Additional ATP Research Articles

Isolation of Arabidopsis extracellular ATP binding proteins by affinity proteomics and identification of PHOSPHOLIPASEC-LIKE1 as an extracellular protein essential for fumonisinB1 toxicity.

ATP is secreted to the extracellular matrix, where it activates plasma membrane receptors for controlling plant growth and stress-adaptive processes. DOES NOT RESPOND TO NUCLEOTIDES1 (DORN1), was the first plant ATP receptor to be identified but key downstream proteins remain sought after. Here, we identified 120 proteins secreted by Arabidopsis cell cultures and screened them for putative stress-responsive proteins using ATP-affinity purification. We report three Arabidopsis proteins isolated by ATP-affinity: PEROXIDASE52, SUBTILASE-LIKE SERINE PROTEASE1.7 and PHOSPHOLIPASEC-LIKE1. In wild-type Arabidopsis, the expression of genes encoding all three proteins responded to fumonisinB1, a cell death-activating mycotoxin. The expression of PEROXIDASE52 and PHOSPHOLIPASEC-LIKE1 was altered in fumonisinB1-resistant salicylic acid induction-deficient (sid2) mutants. Exposure to fumonisinB1 suppressed PHOSPHOLIPASEC-LIKE1 expression in sid2 mutants, suggesting that the inactivation of this gene might provide mycotoxin tolerance. Accordingly, gene knockout mutants of PHOSPHOLIPASEC-LIKE1 were resistant to fumonisinB1-induced death. The activation of PHOSPHOLIPASEC-LIKE1 gene expression by exogenous ATP was not blocked in dorn1 loss-of-function mutants, indicating that DORN1 is not required. Furthermore, exogenous ATP rescued both the wild type and the dorn1 mutants from fumonisin-B1 toxicity, suggesting that different ATP receptor(s) are operational in this process. Our results point to the existence of additional plant ATP receptor(s) and provide crucial downstream targets for use in designing screens to identify these receptors. Finally, PHOSPHOLIPASEC-LIKE1 serves as a convergence point for fumonisinB1 and extracellular ATP signalling, and functions in the Arabidopsis stress response to fumonisinB1.

The Remarkable Metabolism of Vickermania ingenoplastis: Genomic Predictions.

A recently redescribed two-flagellar trypanosomatid Vickermania ingenoplastis is insensitive to the classical inhibitors of respiration and thrives under anaerobic conditions. Using genomic and transcriptomic data, we analyzed its genes of the core metabolism and documented that subunits of the mitochondrial respiratory complexes III and IV are ablated, while those of complexes I, II, and V are all present, along with an alternative oxidase. This explains the previously reported conversion of glucose to acetate and succinate by aerobic fermentation. Glycolytic pyruvate is metabolized to acetate and ethanol by pyruvate dismutation, whereby a unique type of alcohol dehydrogenase (shared only with Phytomonas spp.) processes an excess of reducing equivalents formed under anaerobic conditions, leading to the formation of ethanol. Succinate (formed to maintain the glycosomal redox balance) is converted to propionate by a cyclic process involving three enzymes of the mitochondrial methyl-malonyl-CoA pathway, via a cyclic process, which results in the formation of additional ATP. The unusual structure of the V. ingenoplastis genome and its similarity with that of Phytomonas spp. imply their relatedness or convergent evolution. Nevertheless, a critical difference between these two trypanosomatids is that the former has significantly increased its genome size by gene duplications, while the latter streamlined its genome.

NNT-induced tumor cell "slimming" reverses the pro-carcinogenesis effect of HIF2a in tumors.

BackgroundHIF2a and lipid accumulation play key roles in the progression of clear cell renal cell carcinoma (ccRCC). Tumor cell “slimming” is a new concept in which tumor cells with abnormal lipids efficiently consume lipids to inhibit tumor progression without producing additional ATP. However, their respective regulatory mechanisms are still unclear. The purpose of this study is uncovering the links between these three key elements of ccRCC to elucidate new mechanisms of ccRCC metabolic abnormalities and providing a basis for new drug development for ccRCC.MethodsBioinformatics screening and analyses were performed in ccRCC according to TCGA‐KIRC database. qRT‐PCR, luciferase reporter assay, western blot, chromatin immunoprecipitation (ChIP) assays, and other biological methods were used to explore and verify related pathways. Various cell line models and animal models were used to perform related functional experiments.ResultsScreening based on sequencing data after HIF2a knockdown and three independent mitochondrial metabolism‐related gene sets showed that nicotinamide nucleotide transhydrogenase (NNT) was a mediator between HIF2a and tumor cells “slimming.” Further research showed that NNT had significant prognostic predictive value and was downregulated in ccRCC. It is regulated by HIF2a and can significantly activate lipid browning‐mediated tumor cell “slimming.” Mechanistic investigations indicated that HIF2a enhanced the expression of miR‐455‐5p via binding to HIF2a‐related response elements in the miR‐455‐5p promoter, which suppresses NNT expression by binding to its 3′ untranslated region.ConclusionsOur study revealed a novel mechanism by which HIF2a decreased NNT level through a microRNA that suppressed tumor cell “slimming,” resulting in the progression of ccRCC. This mechanism provides a fresh perspective of lipid accumulation in ccRCC and may help target novel strategies for the treatment of tumors with abnormal lipid metabolism.

Overcoming Energetic Barriers in Acetogenic C1 Conversion.

Currently one of the biggest challenges for society is to combat global warming. A solution to this global threat is the implementation of a CO2-based bioeconomy and a H2-based bioenergy economy. Anaerobic lithotrophic bacteria such as the acetogenic bacteria are key players in the global carbon and H2 cycle and thus prime candidates as driving forces in a H2- and CO2-bioeconomy. Naturally, they convert two molecules of CO2 via the Wood-Ljungdahl pathway (WLP) to one molecule of acetyl-CoA which can be converted to different C2-products (acetate or ethanol) or elongated to C4 (butyrate) or C5-products (caproate). Since there is no net ATP generation from acetate formation, an electron-transport phosphorylation (ETP) module is hooked up to the WLP. ETP provides the cell with additional ATP, but the ATP gain is very low, only a fraction of an ATP per mol of acetate. Since acetogens live at the thermodynamic edge of life, metabolic engineering to obtain high-value products is currently limited by the low energy status of the cells that allows for the production of only a few compounds with rather low specificity. To set the stage for acetogens as production platforms for a wide range of bioproducts from CO2, the energetic barriers have to be overcome. This review summarizes the pathway, the energetics of the pathway and describes ways to overcome energetic barriers in acetogenic C1 conversion.

Data-driven in silico prediction of regulation heterogeneity and ATP demands of Escherichia coli in large-scale bioreactors.

Escherichia coli exposed to industrial-scale heterogeneous mixing conditions respond to external stress by initiating short-term metabolic and long-term strategic transcriptional programs. In native habitats, long-term strategies allow survival insevere stress but are of limited use in large bioreactors, where microenvironmental conditions may change right after said programs are started. Related on/off switching of genes causes additional ATP burden that may reduce the cellular capacity for producing the desired product. Here, we present an agent-based data-driven model linked to computational fluid dynamics, finally allowing to predict additional ATP needs of Escherichiacoli K12 W3110 exposed to realistic large-scale bioreactor conditions. The complex model describes transcriptional up- and downregulation dynamics of about 600 genes starting from subminute range covering 28 h. The data-based approach was extracted from comprehensive scale-down experiments. Simulating mixing and mass transfer conditions in a 54 m3 stirred bioreactor, 120,000 E. coli cells were tracked while fluctuating between different zones of glucose availability. It was found that cellular ATP demands rise between 30%and 45% of growth decoupled maintenance needs, which may limit the production of ATP-intensive product formation accordingly. Furthermore, spatial analysis of individual cell transcriptional patterns reveal very heterogeneous gene amplifications with hot spots of 50%-80% messenger RNA upregulation in the upper region of the bioreactor. The phenomenon reflects the time-delayed regulatory response of the cells that propagatethrough the stirred tank. After 4.2 h, cells adapt to environmental changes but still have to bear an additional 6% ATP demand.

Biophysical and Biochemical Characterization of TP0037, a d-Lactate Dehydrogenase, Supports an Acetogenic Energy Conservation Pathway in Treponema pallidum.

A longstanding conundrum in Treponema pallidum biology concerns how the spirochete generates sufficient energy to fulfill its complex pathogenesis processes during human syphilitic infection. For decades, it has been assumed that the bacterium relies solely on glucose catabolism (via glycolysis) for generation of its ATP. However, the organism's robust motility, believed to be essential for human tissue invasion and dissemination, would require abundant ATP likely not provided by the parsimony of glycolysis. As such, additional ATP generation, either via a chemiosmotic gradient, substrate-level phosphorylation, or both, likely exists in T. pallidum Along these lines, we have hypothesized that T. pallidum exploits an acetogenic energy conservation pathway that relies on the redox chemistry of flavins. Central to this hypothesis is the apparent existence in T. pallidum of an acetogenic pathway for the conversion of d-lactate to acetate. Herein we have characterized the structural, biophysical, and biochemical properties of the first enzyme (d-lactate dehydrogenase [d-LDH]; TP0037) predicted in this pathway. Binding and enzymatic studies showed that recombinant TP0037 consumed d-lactate and NAD+ to produce pyruvate and NADH. The crystal structure of TP0037 revealed a fold similar to that of other d-acid dehydrogenases; residues in the cofactor-binding and active sites were homologous to those of other known d-LDHs. The crystal structure and solution biophysical experiments revealed the protein's propensity to dimerize, akin to other d-LDHs. This study is the first to elucidate the enzymatic properties of T. pallidum's d-LDH, thereby providing new compelling evidence for a flavin-dependent acetogenic energy conservation (ATP-generating) pathway in T. pallidumIMPORTANCE Because T. pallidum lacks a Krebs cycle and the capability for oxidative phosphorylation, historically it has been difficult to reconcile how the syphilis spirochete generates sufficient ATP to fulfill its energy needs, particularly for its robust motility, solely from glycolysis. We have postulated the existence in T. pallidum of a flavin-dependent acetogenic energy conservation pathway that would generate additional ATP for T. pallidum bioenergetics. In the proposed acetogenic pathway, first d-lactate would be converted to pyruvate. Pyruvate would then be metabolized to acetate in three additional steps, with ATP being generated via substrate-level phosphorylation. This study provides structural, biochemical, and biophysical evidence for the first T. pallidum enzyme in the pathway (TP0037; d-lactate dehydrogenase) requisite for the conversion of d-lactate to pyruvate. The findings represent the first experimental evidence to support a role for an acetogenic energy conservation pathway that would contribute to nonglycolytic ATP production in T. pallidum.

Exceeding a "critical" muscle Pi: implications for [Formula: see text] and metabolite slow components, muscle fatigue and the power-duration relationship.

The consequences of the assumption that the additional ATP usage, underlying the slow component of oxygen consumption ([Formula: see text]) and metabolite on-kinetics, starts when cytosolic inorganic phosphate (Pi) exceeds a certain "critical" Pi concentration, and muscle work terminates because of fatigue when Pi exceeds a certain, higher, "peak" Pi concentration are investigated. A previously developed computer model of the myocyte bioenergetic system is used. Simulated time courses of muscle [Formula: see text], cytosolic ADP, pH, PCr and Pi at various ATP usage activities agreed well with experimental data. Computer simulations resulted in a hyperbolic power-duration relationship, with critical power (CP) as an asymptote. CP was increased, and phase II [Formula: see text] on-kinetics was accelerated, by progressive increase in oxygen tension (hyperoxia). Pi is a major factor responsible for the slow component of the [Formula: see text] and metabolite on-kinetics, fatigue-related muscle work termination and hyperbolic power-duration relationship. The successful generation of experimental system properties suggests that the additional ATP usage, underlying the slow component, indeed starts when cytosolic Pi exceeds a "critical" Pi concentration, and muscle work terminates when Pi exceeds a "peak" Pi concentration. The contribution of other factors, such as cytosolic acidification, or glycogen depletion and central fatigue should not be excluded. Thus, a detailed quantitative unifying mechanism underlying various phenomena related to skeletal muscle fatigue and exercise tolerance is offered that was absent in the literature. This mechanism is driven by reciprocal stimulation of Pi increase and additional ATP usage when "critical" Pi is exceeded.

Overproduction of PGR5 enhances the electron sink downstream of photosystem I in a C4 plant, Flaveria bidentis.

C4 plants can fix CO2 efficiently using CO2 -concentrating mechanisms (CCMs), but they require additional ATP. To supply the additional ATP, C4 plants operate at higher rates of cyclic electron transport around photosystem I (PSI), in which electrons are transferred from ferredoxin to plastoquinone. Recently, it has been reported that the NAD(P)H dehydrogenase-like complex (NDH) accumulated in the thylakoid membrane in leaves of C4 plants, making it a candidate for the additional synthesis of ATP used in the CCM. In addition, C4 plants have higher levels of PROTON GRADIENT REGULATION 5 (PGR5) expression, but it has been unknown how PGR5 functions in C4 photosynthesis. In this study, PGR5 was overexpressed in a C4 dicot, Flaveria bidentis. In PGR5-overproducing (OP) lines, PGR5 levels were 2.3- to 3.0-fold greater compared with wild-type plants. PGR5-like PHOTOSYNTHETIC PHENOTYPE 1 (PGRL1), which cooperates with PGR5, increased with PGR5. A spectroscopic analysis indicated that in the PGR5-OP lines, the acceptor side limitation of PSI was reduced in response to a rapid increase in photon flux density. Although it did not affect CO2 assimilation, the overproduction of PGR5 contributed to an enhanced electron sink downstream of PSI.

Modulation of Energy Metabolism Is Important for Low-Oxygen Stress Adaptation in Brassicaceae Species.

Low-oxygen stress, mainly caused by soil flooding, is a serious abiotic stress affecting crop productivity worldwide. To understand the mechanisms of low-oxygen stress responses and adaptation of plants, we characterized and compared low-oxygen responses in six species with different accessions of the Brassicaceae family. Based on the growth and survival responses to submergence or low-oxygen condition, these accessions could be divided into three groups: (i) Highly tolerant species (Rorippa islandica and Arabis stelleri); (ii) moderately tolerant species (Arabidopsis thaliana [esk-1, Ler, Ws and Col-0 ecotype]); and (iii) intolerant species (Thlaspi arvense, Thellungiella salsuginea [Shandong and Yukon ecotype], and Thellungiella parvula). Gene expression profiling using Operon Arabidopsis microarray was carried out with RNA from roots of A. thaliana (Col-0), A. stelleri, R. islandica, and T. salsuginea (Shandong) treated with low-oxygen stress (0.1% O2/99.9% N2) for 0, 1, 3, 8, 24, and 72 h. We performed a comparative analysis of the gene expression profiles using the gene set enrichment analysis (GSEA) method. Our comparative analysis suggested that under low-oxygen stress each species distinctively reconfigures the energy metabolic pathways including sucrose–starch metabolism, glycolysis, fermentation and nitrogen metabolism, tricarboxylic acid flow, and fatty acid degradation via beta oxidation and glyoxylate cycle. In A. thaliana, a moderately tolerant species, the dynamical reconfiguration of energy metabolisms occurred in the early time points of low-oxygen treatment, but the energy reconfiguration in the late time points was not as dynamic as in the early time points. Highly tolerant A. stelleri appeared to have high photosynthesis capacity that could produce more O2 and in turn additional ATP energy to cope with energy depletion caused by low-oxygen stress. R. islandica seemed to retain some ATP energy produced by anaerobic energy metabolism during a prolonged period of low-oxygen conditions. Intolerant T. salsuginea did not show significant changes in the expression of genes involved in anaerobic energy metabolisms. These results indicate that plants developed different energy metabolisms to cope with the energy crisis caused by low-oxygen stress.

Fluxomic Analysis Reveals Central Carbon Metabolism Adaptation for Diazotroph Azotobacter vinelandii Ammonium Excretion

Diazotrophic bacteria are an attractive biological alternative to synthetic nitrogen fertilizers due to their remarkable capacity to fix atmospheric nitrogen gas to ammonium via nitrogenase enzymes. However, how diazotrophic bacteria tailor central carbon catabolism to accommodate the energy requirement for nitrogenase activity is largely unknown. In this study, we used Azotobacter vinelandii DJ and an ammonium excreting mutant, AV3 (ΔNifL), to investigate central carbon metabolism fluxes and central cell bioenergetics in response to ammonium availability and nitrogenase activity. Enabled by the powerful and reliable methodology of 13C-metabolic flux analysis, we show that the respiratory TCA cycle is upregulated in association with increased nitrogenase activity and causes a monotonic decrease in specific growth rate. Whereas the activity of the glycolytic Entner–Doudoroff pathway is positively correlated with the cell growth rate. These new observations are formulated into a 13C-metabolic flux model which further improves the understanding and interpretation of intracellular bioenergetics. This analysis leads to the conclusion that, under aerobic conditions, respiratory TCA metabolism is responsible for the supply of additional ATP and reducing equivalents required for elevated nitrogenase activity. This study provides a quantitative relationship between central carbon and nitrogen metabolism in an aerobic diazotroph for the first time.

Cyclic electron flow and light partitioning between the two photosystems in leaves of plants with different functional types

Cyclic electron flow (CEF) around photosystem I (PSI) is essential for generating additional ATP and enhancing efficient photosynthesis. Accurate estimation of CEF requires knowledge of the fractions of absorbed light by PSI (fI) and PSII (fII), which are only known for a few model species such as spinach. No measures of fI are available for C4 grasses under different irradiances. We developed a new method to estimate (1) fII in vivo by concurrently measuring linear electron flux through both photosystems left( {{text{LEF}}_{{{text{O}}_{ 2} }} } right) in leaf using membrane inlet mass spectrometry (MIMS) and total electron flux through PSII (ETR2) using chlorophyll fluorescence by a Dual-PAM at low light and (2) CEF as ETR1—{text{LEF}}_{{{text{O}}_{ 2} }}. For a C3 grass, fI was 0.5 and 0.4 under control (high light) and shade conditions, respectively. C4 species belonging to NADP-ME and NAD-ME subtypes had fI of 0.6 and PCK subtype had 0.5 under control. All shade-grown C4 species had fI of 0.6 except for NADP-ME grass which had 0.7. It was also observed that fI ranged between 0.3 and 0.5 for gymnosperm, liverwort and fern species. CEF increased with irradiance and was induced at lower irradiances in C4 grasses and fern relative to other species. CEF was greater in shade-grown plants relative to control plants except for C4 NADP-ME species. Our study reveals a range of CEF and fI values in different plant functional groups. This variation must be taken into account for improved photosynthetic calculations and modelling.

Can Galactose Be Converted to Glucose in HepG2 Cells? Improving the in Vitro Mitochondrial Toxicity Assay for the Assessment of Drug Induced Liver Injury.

Human hepatocellular carcinoma cells, HepG2, are often used for drug mediated mitochondrial toxicity assessments. Glucose in HepG2 culture media is replaced by galactose to reveal drug-induced mitochondrial toxicity as a marked shift of drug IC50 values for the reduction of cellular ATP. It has been postulated that galactose sensitizes HepG2 mitochondria by the additional ATP consumption demand in the Leloir pathway. However, our NMR metabolomics analysis of HepG2 cells and culture media showed very limited galactose metabolism. To clarify the role of galactose in HepG2 cellular metabolism, U-13C6-galactose or U-13C6-glucose was added to HepG2 culture media to help specifically track the metabolism of those two sugars. Conversion to U-13C3-lactate was hardly detected when HepG2 cells were incubated with U-13C6-galactose, while an abundance of U-13C3-lactate was produced when HepG2 cells were incubated with U-13C6-glucose. In the absence of glucose, HepG2 cells increased glutamine consumption as a bioenergetics source. The requirement of additional glutamine almost matched the amount of glucose needed to maintain a similar level of cellular ATP in HepG2 cells. This improved understanding of galactose and glutamine metabolism in HepG2 cells helped optimize the ATP-based mitochondrial toxicity assay. The modified assay showed 96% sensitivity and 97% specificity in correctly discriminating compounds known to cause mitochondrial toxicity from those with prior evidence of not being mitochondrial toxicants. The greatest significance of the modified assay was its improved sensitivity in detecting the inhibition of mitochondrial fatty acid β-oxidation (FAO) when glutamine was withheld. Use of this improved assay for an empirical prediction of the likely contribution of mitochondrial toxicity to human DILI (drug induced liver injury) was attempted. According to testing of 65 DILI positive compounds representing numerous mechanisms of DILI together with 55 DILI negative compounds, the overall prediction of mitochondrial mechanism-related DILI showed 25% sensitivity and 95% specificity.

Studying the phosphoryl transfer mechanism of the E. coli phosphofructokinase-2: from X-ray structure to quantum mechanics/molecular mechanics simulations.

Phosphofructokinases (Pfks) catalyze the ATP-dependent phosphorylation of fructose-6-phosphate (F6P) and they are regulated in a wide variety of organisms. Although numerous aspects of the kinetics and regulation have been characterized for Pfks, the knowledge about the mechanism of the phosphoryl transfer reaction and the transition state lags behind. In this work, we describe the X-ray crystal structure of the homodimeric Pfk-2 from E. coli, which contains products in one site and reactants in the other, as well as an additional ATP molecule in the inhibitory allosteric site adjacent to the reactants. This complex was previously predicted when studying the kinetic mechanism of ATP inhibition. After removing the allosteric ATP, molecular dynamic (MD) simulations revealed conformational changes related to domain packing, as well as stable interactions of Lys27 and Asp256 with donor (ATP) and acceptor (fructose-6-) groups, and of Asp166 with Mg2+. The phosphoryl transfer reaction mechanism catalyzed by Pfk-2 was investigated through Quantum Mechanics/Molecular Mechanics (QM/MM) simulations using a combination of the string method and a path-collective variable for the exploration of its free energy surface. The calculated activation free energies showed that a dissociative mechanism, occurring with a metaphosphate intermediate formation followed by a proton transfer to Asp256, is more favorable than an associative one. The structural analysis reveals the role of Asp256 acting as a catalytic base and Lys27 stabilizing the transition state of the dissociative mechanism.

Muscle V˙O2-power output nonlinearity in constant-power, step-incremental, and ramp-incremental exercise: magnitude and underlying mechanisms

A computer model of the skeletal muscle bioenergetic system was used to simulate time courses of muscle oxygen consumption (V˙O2), cytosolic metabolite (ADP, PCr, Pi, and ATP) concentrations, and pH during whole‐body constant‐power exercise (CPE) (6 min), step‐incremental exercise (SIE) (30 W/3 min), and slow (10 W/min), medium (30 W/min), and fast (50 W/min) ramp‐incremental exercise (RIE). Different ESA (each‐step activation) of oxidative phosphorylation (OXPHOS) intensity‐ATP usage activity relationships, representing different muscle fibers recruitment patterns, gave best agreement with experimental data for CPE, and for SIE and RIE. It was assumed that the muscle V˙O2‐power output (PO) nonlinearity is related to a time‐ and PO‐dependent increase in the additional ATP usage underlying the slow component of the V˙O2 on‐kinetics minus the increase in ATP supply by anaerobic glycolysis leading to a decrease in V˙O2. The muscle V˙O2‐PO relationship deviated upward (+) or downward (−) from linearity above critical power (CP), and the nonlinearity equaled +16% (CPE),+12% (SIE), +8% (slow RIE), +1% (moderate RIE), and −2% (fast RIE) at the end of exercise, in agreement with experimental data. During SIE and RIE, changes in PCr and Pi accelerated moderately above CP, while changes in ADP and pH accelerated significantly with time and PO. It is postulated that the intensity of the additional ATP usage minus ATP supply by anaerobic glycolysis determines the size of the muscle V˙O2‐PO nonlinearity. It is proposed that the extent of the additional ATP usage is proportional to the time integral of PO ‐ CP above CP.

Structure of a PSI–LHCI–cyt b6f supercomplex in Chlamydomonas reinhardtii promoting cyclic electron flow under anaerobic conditions

Photosynthetic linear electron flow (LEF) produces ATP and NADPH, while cyclic electron flow (CEF) exclusively drives photophosphorylation to supply extra ATP. The fine-tuning of linear and cyclic electron transport levels allows photosynthetic organisms to balance light energy absorption with cellular energy requirements under constantly changing light conditions. As LEF and CEF share many electron transfer components, a key question is how the same individual structural units contribute to these two different functional modes. Here, we report the structural identification of a photosystem I (PSI)-light harvesting complex I (LHCI)-cytochrome (cyt) b6f supercomplex isolated from the unicellular alga Chlamydomonas reinhardtii under anaerobic conditions, which induces CEF. This provides strong evidence for the model that enhanced CEF is induced by the formation of CEF supercomplexes, when stromal electron carriers are reduced, to generate additional ATP. The additional identification of PSI-LHCI-LHCII complexes is consistent with recent findings that both CEF enhancement and state transitions are triggered by similar conditions, but can occur independently from each other. Single molecule fluorescence correlation spectroscopy indicates a physical association between cyt b6f and fluorescent chlorophyll containing PSI-LHCI supercomplexes. Single particle analysis identified top-view projections of the corresponding PSI-LHCI-cyt b6f supercomplex. Based on molecular modeling and mass spectrometry analyses, we propose a model in which dissociation of LHCA2 and LHCA9 from PSI supports the formation of this CEF supercomplex. This is supported by the finding that a Δlhca2 knockout mutant has constitutively enhanced CEF.

Engineering of Escherichia coli for Krebs cycle-dependent production of malic acid

BackgroundMalate is a C4-dicarboxylic acid widely used as an acidulant in the food and beverage industry. Rational engineering has been performed in the past for the development of microbial strains capable of efficient production of this metabolite. However, as malate can be a precursor for specialty chemicals, such as 2,4-dihydroxybutyric acid, that require additional cofactors NADP(H) and ATP, we set out to reengineer Escherichia coli for Krebs cycle-dependent production of malic acid that can satisfy these requirements.ResultsWe found that significant malate production required at least simultaneous deletion of all malic enzymes and dehydrogenases, and concomitant expression of a malate-insensitive PEP carboxylase. Metabolic flux analysis using 13C-labeled glucose indicated that malate-producing strains had a very high flux over the glyoxylate shunt with almost no flux passing through the isocitrate dehydrogenase reaction. The highest malate yield of 0.82 mol/mol was obtained with E. coli Δmdh Δmqo ΔmaeAB ΔiclR ΔarcA which expressed malate-insensitive PEP carboxylase PpcK620S and NADH-insensitive citrate synthase GltAR164L. We also showed that inactivation of the dicarboxylic acid transporter DcuA strongly reduced malate production arguing for a pivotal role of this permease in malate export.ConclusionsSince more NAD(P)H and ATP cofactors are generated in the Krebs cycle-dependent malate production when compared to pathways which depend on the function of anaplerotic PEP carboxylase or PEP carboxykinase enzymes, the engineered strain developed in this study can serve as a platform to increase biosynthesis of malate-derived metabolites such as 2,4-dihydroxybutyric acid.

Computational analysis of kinase inhibitor selectivity using structural knowledge.

Kinases play a significant role in diverse disease signaling pathways and understanding kinase inhibitor selectivity, the tendency of drugs to bind to off-targets, remains a top priority for kinase inhibitor design and clinical safety assessment. Traditional approaches for kinase selectivity analysis using biochemical activity and binding assays are useful but can be costly and are often limited by the kinases that are available. On the other hand, current computational kinase selectivity prediction methods are computational intensive and can rarely achieve sufficient accuracy for large-scale kinome wide inhibitor selectivity profiling. Here, we present a KinomeFEATURE database for kinase binding site similarity search by comparing protein microenvironments characterized using diverse physiochemical descriptors. Initial selectivity prediction of 15 known kinase inhibitors achieved an >90% accuracy and demonstrated improved performance in comparison to commonly used kinase inhibitor selectivity prediction methods. Additional kinase ATP binding site similarity assessment (120 binding sites) identified 55 kinases with significant promiscuity and revealed unexpected inhibitor cross-activities between PKR and FGFR2 kinases. Kinome-wide selectivity profiling of 11 kinase drug candidates predicted novel as well as experimentally validated off-targets and suggested structural mechanisms of kinase cross-activities. Our study demonstrated potential utilities of our approach for large-scale kinase inhibitor selectivity profiling that could contribute to kinase drug development and safety assessment. The KinomeFEATURE database and the associated scripts for performing kinase pocket similarity search can be downloaded from the Stanford SimTK website (https://simtk.org/projects/kdb). Supplementary data are available at Bioinformatics online.

A Phytoplankton Model for the Allocation of Gross Photosynthetic Energy Including the Trade‐Offs of Diazotrophy

AbstractGross photosynthetic activity by phytoplankton is directed to linear and alternative electron pathways that generate ATP, reductant, and fix carbon. Ultimately less than half is directed to net growth. Here we present a phytoplankton cell allocation model that explicitly represents a number of cell metabolic processes and functional pools with the goal of evaluating ATP and reductant demands as a function of light, nitrate, iron, oxygen, and temperature for diazotrophic versus nondiazotrophic growth. We employ model analogues of Synechoccocus and Crocosphaera watsonii, to explore the trade‐offs of diazotrophy over a range of environmental conditions. Model analogues are identical in construction, except for an iron quota associated with nitrogenase, an additional respiratory demand to remove oxygen in order to protect nitrogenase and an additional ATP demand to split dinitrogen. We find that these changes explain observed differences in growth rate and iron limitation between diazotrophs and nondiazotrophs. Oxygen removal imparted a significantly larger metabolic cost to diazotrophs than ATP demand for fixing nitrogen. Results suggest that diazotrophs devote a much smaller fraction of gross photosynthetic energy to growth than nondiazotrophs. The phytoplankton cell allocation model model provides a predictive framework for how photosynthate allocation varies with environmental conditions in order to balance cellular demands for ATP and reductant across phytoplankton functional groups.

The Origin Recognition Complex: where it all begins

Binding of the Origin Recognition Complex (ORC) to origins of replication marks the first step in the initiation of replication of the genome in all eukaryotic cells. I will present the structure of the active form of human ORC determined by X‐ray crystallography and cryo‐electron microscopy. The complex is composed of an ORC1/4/5 motor module lobe in an organization reminiscent of the DNA polymerase clamp loader complexes. A second lobe contains the ORC2/3 subunits. The complex is organized as a double‐layered shallow corkscrew, with the AAA+ and AAA+‐like domains forming one layer, and the winged‐helix domains (WHDs) forming a top layer. CDC6 fits easily between ORC1 and ORC2, completing the ring and the DNA‐binding channel, forming an additional ATP hydrolysis site. Analysis of the ATPase activity of the complex provides a basis for understanding ORC activity as well as molecular defects observed in Meier‐Gorlin Syndrome mutations.Support or Funding InformationHoward Hughes Medical InstituteNational Institutes of HealthThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

An autocrine ATP release mechanism regulates basal ciliary activity in airway epithelium.

Extracellular ATP, in association with [Ca2+ ]i regulation, is required to maintain basal ciliary beat frequency. Increasing extracellular ATP levels increases ciliary beating in airway epithelial cells, maintaining a sustained response by inducing the release of additional ATP. Extracellular ATP levels in the millimolar range, previously associated with pathophysiological conditions of the airway epithelium, produce a transient arrest of ciliary activity. The regulation of ciliary beat frequency is dependent on ATP release by hemichannels (connexin/pannexin) and P2X receptor activation, the blockage of which may even stop ciliary movement. The force exerted by cilia, measured by atomic force microscopy, is reduced following extracellular ATP hydrolysis. This result complements the current understanding of the ciliary beating regulatory mechanism, with special relevance to inflammatory diseases of the airway epithelium that affect mucociliary clearance. Extracellular nucleotides, including ATP, are locally released by the airway epithelium and stimulate ciliary activity in a [Ca2+ ]i -dependent manner after mechanical stimulation of ciliated cells. However, it is unclear whether the ATP released is involved in regulating basal ciliary activity and mediating changes in ciliary activity in response to chemical stimulation. In the present study, we evaluated ciliary beat frequency (CBF) and ciliary beating forces in primary cultures from mouse tracheal epithelium, using videomicroscopy and atomic force microscopy (AFM), respectively. Extracellular ATP levels and [Ca2+ ]i were measured by luminometric and fluorimetric assays, respectively. Uptake of ethidium bromide was measured to evaluate hemichannel functionality. We show that hydrolysis of constitutive extracellular ATP levels with apyrase (50Uml-1 ) reduced basal CBF by 45% and ciliary force by 67%. The apyrase effect on CBF was potentiated by carbenoxolone, a hemichannel inhibitor, and oxidized ATP, an antagonist used to block P2X7 receptors, which reduced basal CBF by 85%. Additionally, increasing extracellular ATP levels (0.1-100μm) increased CBF, maintaining a sustained response that was suppressed in the presence of carbenoxolone. We also show that high levels of ATP (1mm), associated with inflammatory conditions, lowered basal CBF by reducing [Ca2+ ]i and hemichannel functionality. In summary, we provide evidence indicating that airway epithelium ATP release is the molecular autocrine mechanism regulating basal ciliary activity and is also the mediator of the ciliary response to chemical stimulation.