Substrate ATP Research Articles

Stochastic nature and physiological implications of 5'-NAD RNA cap in bacteria.

RNA 5'-modification with NAD+/NADH (oxidized/reduced nicotinamide adenine dinucleotide) has been found in bacteria, eukaryotes and viruses. 5'-NAD is incorporated into RNA by RNA polymerases (RNAPs) during the initiation of synthesis. It is unknown (i) which factors and physiological conditions permit substantial NAD incorporation into RNA in vivo and (ii) how 5'-NAD impacts gene expression and the fate of RNA in bacteria. Here we show in Escherichia coli that RNA NADylation is stimulated by low cellular concentration of the competing substrate ATP, and by weakening ATP contacts with RNAP active site. Additionally, RNA NADylation may be influenced by DNA supercoiling. RNA NADylation does not interfere with posttranscriptional RNA processing by major ribonuclease RNase E. It does not impact the base-pairing between RNAI, the repressor of plasmid replication, and its antisense target, RNAII. Leaderless NADylated model mRNA cI-lacZ is recognized by the 70S ribosome and is translated with the same efficiency as triphosphorylated cI-lacZ mRNA. Translation exposes the 5'-NAD of this mRNA to de-capping by NudC enzyme. We suggest that NADylated mRNAs are rapidly degraded, consistent with their low abundance in published datasets. Furthermore, we observed that ppGpp inhibits NudC de-capping activity, contributing to the growth phase-dependency of NADylated RNA levels.

Oxidative Stress Markers and Na,K-ATPase Enzyme Kinetics Are Altered in the Cerebellum of Zucker Diabetic Fatty fa/fa Rats: A Comparison with Lean fa/+ and Wistar Rats.

Type 2 diabetes mellitus has been referred to as being closely related to oxidative stress, which may affect brain functions and brain glucose metabolism due to its high metabolic activity and lipid-rich content. Na,K-ATPase is an essential enzyme maintaining intracellular homeostasis, with properties that can sensitively mirror various pathophysiological conditions such as diabetes. The goal of this study was to determine oxidative stress markers as well as Na,K-ATPase activities in the cerebellum of Zucker diabetic fatty (ZDF) rats depending on diabetes severity. The following groups of male rats were used: Wistar, ZDF Lean (fa/+), and ZDF (fa/fa) rats, arbitrarily divided according to glycemia into ZDF obese (ZO, less severe diabetes) and ZDF diabetic (ZOD, advanced diabetes) groups. In addition to basic biometry and biochemistry, oxidative stress markers were assessed in plasma and cerebellar tissues. The Na, K-ATPase enzyme activity was measured at varying ATP substrate concentrations. The results indicate significant differences in basic biometric and biochemical parameters within all the studied groups. Furthermore, oxidative damage was greater in the cerebellum of both ZDF (fa/fa) groups compared with the controls. Interestingly, Na,K-ATPase enzyme activity was highest to lowest in the following order: ZOD > ZO > Wistar > ZDF lean rats. In conclusion, an increase in systemic oxidative stress resulting from diabetic conditions has a significant impact on the cerebellar tissue independently of diabetes severity. The increased cerebellar Na,K-ATPase activity may reflect compensatory mechanisms in aged ZDF (fa/fa) animals, rather than indicating cerebellar neurodegeneration: a phenomenon that warrants further investigation.

Enzyme activation by urea reveals the interplay between conformational dynamics and substrate binding: a single-molecule FRET study.

Proteins often harness extensive motions of domains and subunits to promote their function. Deciphering how these movements impact activity is key for understanding life's molecular machinery. The enzyme adenylate kinase is an intriguing example for this relationship; it ensures efficient catalysis by large-scale domain motions that lead to the enclosure of the bound substrates ATP and AMP. At high concentrations, AMP also operates as an allosteric inhibitor of the protein. Surprisingly, the enzyme is activated by urea, a compound commonly acting as a denaturant. Combining single-molecule FRET spectroscopy and enzymatic activity studies, we find that urea interferes with two key mechanisms that contribute to enzyme efficacy. First, urea promotes the open conformation of the enzyme, aiding the proper positioning of the substrates. Second, urea decreases AMP affinity, paradoxically facilitating a more efficient progression towards the catalytically active complex. These results signify the important interplay between conformational dynamics and chemical steps, including binding, in the activity of enzymes. State-of-the-art tools, such as single-molecule fluorescence spectroscopy, offer new insights into how enzymes balance different conformations to regulate activity.

Structural basis for substrate binding and selection by human mitochondrial RNA polymerase

The mechanism by which RNAP selects cognate substrates and discriminates between deoxy and ribonucleotides is of fundamental importance to the fidelity of transcription. Here, we present cryo-EM structures of human mitochondrial transcription elongation complexes that reveal substrate ATP bound in Entry and Insertion Sites. In the Entry Site, the substrate binds along the O helix of the fingers domain of mtRNAP but does not interact with the templating DNA base. Interactions between RNAP and the triphosphate moiety of the NTP in the Entry Site ensure discrimination against nucleosides and their diphosphate and monophosphate derivatives but not against non-cognate rNTPs and dNTPs. Closing of the fingers domain over the catalytic site results in delivery of both the templating DNA base and the substrate into the Insertion Site and recruitment of the catalytic magnesium ions. The cryo-EM data also reveal a conformation adopted by mtRNAP to reject a non-cognate substrate from its active site. Our findings establish a structural basis for substrate binding and suggest a unified mechanism of NTP selection for single-subunit RNAPs.

Structural Characterization of Heat Shock Protein 90β and Molecular Interactions with Geldanamycin and Ritonavir: A Computational Study.

Drug repositioning is an important therapeutic strategy for treating breast cancer. Hsp90β chaperone is an attractive target for inhibiting cell progression. Its structure has a disordered and flexible linker region between the N-terminal and central domains. Geldanamycin was the first Hsp90β inhibitor to interact specifically at the N-terminal site. Owing to the toxicity of geldanamycin, we investigated the repositioning of ritonavir as an Hsp90β inhibitor, taking advantage of its proven efficacy against cancer. In this study, we used molecular modeling techniques to analyze the contribution of the Hsp90β linker region to the flexibility and interaction between the ligands geldanamycin, ritonavir, and Hsp90β. Our findings indicate that the linker region is responsible for the fluctuation and overall protein motion without disturbing the interaction between the inhibitors and the N-terminus. We also found that ritonavir established similar interactions with the substrate ATP triphosphate, filling the same pharmacophore zone.

BsuMI regulates DNA transformation in Bacillus subtilis besides the defense system and the constructed strain with BsuMI-absence is applicable as a universal transformation platform for wild-type Bacillus

BackgroundTo effectively introduce plasmids into Bacillus species and conduct genetic manipulations in Bacillus chassis strains, it is essential to optimize transformation methods. These methods aim to extend the period of competence and enhance the permeability of the cell membrane to facilitate the entry of exogenous DNA. Although various strategies have been explored, few studies have delved into identifying metabolites and pathways associated with enhanced competence. Additionally, derivative Bacillus strains with non-functional restriction-modification systems have demonstrated superior efficiency in transforming exogenous DNA, lacking more explorations in the regulation conducted by the restriction-modification system to transformation process.ResultsTranscriptomic comparisons were performed to discover the competence forming mechanism and the regulation pathway conducted by the BsuMI methylation modification group in Bacillus. subtilis 168 under the Spizizen transformation condition, which were speculated to be the preferential selection of carbon sources by the cells and the preference for specific metabolic pathway when utilizing the carbon source. The cells were found to utilize the glycolysis pathway to exploit environmental glucose while reducing the demand for other phosphorylated precursors in this pathway. The weakening of these ATP-substrate competitive metabolic pathways allowed more ATP substrates to be distributed into the auto-phosphorylation of the signal transduction factor ComP during competence formation, thereby increasing the expression level of the key regulatory protein ComK. The expression of ComK upregulated the expression of the negative regulator SacX of starch and sucrose in host cells, reinforcing the preference for glucose as the primary carbon source. The methylation modification group of the primary protein BsuMI in the restriction-modification system was associated with the functional modification of key enzymes in the oxidative phosphorylation pathway. The absence of the BsuMI methylation modification group resulted in a decrease in the expression of subunits of cytochrome oxidase, leading to a weakening of the oxidative phosphorylation pathway, which promoted the glycolytic rate of cells and subsequently improved the distribution of ATP molecules into competence formation. A genetic transformation platform for wild-type Bacillus strains was successfully established based on the constructed strain B. subtilis 168-R−M− without its native restriction-modification system. With this platform, high plasmids transformation efficiencies were achieved with a remarkable 63-fold improvement compared to the control group and an increased universality in Bacillus species was also obtained.ConclusionsThe enhanced competence formation mechanism and the regulation pathway conducted by the functional protein BsuMI of the restriction-modification system were concluded, providing a reference for further investigation. An effective transformation platform was established to overcome the obstacles in DNA transformations in wild-type Bacillus strains.Graphical

The Role of SF1 and SF2 Helicases in Biotechnological Applications.

Helicases, which utilize ATP hydrolysis to separate nucleic acid duplexes, play crucial roles in DNA and RNA replication, repair, recombination, and transcription. Categorized into the major groups superfamily 1 (SF1) and superfamily 2 (SF2), alongside four minor groups, these proteins exhibit a conserved catalytic core indicative of a shared evolutionary origin while displaying functional diversity through interactions with various substrates. This review summarizes the structures, functions and mechanisms of SF1 and SF2 helicases, with an emphasis on conserved ATPase sites and RecA-like domains essential for their enzymatic and nucleic acid binding capabilities. It highlights the unique 1B and 2B domains in SF1 helicases and their impact on enzymatic activity. The DNA unwinding process is detailed, covering substrate recognition, ATP hydrolysis, and conformational changes, while addressing debates over the active form of UvrD helicase and post-unwinding dissociation. More importantly, this review discusses the biotechnological potential of helicases in emerging technologies such as nanopore sequencing, protein sequencing, and isothermal amplification, focusing on their use in pathogen detection, biosensor enhancement, and cancer treatment. As understanding deepens, innovative applications in genome editing, DNA sequencing, and synthetic biology are anticipated.

Effects of substrate availability and mitochondrial disruption on oxidative metabolism and sperm motility in fertile dogs.

In several mammalian species, the measurement of mitochondrial oxygen consumption (MITOX) under different metabolic conditions has demonstrated a positive correlation with sperm motility and may be a sensitive indicator of mitochondrial health. In general, the maintenance of sperm motility and many key sperm functions and fertilizing events are heavily energy-dependent processes, and some species-specific substrate preferences exist. Although canine sperm have been known to undergo capacitation and maintain motility with supplementation of a wide range of energy substrates, the relationship between mitochondrial function, and the maintenance of oxidative metabolism and sperm motility remain unclear. The objective of this study was to explore the metabolic flexibility of canine sperm, and to investigate the relationship between mitochondrial function, and maintenance of motility under differing nutrient conditions. We explored substrate preferences and the bioenergetics underlying maintenance of canine sperm motility by monitoring mitochondrial oxidative function and sperm kinematics in the presence of mitochondrial effector drug treatments: FCCP, antimycin (ANTI), and oligomycin (OLIGO). We hypothesized that canine sperm possess the ability to use compensatory pathways and utilize diverse nutrient sources in the maintenance of motility. Oxygen consumption (change in pO2, oxygen partial pressure) and sperm kinematics (CASA) were measured concurrently (t0-t30) to assess the relationship between oxidative metabolism and maintenance of sperm motility in dogs. Four media were tested: containing glucose, lactate, and pyruvate (GLP), containing glucose (G), fructose (F), or lactate and pyruvate (LP). In the absence of pharmacological inhibition of the electron transport chain, energetic substrate had no effect on sperm kinematics in fertile dogs. Following mitochondrial disruption by ANTI and OLIGO, mitochondrial oxygen consumption was negatively correlated with several sperm motility parameters in GLP, G, F, and LP media. In every media, FCCP treatment quickly induced significantly higher oxygen consumption than in untreated sperm, and spare respiratory capacity, the maximal inducible oxidative metabolism, was high. With respiratory control ratios RCR >1 there was no indication of bioenergetic dysfunction in any media type, indicating that sperm mitochondria of fertile dogs have a high capacity for substrate oxidation and ATP turnover regardless of substrate. Our results suggest MITOX assessment is a valuable tool for assessing mitochondrial functionality, and that canine sperm employ flexible energy management systems which may be exploited to improve sperm handling and storage.

Probing for optimal photoaffinity linkers of benzophenone-based photoaffinity probes for adenylating enzymes

The adenylation (A) domain of non-ribosomal peptide synthetases (NRPSs) catalyzes the adenylation reaction with substrate amino acids and ATP. Leveraging the distinct substrate specificity of A-domains, we previously developed photoaffinity probes for A-domains based on derivatization with a 5′-O-N-(aminoacyl)sulfamoyl adenosine (aminoacyl-AMS)-appended clickable benzophenone. Although our photoaffinity probes with different amino acid warheads enabled selective detection, visualization, and enrichment of target A-domains in proteomic environments, the effects of photoaffinity linkers have not been investigated. To explore the optimal benzophenone-based linker scaffold, we designed seven photoaffinity probes for the A-domains with different lengths, positions, and molecular shapes. Using probes 2–8 for the phenylalanine-activating A-domain of gramicidin S synthetase A (GrsA), we systematically investigated the binding affinity and labeling efficiency of the endogenous enzyme in a live producer cell. Our results indicated that the labeling efficiencies of probes 2–8 tended to depend on their binding affinities rather than on the linker length, flexibility, or position of the photoaffinity group. We also identified that probe 2 with a 4,4′-diaminobenzophenone linker exhibits the highest labeling efficiency for GrsA with fewer non-target labeling properties in live cells.

Abstract 2472: Inhibition of ENPP1 catalyzed ATP and cGAMP hydrolysis and in vivo antitumor efficacy of RBS2418 in the Hepa1-6 and GL261-luc syngeneic liver and glioblastoma mouse models

Abstract Background: ENPP1, a nucleotide pyrophosphatase and phosphodiesterase, has emerged as an important modulator of anti-tumor immune activation, as it regulates the concentrations of cGAMP and ATP in the TME. ENPP1 expression in tumors is common and correlates with a cold tumor phenotype. Its loss has been reported to suppress metastasis, restore immune cell infiltration into the tumor and potentiate the response to immune checkpoint blockade, while, conversely, the overexpression of ENPP1 promoted metastasis and rendered tumors resistant to checkpoint blockade. RBS2418 was designed as a potent and selective inhibitor of ENPP1 and is in clinical development. Methods: ENPP1 enzyme inhibition was measured in vitro using cGAMP and ATP substrates. Effects of pH and serum and selectivity against related enzymes were evaluated. In vivo efficacy was assessed in Hepa1-6, and GL261-luc syngeneic liver and glioblastoma mouse models. Compound exposures were determined in tumor and blood samples. Cures were confirmed by re-challenge of cured animals with the same cancer cells. Results: RBS2418 inhibited hydrolysis of cGAMP and ATP in vitro with similar potency (Ki =0.14 and 0.13 nM). Inhibition was also similar in human serum, consistent with low binding affinity to human serum albumin. In humans, oral dosing at doses of 100 mg could inhibit ENPP1 enzyme activity during the full dosing interval. Dosing of RBS2418 was performed in mice to achieve trough concentrations of RBS2418 in tumors and plasma exceeding ENPP1 EC90 levels for the complete dosing interval. In the Hepa1-6 model treatment with RBS2418 for either 2 or 10 days resulted in significant reduction of tumor growth and prolongation of survival as compared to vehicle control. There was no significant difference between the RBS2418 groups. Complete tumor regression was observed in 44% of treated mice across the two treatment groups. These mice were resistant to rechallenge with Hepa1-6 tumor cells. In the GL261-luc model treatment with RBS2418 showed significant reduction of tumor burden and prolongation of survival as compared to vehicle control. The mice that achieved complete tumor regression were resistant to rechallenge with GL261-luc tumor cells. Conclusions: RBS2418 inhibited ATP and cGAMP hydrolysis by ENPP1 with similar high potency. The pharmacology profile was optimized to achieve low serum protein binding and oral bioavailability in humans. RBS2418 treatment was well tolerated in mice under conditions where tumor and plasma levels exceeding ENPP1 serum EC90 levels were achieved. In the syngeneic mouse models Hepa1-6 and GL261-luc significant tumor growth reductions, complete tumor regression and cures were observed. Resistance to cancer cell rechallenge was consistent with treatment-induced anti-tumor immunity. Citation Format: Ningwu Huang, Junjun Cheng, Ron Hawley, Klaus Klumpp. Inhibition of ENPP1 catalyzed ATP and cGAMP hydrolysis and in vivo antitumor efficacy of RBS2418 in the Hepa1-6 and GL261-luc syngeneic liver and glioblastoma mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2472.

Discovering potential WRN inhibitors from natural product database through computational methods

Microsatellite instability (MSI) is a relatively common feature associated with multiple cancers, and Werner syndrome (WRN) ATP-dependent helicase has been recognized as a novel target for treating MSI cancers, such as colorectal cancer. A small-molecule inhibitor targeting WRN would be a promising strategy for treating colorectal cancer with high MSI expression. In this study, we employed a computer-assisted drug discovery strategy to screen over 30,000 natural product molecules. By using a combination of docking, ligand efficiency, Molecular Mechanics/Generalized Born Surface Area (MM/GBSA), and thermodynamic integration (TI) calculations, we identified MOL008980, MOL010740, MOL011832, T4743, TN1166, and TNP-002173 as potential WRN inhibitors. Subsequent molecular dynamics simulation revealed that these screened natural products possessed better binding dynamic characteristics than ATP substrate and were capable of inhibiting the dynamic process of WRN, making them potential strong ATP competitive inhibitors. In conclusion, our computational approach revealed potential WRN inhibitors from a natural product database, providing a theoretical basis for future research.

Linking ATP and allosteric sites to achieve superadditive binding with bivalent EGFR kinase inhibitors

Bivalent molecules consisting of groups connected through bridging linkers often exhibit strong target binding and unique biological effects. However, developing bivalent inhibitors with the desired activity is challenging due to the dual motif architecture of these molecules and the variability that can be introduced through differing linker structures and geometries. We report a set of alternatively linked bivalent EGFR inhibitors that simultaneously occupy the ATP substrate and allosteric pockets. Crystal structures show that initial and redesigned linkers bridging a trisubstituted imidazole ATP-site inhibitor and dibenzodiazepinone allosteric-site inhibitor proved successful in spanning these sites. The re-engineered linker yielded a compound that exhibited significantly higher potency (~60 pM) against the drug-resistant EGFR L858R/T790M and L858R/T790M/C797S, which was superadditive as compared with the parent molecules. The enhanced potency is attributed to factors stemming from the linker connection to the allosteric-site group and informs strategies to engineer linkers in bivalent agent design.

Ovarian cancer cells regulate their mitochondrial content and high mitochondrial content is associated with a poor prognosis

Most cancer patients ultimately die from the consequences of distant metastases. As metastasis formation consumes energy mitochondria play an important role during this process as they are the most important cellular organelle to synthesise the energy rich substrate ATP, which provides the necessary energy to enable distant metastasis formation. However, mitochondria are also important for the execution of apoptosis, a process which limits metastasis formation. We therefore wanted to investigate the mitochondrial content in ovarian cancer cells and link its presence to the patient’s prognosis in order to analyse which of the two opposing functions of mitochondria dominates during the malignant progression of ovarian cancer. Monoclonal antibodies directed against different mitochondrial specific proteins, namely heat shock proteins 60 (HSP60), fumarase and succinic dehydrogenase, were used in immunohistochemistry in preliminary experiments to identify the antibody most suited to detect mitochondria in ovarian cancer cells in clinical tissue samples. The clearest staining pattern, which even delineated individual mitochondria, was seen with the anti-HSP60 antibody, which was used for the subsequent clinical study staining primary ovarian cancers (n = 155), borderline tumours (n = 24) and recurrent ovarian cancers (n = 26). The staining results were semi-quantitatively scored into three groups according to their mitochondrial content: low (n = 26), intermediate (n = 50) and high (n = 84). Survival analysis showed that high mitochondrial content correlated with a statistically significant overall reduced survival rate In addition to the clinical tissue samples, mitochondrial content was analysed in ovarian cancer cells grown in vitro (cell lines: OVCAR8, SKOV3, OVCAR3 and COV644) and in vivo in severe combined immunodeficiency (SCID) mice.In in vivo grown SKOV3 and OVCAR8 cells, the number of mitochondria positive cells was markedly down-regulated compared to the in vitro grown cells indicating that mitochondrial number is subject to regulatory processes. As high mitochondrial content is associated with a poor prognosis, the provision of high energy substrates by the mitochondria seems to be more important for metastasis formation than the inhibition of apoptotic cell death, which is also mediated by mitochondria. In vivo and in vitro grown human ovarian cancer cells showed that the mitochondrial content is highly adaptable to the growth condition of the cancer cells.

Identification of FtfL as a novel target of berberine in intestinal bacteria

BackgroundBerberine (BBR) is a commonly used anti-intestinal inflammation drug, and its anti-cancer activity has been found recently. BBR can intervene and control malignant colorectal cancer (CRC) through intestinal microbes, but the direct molecular target and related mechanism are unclear. This study aimed to identify the target of BBR and dissect related mechanisms against the occurrence and development of CRC from the perspective of intestinal microorganisms.ResultsHere, we found that BBR inhibits the growth of several CRC-driving bacteria, especially Peptostreptococcus anaerobius. By using a biotin-conjugated BBR derivative, we identified the protein FtfL (formate tetrahydrofolate ligase), a key enzyme in C1 metabolism, is the molecular target of BBR in P. anaerobius. BBR exhibits strong binding affinity and potent inhibition on FtfL. Based on this, we determined the crystal structure of PaFtfL (P. anaerobius FtfL)-BBR complex and found that BBR can not only interfere with the conformational flexibility of PaFtfL tetramer by wedging the tetramer interface but also compete with its substrate ATP for binding within the active center. In addition, the enzymatic activities of FtfL homologous proteins in human tumor cells can also be inhibited by BBR.ConclusionsIn summary, our study has identified FtfL as a direct target of BBR and uncovered molecular mechanisms involved in the anti-CRC of BBR. BBR interferes with intestinal pathogenic bacteria by targeting FtfLs, suggesting a new means for controlling the occurrence and development of CRC.

Selenite ameliorates the ATP hydrolysis of mitochondrial F1FO-ATPase by changing the redox state of thiol groups and impairs the ADP phosphorylation

Selenite as an inorganic form of selenium can affect the redox state of mitochondria by modifying the thiol groups of cysteines. The F1FO-ATPase has been identified as a mitochondrial target of this compound. Indeed, the bifunctional mechanism of ATP turnover of F1FO-ATPase was differently modified by selenite. The activity of ATP hydrolysis was stimulated, whereas the ADP phosphorylation was inhibited. We ascertain that a possible new protein adduct identified as seleno-dithiol (-S-Se-S-) mercaptoethanol-sensitive caused the activation of F-ATPase activity and the oxidation of free –SH groups in mitochondria. Conversely, the inhibition of ATP synthesis by selenite might be irreversible. The kinetic analysis of the activation mechanism was an uncompetitive mixed type with respect to the ATP substrate. Selenite bound more selectively to the F1FO-ATPase loaded with the substrate by preferentially forming a tertiary (enzyme-ATP-selenite) complex. Otherwise, the selenite was a competitive mixed-type activator with respect to the Mg2+ cofactor. Thus, selenite more specifically bound to the free enzyme forming the complex enzyme-selenite. However, even if the selenite impaired the catalysis of F1FO-ATPase, the mitochondrial permeability transition pore phenomenon was unaffected. Therefore, the reversible energy transduction mechanism of F1FO-ATPase can be oppositely regulated by selenite.

Elucidating Events within the Black Box of Enzyme Catalysis in Energy Metabolism: Insights into the Molecular Mechanism of ATP Hydrolysis by F1-ATPase.

Oxygen exchange reactions occurring at β-catalytic sites of the FOF1-ATP synthase/F1-ATPase imprint a unique record of molecular events during the catalytic cycle of ATP synthesis/hydrolysis. This work presents a new theory of oxygen exchange and tests it on oxygen exchange data recorded on ATP hydrolysis by mitochondrial F1-ATPase (MF1). The apparent rate constant of oxygen exchange governing the intermediate Pi-HOH exchange accompanying ATP hydrolysis is determined by kinetic analysis over a ~50,000-fold range of substrate ATP concentration (0.1-5000 μM) and a corresponding ~200-fold range of reaction velocity (3.5-650 [moles of Pi/{moles of F1-ATPase}-1 s-1]). Isotopomer distributions of [18O]Pi species containing 0, 1, 2, and 3 labeled oxygen atoms predicted by the theory have been quantified and shown to be in perfect agreement with the experimental distributions over the entire range of medium ATP concentrations without employing adjustable parameters. A novel molecular mechanism of steady-state multisite ATP hydrolysis by the F1-ATPase has been proposed. Our results show that steady-state ATP hydrolysis by F1-ATPase occurs with all three sites occupied by Mg-nucleotide. The various implications arising from models of energy coupling in ATP synthesis/hydrolysis by the ATP synthase/F1-ATPase have been discussed. Current models of ATP hydrolysis by F1-ATPase, including those postulated from single-molecule data, are shown to be effectively bisite models that contradict the data. The trisite catalysis formulated by Nath's torsional mechanism of energy transduction and ATP synthesis/hydrolysis since its first appearance 25 years ago is shown to be in better accord with the experimental record. The total biochemical information on ATP hydrolysis is integrated into a consistent model by the torsional mechanism of ATP synthesis/hydrolysis and shown to elucidate the elementary chemical and mechanical events within the black box of enzyme catalysis in energy metabolism by F1-ATPase.

Electrically stimulated in vitro heart cell mimic of acute exercise reveals novel immediate cellular responses to exercise: Reduced contractility and metabolism, but maintained calcium cycling and increased myofilament calcium sensitivity.

Cardiac cellular responses to acute exercise remain undescribed. We present a model for mimicking acute aerobic endurance exercise to freshly isolated cardiomyocytes by evoking exercise-like contractions over prolonged periods of time with trains of electrical twitch stimulations. We then investigated immediate contractile, Ca2+ , and metabolic responses to acute exercise in perfused freshly isolated left ventricular rat cardiomyocytes, after a matrix-design optimized protocol and induced a mimic for acute aerobic endurance exercise by trains of prolonged field twitch stimulations. Acute exercise decreased cardiomyocyte fractional shortening 50%-80% (p < .01). This was not explained by changes to intracellular Ca2+ handling (p > .05); rather, we observed a weak insignificant Ca2+ transient increase (p = .11), while myofilament Ca2+ sensitivity increased 20%-70% (p < .05). Acidic pH 6.8 decreased fractional shortening 20%-70% (p < .05) because of 20%-30% decreased Ca2+ transients (p < .05), but no difference occurred between control and acute exercise (p > .05). Addition of 1 or 10 mM La- increased fractional shortening in control (1 mM La- : no difference, p > .05; 10 mM La- : 20%-30%, p < .05) and acute exercise (1 mM La- : 40%-90%, p < .01; 10 mM La- : 50%-100%, p < .01) and rendered acute exercise indifferent from control (p > .05). Intrinsic autofluorescence showed a resting NADstate of 0.59 ± 0.04 and FADstate of 0.17 ± 0.03, while acute exercise decreased NADH/FAD ratio 8% (p < .01), indicating intracellular oxidation. In conclusion, we show a novel approach for studying immediate acute cardiomyocyte responses to aerobic endurance exercise. We find that acute exercise in cardiomyocytes decreases contraction, but Ca2+ handling and myofilament Ca2+ sensitivity compensate for this, while acidosis and reduced energy substrate and mitochondrial ATP generation explain this.

Catalytic process of anhydro-N-acetylmuramic acid kinase from Pseudomonas aeruginosa

The bacterial cell envelope is the structure with which the bacterium engages with, and is protected from, its environment. Within this envelop is a conserved peptidoglycan polymer which confers shape and strength to the cell envelop. The enzymatic processes that build, remodel, and recycle the chemical components of this cross-linked polymer are preeminent targets of antibiotics and exploratory targets for emerging antibiotic structures. We report a comprehensive kinetic and structural analysis for one such enzyme, the Pseudomonas aeruginosa anhydro-N-acetylmuramic acid (anhNAM) kinase (AnmK). AnmK is an enzyme in the peptidoglycan-recycling pathway of this pathogen. It catalyzes the pairing of hydrolytic ring opening of anhNAM with concomitant ATP-dependent phosphoryl transfer. AnmK follows a random-sequential kinetic mechanism with respect to its anhNAM and ATP substrates. Crystallographic analyses of four distinct structures (apo AnmK, AnmK:AMPPNP, AnmK:AMPPNP:anhNAM, and AnmK:ATP:anhNAM) demonstrate that both substrates enter the active site independently in an ungated conformation of the substrate subsites, with protein loops acting as gates for anhNAM binding. Catalysis occurs within a closed conformational state for the enzyme. We observe this state crystallographically using ATP-mimetic molecules. A remarkable X-ray structure for dimeric AnmK sheds light on the precatalytic and postcatalytic ternary complexes. Computational simulations in conjunction with the high-resolution X-ray structures reveal the full catalytic cycle. We further report that a P.aeruginosa strain with disrupted anmK gene is more susceptible to the β-lactam imipenem compared to the WT strain. These observations position AnmK for understanding the nexus among peptidoglycan recycling, susceptibility to antibiotics, and bacterial virulence.

Structure elucidation and identification of novel lead molecules against Sulfur import protein cysA of Mycobacterium tuberculosis.

The present work considers the Sulphate import ABC transporter protein (cysA) as a potential drug target for the identification of inhibitors for the protein. The ABC (ATP binding cassette) transporters play a crucial role in the survival and virulence of Mycobacterium tuberculosis by the acquisition of micronutrients from host tissue. The 3D structural features of the cysA protein are built. Molecular scaffolds are identified by implementing active site identification, ADME properties, Virtual Screening, and a few other computational techniques. The theoretical model of cysA is predicted using homology modeling protocols, and the structure is validated by various validation methods. The prediction of partial dimer formation through protein-protein docking methods gave insight into the conformational changes taking place in the cysA protein. The natural substrate ATP is docked with cysA protein that confirms the ATP binding site. To find the drug-like compounds, virtual screening studies were carried out around the active site by several ligand databases. The findings demonstrate the significance of residues SER41, GLY42, ARG50, GLN85, HIS86, LYS91, ARG142, and ASP161 in drug-target interactions. The docking studies of existing TB drugs against cysA were also performed. The result analysis shows that none of the existing drugs inhibits the ATP active site, which confirms cysA as a promising drug target. Using in-silico methods, the ADME parameters of a few chosen ligand molecules are predicted and contrasted with the ADME characteristics of the available TB medications. The results revealed the values of ADME parameters of selected ligand molecules are more permissible than existing TB drugs, which emphasizes the drug-like activity of ligand molecules by inhibition of cysA proteins. The structural data, active site information, and selected ligand molecules help in the identification of new therapeutic scaffolds for Tuberculosis.

Phytochemical and biological characterization of aqueous extract of Vassobia breviflora on proliferation and viability of melanoma cells: involvement of purinergic pathway

ABSTRACT Vassobia breviflora belongs to the Solanaceae family, possessing biological activity against tumor cells and is a promising alternative for therapy. The aim of this investigation was to determine the phytochemical properties V. breviflora using ESI-ToF-MS. The cytotoxic effects of this extract were examined in B16-F10 melanoma cells and the relationship if any to purinergic signaling was involved. The antioxidant activity of total phenols, (2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) was analyzed, as well as production of reactive oxygen species (ROS) and nitric oxide (NO) was determined. Genotoxicity was assessed by DNA damage assay. Subsequently, the structural bioactive compounds were docked against purinoceptors P2X7 and P2Y1 receptors. The bioactive compounds found in V. breviflora were N-methyl-(2S,4 R)-trans-4-hydroxy-L-proline, calystegine B, 12-O-benzoyl- tenacigenin A and bungoside B. In vitro cytotoxicity was demonstrated at concentration ranges of 0.1–10 mg/ml, and plasmid DNA breaks only at the concentration of 10 mg/ml. V. breviflora extracts affected hydrolysis by ectoenzymes, such as ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) and ectoadenosine deaminase (E-ADA) which control levels of degradation and formation of nucleosides and nucleotides. In the presence of substrates ATP, ADP, AMP and adenosine, the activities of E-NTPDase, 5´-NT or E-ADA were significantly modulated by V. breviflora. N-methyl-(2S,4 R)-trans-4-hydroxy-L-proline presented higher binding affinity (according to receptor-ligand complex estimated binding affinity as evidenced by ∆G values) to bind to both P2X7 and P2Y1purinergic receptors. Our results suggest a putative interaction of V. breviflora bioactive compounds with growth inhibitory potential in B16-F10 melanoma and suggest that may be considered as promising compounds in melanoma and cancer treatment