different media. The novel aspect of this research is the selective inhibition of ATP synthase in bacteria. The selective inhibition could ultimately help combat the issue of increasing antibiotic resistant microbial infections.

Inhibitors of F1F0-ATP synthase enzymes for the treatment of tuberculosis and cancer.

The spectacular success of the mycobacterial F1F0-ATP synthase inhibitor bedaquiline for the treatment of drug-resistant tuberculosis has generated wide interest in the development of other inhibitors of this enzyme. Work in this realm has included close analogues of bedaquiline with better safety profiles and 'bedaquiline-like'compounds, some of which show potent antibacterial activity in vitro although none have yet progressed to clinical trials. The search has lately extended to a range of new scaffolds as potential inhibitors, including squaramides, diaminoquinazolines, chloroquinolines, dihydropyrazolo[1,5-a]pyrazin-4-ones, thiazolidinediones, diaminopyrimidines and tetrahydroquinolines. Because of the ubiquitous expression of ATP synthase enzymes, there has also been interest in inhibitors of other bacterial ATP synthases, as well as inhibitors of human mitochondrial ATP synthase for cancer therapy. The latter encompass bothcomplex natural products and simpler small molecules. The review seeks to demonstrate the breadth of the structural types of molecules able to effectively inhibit the function of variants of this intriguing enzyme.

Regulation of bacterial ATP synthase activity: A gear-shifting or a pawl-ratchet mechanism?

The F1 Fo -ATP synthase, a widely distributed nanomotor responsible of ATP synthesis, rotates its central rotor reversibly: In the clockwise direction when viewed from the Fo (with the observer facing the positive side of the energy transducing membrane and looking down into the negative side of the membrane), it functions as ATP synthase, while in counterclockwise sense, it operates as a proton-pumping ATP hydrolase. Regulation exerted by naturally occurring inhibitory proteins of the enzyme appears to function by avoiding ATP hydrolysis while preserving ATP synthesis. The work of Liu et al. describes an unbiased, elegant analytical pipeline that provides important insights into the inhibitory role of the ε-subunit of the bacterial F1 Fo -ATP synthase invivo. We discuss if a gear-shifting versus a pawl-ratchet mechanism may explain the regulatory role of the ε-subunit.

A high-throughput genetically directed protein crosslinking analysis reveals the physiological relevance of the ATP synthase 'inserted' state.

ATP synthase, a highly conserved protein complex that has a subunit composition of α3 β3 γδεab2 c8-15 for the bacterial enzyme, is a key player in supplying energy to living organisms. This protein complex consists of a peripheral F1 sector (α3 β3 γδε) and a membrane-integrated Fo sector (ab2 c8-15 ). Structural analyses of the isolated protein components revealed that, remarkably, the C-terminal domain of its ε-subunit seems to adopt two dramatically different structures, but the physiological relevance of this conformational change remains largely unknown. In an attempt to decipher this, we developed a high-throughput invivo protein photo-cross-linking analysis pipeline based on the introduction of the unnatural amino acid into the target protein via the scarless genome-targeted site-directed mutagenesis technique, and probing the cross-linked products via the high-throughput polyacrylamide gel electrophoresis technique. Employing this pipeline, we examined the interactions involving the C-terminal helix of the ε-subunit in cells living under a variety of experimental conditions. These studies enabled us to uncover that the bacterial ATP synthase exists as an equilibrium between the 'inserted' and 'noninserted' state in cells, maintaining a moderate but significant level of net ATP synthesis when shifting to the former upon exposing to unfavorable energetically stressful conditions. Such a mechanism allows the bacterial ATP synthases to proportionally and instantly switch between two reversible functional states in responding to changing environmental conditions. Importantly, this high-throughput approach could allow us to decipher the physiological relevance of protein-protein interactions identified under invitro conditions or to unveil novel physiological context-dependent protein-protein interactions that are unknown before.

A second shell residue modulates a conserved ATP-binding site with radically different affinities for ATP

BackgroundPrediction of ligand binding and design of new function in enzymes is a time-consuming and expensive process. Crystallography gives the impression that proteins adopt a fixed shape, yet enzymes are functionally dynamic. Molecular dynamics offers the possibility of probing protein movement while predicting ligand binding. Accordingly, we choose the bacterial F1Fo ATP synthase ε subunit to unravel why ATP affinity by ε subunits from Bacillus subtilis and Bacillus PS3 differs ~500-fold, despite sharing identical sequences at the ATP-binding site. MethodsWe first used the Bacillus PS3 ε subunit structure to model the B. subtilis ε subunit structure and used this to explore the utility of molecular dynamics (MD) simulations to predict the influence of residues outside the ATP binding site. To verify the MD predictions, point mutants were made and ATP binding studies were employed. ResultsMD simulations predicted that E102 in the B. subtilis ε subunit, outside of the ATP binding site, influences ATP binding affinity. Engineering E102 to alanine or arginine revealed a ~10 or ~54 fold increase in ATP binding, respectively, confirming the MD prediction that E102 drastically influences ATP binding affinity. ConclusionsThese findings reveal how MD can predict how changes in the “second shell” residues around substrate binding sites influence affinity in simple protein structures. Our results reveal why seemingly identical ε subunits in different ATP synthases have radically different ATP binding affinities. General significanceThis study may lead to greater utility of molecular dynamics as a tool for protein design and exploration of protein design and function.

The Molecular Basis for Purine Binding Selectivity in the Bacterial ATP Synthase ϵ Subunit.

The ϵ subunit of ATP synthases has been proposed to regulate ATP hydrolysis in bacteria. Prevailing evidence supports the notion that when the ATP concentration falls below a certain threshold, the ϵ subunit changes its conformation from a non-inhibitory down-state to an extended up-state that then inhibits enzymatic ATP hydrolysis by binding to the catalytic domain. It has been demonstrated that the ϵ subunit from Bacillus PS3 is selective for ATP over other nucleotides, including GTP. In this study, the purine triphosphate selectivity is rationalized by using results from MD simulations and free energy calculations for the R103A/R115A mutant of the ϵ subunit from Bacillus PS3, which binds ATP more strongly than the wild-type protein. Our results are in good agreement with experimental data, and the elucidated molecular basis for selectivity could help to guide the design of novel GTP sensors.

The ASCT/SCS cycle fuels mitochondrial ATP and acetate production in Trypanosoma brucei

Acetate:succinate CoA transferase (ASCT) is a mitochondrial enzyme that catalyzes the production of acetate and succinyl-CoA, which is coupled to ATP production with succinyl-CoA synthetase (SCS) in a process called the ASCT/SCS cycle. This cycle has been studied in Trypanosoma brucei (T. brucei), a pathogen of African sleeping sickness, and is involved in (i) ATP and (ii) acetate production and proceeds independent of oxygen and an electrochemical gradient. Interestingly, knockout of ASCT in procyclic form (PCF) of T. brucei cause oligomycin A-hypersensitivity phenotype indicating that ASCT/SCS cycle complements the deficiency of ATP synthase activity. In bloodstream form (BSF) of T. brucei, ATP synthase works in reverse to maintain the electrochemical gradient by hydrolyzing ATP. However, no information has been available on the source of ATP, although ASCT/SCS cycle could be a potential candidate. Regarding mitochondrial acetate production, which is essential for fatty acid biosynthesis and growth of T. brucei, ASCT or acetyl-CoA hydrolase (ACH) are known to be its source. Despite the importance of this cycle, direct evidence of its function is lacking, and there are no comprehensive biochemical or structural biology studies reported so far. Here, we show that in vitro–reconstituted ASCT/SCS cycle is highly specific towards acetyl-CoA and has a higher kcat than that of yeast and bacterial ATP synthases. Our results provide the first biochemical basis for (i) rescue of ATP synthase-deficient phenotype by ASCT/SCS cycle in PCF and (ii) a potential source of ATP for the reverse reaction of ATP synthase in BSF.