ATP Cycling Research Articles

Production of 1,4-Butanediol from Succinic Acid Using Escherichia Coli Whole-Cell Catalysis.

The widespread attention towards 1,4-butanediol (BDO) as a key chemical raw material stems from its potential in producing biodegradable plastics. However, the efficiency of its biosynthesis via current bioprocesses is limited. In this study, a dual-pathway approach for 1,4-BDO production from succinic acid was developed. Specifically, a double-enzyme catalytic pathway involving carboxylic acid reductase and ethanol dehydrogenase was proposed. Optimization of the expression levels of the pathway enzymes led to a significant 318 % increase in 1,4-BDO titer. Additionally, the rate-limiting enzyme MmCAR was engineered to enhance the kcat/KM values by 50 % and increase 1,4-BDO titer by 46.7 %. To address cofactor supply limitations, an NADPH and ATP cycling system was established, resulting in a 48.9 % increase in 1,4-BDO production. Ultimately, after 48 hours, 1,4-BDO titers reached 201 mg/L and 1555 mg/L in shake flask and 5 L fermenter, respectively. This work represents a significant advancement in 1,4-BDO synthesis from succinic acid, with potential applications in the organic chemical and food industries.

The effect of small molecule myosin inhibitors on ATP cycling in porcine cardiac muscle using stopped-flow spectroscopy and in vitro motility

The effect of small molecule myosin inhibitors on ATP cycling in porcine cardiac muscle using stopped-flow spectroscopy and in vitro motility

Optimal energy and redox metabolism in the cyanobacterium Synechocystis sp. PCC 6803

Understanding energy and redox homeostasis and carbon partitioning is crucial for systems metabolic engineering of cell factories. Carbon metabolism alone cannot achieve maximal accumulation of metabolites in production hosts, since an efficient production of target molecules requires energy and redox balance, in addition to carbon flow. The interplay between cofactor regeneration and heterologous production in photosynthetic microorganisms is not fully explored. To investigate the optimality of energy and redox metabolism, while overproducing alkenes—isobutene, isoprene, ethylene and 1-undecene, in the cyanobacterium Synechocystis sp. PCC 6803, we applied stoichiometric metabolic modelling. Our network-wide analysis indicates that the rate of NAD(P)H regeneration, rather than of ATP, controls ATP/NADPH ratio, and thereby bioproduction. The simulation also implies that energy and redox balance is interconnected with carbon and nitrogen metabolism. Furthermore, we show that an auxiliary pathway, composed of serine, one-carbon and glycine metabolism, supports cellular redox homeostasis and ATP cycling. The study revealed non-intuitive metabolic pathways required to enhance alkene production, which are mainly driven by a few key reactions carrying a high flux. We envision that the presented comparative in-silico metabolic analysis will guide the rational design of Synechocystis as a photobiological production platform of target chemicals.

Translational Regulation of PHOSPHATE1 Affects Shoot Phosphate Levels and Biomass.

Phosphorus (P) is an essential plant nutrient that makes up part of membranes, DNA, and RNA; it also serves as energy currency in the form of ATP cycling. It is involved in many signaling processes that enable proper growth and development. The availability of P in the soil is often limited, and to

Modulators of actin-myosin dissociation: basis for muscle type functional differences during fatigue

The muscle types present with variable fatigue tolerance, in part due to the myosin isoform expressed. However, the critical steps that define “fatigability” in vivo of fast vs. slow myosin isoforms, at the molecular level, are not yet fully understood. We examined the modulation of the ATP-induced myosin subfragment 1 (S1) dissociation from pyrene-actin by inorganic phosphate (Pi), pH, and temperature using a specially modified stopped-flow system that allowed fast kinetics measurements at physiological temperature. We contrasted the properties of rabbit psoas (fast) and bovine masseter (slow) myosins (obtained from samples collected from New Zealand rabbits and from a licensed abattoir, respectively, according to institutional and national ethics permits). To identify ATP cycling biochemical intermediates, we assessed ATP binding to a preequilibrated mixture of actomyosin and variable [ADP], pH (pH 7 vs. pH 6.2), and Pi (zero, 15, or 30 added mM Pi) in a range of temperatures (5 to 45°C). Temperature and pH variations had little, if any, effect on the ADP dissociation constant (KADP) for fast S1, but for slow S1, KADP was weakened with increasing temperature or low pH. In the absence of ADP, the dissociation constant for phosphate (KPi) was weakened with increasing temperature for fast S1. In the presence of ADP, myosin type differences were revealed at the apparent phosphate affinity, depending on pH and temperature. Overall, the newly revealed kinetic differences between myosin types could help explain the in vivo observed muscle type functional differences at rest and during fatigue.

Transcriptomic and microRNAomic profiling reveals multi-faceted mechanisms to cope with phosphate stress in a dinoflagellate.

Although gene regulation can occur at both transcriptional and epigenetic (microRNA) levels, combined transcriptomic and microRNAomic responses to environmental stress are still largely unexplored for marine plankton. Here, we conducted transcriptome and microRNAome sequencing for Prorocentrum donghaiense to understand the molecular mechanisms by which this dinoflagellate copes with phosphorus (P) deficiency. Under P-depleted conditions, G1/S specific cyclin gene was markedly downregulated, consistent with growth inhibition, and genes related to dissolved organic phosphorus (DOP) hydrolysis, carbon fixation, nitrate assimilation, glycolysis, and cellular motility were upregulated. The elevated expression of ATP-generating genes (for example, rhodopsin) and ATP-consuming genes suggests some metabolic reconfiguration towards accelerated ATP recycling under P deficiency. MicroRNAome sequencing revealed 17 microRNAs, potentially regulating 3268 protein-coding genes. Functional enrichment analysis of these microRNA-targeted genes predicted decreases in sulfatide (sulfolipid) catabolism under P deficiency. Strikingly, we detected a significant increase in sulfolipid sulfatide content (but not in sulphoquinovosyldiacylglycerol content) and its biosynthesis gene expression, indicating a different sulfolipid-substituting-phospholipid mechanism in this dinoflagellate than other phytoplankters studied previously. Taken together, our integrative transcriptomic and microRNAomic analyses show that enhanced DOP utilization, accelerated ATP cycling and repressed sulfolipid degradation constitute a comprehensive strategy to cope with P deficiency in a model dinoflagellate.

Decline of Phosphotransfer and Substrate Supply Metabolic Circuits Hinders ATP Cycling in Aging Myocardium.

Integration of mitochondria with cytosolic ATP-consuming/ATP-sensing and substrate supply processes is critical for muscle bioenergetics and electrical activity. Whether age-dependent muscle weakness and increased electrical instability depends on perturbations in cellular energetic circuits is unknown. To define energetic remodeling of aged atrial myocardium we tracked dynamics of ATP synthesis-utilization, substrate supply, and phosphotransfer circuits through adenylate kinase (AK), creatine kinase (CK), and glycolytic/glycogenolytic pathways using 18O stable isotope-based phosphometabolomic technology. Samples of intact atrial myocardium from adult and aged rats were subjected to 18O-labeling procedure at resting basal state, and analyzed using the 18O-assisted HPLC-GC/MS technique. Characteristics for aging atria were lower inorganic phosphate Pi[18O], γ-ATP[18O], β-ADP[18O], and creatine phosphate CrP[18O] 18O-labeling rates indicating diminished ATP utilization-synthesis and AK and CK phosphotransfer fluxes. Shift in dynamics of glycolytic phosphotransfer was reflected in the diminished G6P[18O] turnover with relatively constant glycogenolytic flux or G1P[18O] 18O-labeling. Labeling of G3P[18O], an indicator of G3P-shuttle activity and substrate supply to mitochondria, was depressed in aged myocardium. Aged atrial myocardium displayed reduced incorporation of 18O into second (18O2), third (18O3), and fourth (18O4) positions of Pi[18O] and a lower Pi[18O]/γ-ATP[18 O]-labeling ratio, indicating delayed energetic communication and ATP cycling between mitochondria and cellular ATPases. Adrenergic stress alleviated diminished CK flux, AK catalyzed β-ATP turnover and energetic communication in aging atria. Thus, 18O-assisted phosphometabolomics uncovered simultaneous phosphotransfer through AK, CK, and glycolytic pathways and G3P substrate shuttle deficits hindering energetic communication and ATP cycling, which may underlie energetic vulnerability of aging atrial myocardium.

Mutations in the Yeast Hsp70, Ssa1, at P417 Alter ATP Cycling, Interdomain Coupling, and Specific Chaperone Functions

The major cytoplasmic Hsp70 chaperones in the yeast Saccharomyces cerevisiae are the Ssa proteins, and much of our understanding of Hsp70 biology has emerged from studying ssa mutant strains. For example, Ssa1 catalyzes multiple cellular functions, including protein transport and degradation, and to this end, the ssa1-45 mutant has proved invaluable. However, the biochemical defects associated with the corresponding Ssa1-45 protein (P417L) are unknown. Consequently, we characterized Ssa1 P417L, as well as a P417S variant, which corresponds to a mutation in the gene encoding the yeast mitochondrial Hsp70. We discovered that the P417L and P417S proteins exhibit accelerated ATPase activity that was similar to the Hsp40-stimulated rate of ATP hydrolysis of wild-type Ssa1. We also found that the mutant proteins were compromised for peptide binding. These data are consistent with defects in peptide-stimulated ATPase activity and with results from limited proteolysis experiments, which indicated that the mutants' substrate binding domains were highly vulnerable to digestion. Defects in the reactivation of heat-denatured luciferase were also evident. Correspondingly, yeast expressing P417L or P417S as the only copy of Ssa were temperature sensitive and exhibited defects in Ssa1-dependent protein translocation and misfolded protein degradation. Together, our studies suggest that the structure of the substrate binding domain is altered and that coupling between this domain and the nucleotide binding domain is disabled when the conserved P417 residue is mutated. Our data also provide new insights into the nature of the many cellular defects associated with the ssa1-45 allele.

Inhibitors of Difficult Protein–Protein Interactions Identified by High-Throughput Screening of Multiprotein Complexes

Protein-protein interactions (PPIs) are important in all aspects of cellular function, and there is interest in finding inhibitors of these contacts. However, PPIs with weak affinities and/or large interfaces have traditionally been more resistant to the discovery of inhibitors, partly because it is more challenging to develop high-throughput screening (HTS) methods that permit direct measurements of these physical interactions. Here, we explored whether the functional consequences of a weak PPI might be used as a surrogate for binding. As a model, we used the bacterial ATPase DnaK and its partners DnaJ and GrpE. Both DnaJ and GrpE bind DnaK and catalytically accelerate its ATP cycling, so we used stimulated nucleotide turnover to indirectly report on these PPIs. In pilot screens, we identified compounds that block activation of DnaK by either DnaJ or GrpE. Interestingly, at least one of these molecules blocked binding of DnaK to DnaJ, while another compound disrupted allostery between DnaK and GrpE without altering the physical interaction. These findings suggest that the activity of a reconstituted multiprotein complex might be used in some cases to identify allosteric inhibitors of challenging PPIs.

A Gradient of ATP Affinities Generates an Asymmetric Power Stroke Driving the Chaperonin TRIC/CCT Folding Cycle

The eukaryotic chaperonin TRiC/CCT uses ATP cycling to fold many essential proteins that other chaperones cannot fold. This 1 MDa hetero-oligomer consists of two identical stacked rings assembled from eight paralogous subunits, each containinga conserved ATP-binding domain. Here, we report a dramatic asymmetry in the ATP utilization cycle of this ring-shaped chaperonin, despite its apparently symmetric architecture. Only four of the eight different subunits bind ATP at physiological concentrations. ATP binding and hydrolysis by the low-affinity subunits is fully dispensable for TRiC function invivo. The conserved nucleotide-binding hierarchy among TRiC subunits is evolutionarily modulated through differential nucleoside contacts. Strikingly, high- and low-affinity subunits are spatially segregated within two contiguous hemispheres in the ring, generating an asymmetric power stroke that drives the folding cycle. This unusual mode of ATP utilization likely serves to orchestrate a directional mechanism underlying TRiC/CCT's unique ability to fold complex eukaryotic proteins.

The Molecular Architecture of the Eukaryotic Chaperonin TRiC/CCT

TRiC/CCT is a highly conserved and essential chaperonin that uses ATP cycling to facilitate folding of approximately 10% of the eukaryotic proteome. This 1 MDa hetero-oligomeric complex consists of two stacked rings of eight paralogous subunits each. Previously proposed TRiC models differ substantially in their subunit arrangements and ring register. Here, we integrate chemical crosslinking, mass spectrometry, and combinatorial modeling to reveal the definitive subunit arrangement of TRiC. In vivo disulfide mapping provided additional validation for the crosslinking-derived arrangement as the definitive TRiC topology. This subunit arrangement allowed the refinement of a structural model using existing X-ray diffraction data. The structure described here explains all available crosslink experiments, provides a rationale for previously unexplained structural features, and reveals a surprising asymmetry of charges within the chaperonin folding chamber.

Flaviviral helicase: Insights into the mechanism of action of a motor protein

Motor proteins are involved in crucial cell activities, such as cargo transport or nucleic acid remodeling, by converting the free energy of ATP hydrolysis into motion or mechanical work. Flavivirus helicase is a motor protein involved in dsRNA separation during viral replication, thus essential for virus infection. Since a clear vision of the protein activity, in particular of the relationship between ATP cycling and dynamics, is missing, we carried over a molecular dynamics study on Dengue virus helicase in its ATP bound and unbound states. Our simulations show different opening levels of the ssRNA access site to the helicase core. Specifically, we show that ATP induces a closed state into the ssRNA access site, likely involved in the helicase unwinding activity.

Single Molecule FRET Study of Nucleotide Binding Effects on Muts Proteins

The DNA mismatch repair (MMR) system is an essential component of the cellular DNA replication process that ensures high overall fidelity. The protein MutS initiates MMR by binding specifically to base-base mismatches and insertion-deletion mismatches in double stranded DNA. The active MutS dimer includes two ATP binding sites that hydrolyze ATP in both DNA bound and free states. ATP is also hydrolyzed as part of the MMR cascade involving MutS. Biochemical characterizations have shown that distinct nucleotides produce different interactions between MutS and mismatched DNA substrates, but the details of the conformations of MutS-mismatched DNA complexes under exposure to different nucleotides are not well known. We used single molecule fluorescence resonance energy transfer (smFRET) to study MutS from Thermus aquaticus in isolation and in complex with mismatched DNA substrates when exposed to a variety of different nucleotide conditions. We report results using intramolecular FRET from MutS or the DNA substrate as well as intermolecular FRET between MutS and the DNA. Our results allow MutS-DNA conformations resulting from specific nucleotide bound states to be characterized. We suggest possible roles for ATP cycling that could regulate the function of MutS in DNA MMR.

ATP Binding to Hsp90 Is Sufficient for Effective Chaperoning of p53 Protein

Hsp90 is a ubiquitous, ATP-dependent chaperone, essential for eukaryotes. It possesses a broad spectrum of substrates, among which is the p53 transcription factor, encoded by a tumor-suppressor gene. Here, we elucidate the role of the adenine nucleotide in the Hsp90 chaperone cycle, by taking advantage of a unique in vitro assay measuring Hsp90-dependent p53 binding to the promoter sequence. E42A and D88N Hsp90β variants bind but do not hydrolyze ATP, whereas E42A has increased and D88N decreased ATP affinity, compared with WT Hsp90β. Nevertheless, both of these mutants interact with WT p53 with a similar affinity. Surprisingly, in the case of WT, but also E42A Hsp90β, the presence of ATP stimulates dissociation of Hsp90-p53 complexes and results in p53 binding to the promoter sequence. D88N Hsp90β is not efficient in both of these reactions. Using a trap version of the chaperonin GroEL, which irreversibly captures unfolded proteins, we show that Hsp90 chaperone action on WT p53 results in a partial unfolding of the substrate. The ATP-dependent dissociation of p53-Hsp90 complex allows further folding of p53 protein to an active conformation, able to bind to the promoter sequence. Furthermore, in support of these results, the overproduction of WT or E42A Hsp90β stimulates transcription from the WAF1 gene promoter in H1299 cells. Altogether, our research indicates that ATP binding to Hsp90β is a sufficient step for effective WT p53 client protein chaperoning.

Ion homeostasis and the functional roles of SERCA reactions in stimulus–secretion coupling of the pancreatic β-cell: A mathematical simulation

The present paper concerns with ion homeostatic reactions in view of stimulus–secretion coupling of the β-cell, including Ca 2+ fluxes of the endoplasmatic reticulum (ER). A steady state of cytosolic sodium and potassium ion concentrations ([Na +] c and [K +] c, respectively), and of the membrane potential (Δ cϕ) can be attained only, if the flux through the electrogenic Na-K pump ( J NaK) is balanced electrically, and if J NaK is rather high (about 25% of total ATP consumption at 10 mM glucose). Metabolically caused changes of cellular pH are unlikely, because, on the one hand, CO 2 can rapidly leave the cell through cellular membranes, and because ATP cycling cannot produce nor consume protons. A slight decrease of pH c during cellular activity is caused mainly by an increased Ca-H exchange flux through the plasma membrane Ca 2+ pump ( J PMCA), which might be overcome, however, by H + transport into secretory granules. The present simulations show that the conductance of ATP-sensitive K + channels (K ATP) is highly susceptible to changes of [Mg 2+] c. As a physical link between the Ca 2+ filling state of the ER and the initiation of a depolarising, Ca 2+ release-activated current ( I CRAN), a metabolite (inositol 1,4,-diphosphate (IP 2)) of the inositol 1,4,5-triphosphate (IP 3) cycle is introduced. Sufficient ATP for insulin secretion is made available during glucose activation by [IP 2] inhibition of a parallel [ATP] c consuming flux through protein biosynthesis ( J Pbs). This leads to fast oscillations with a triphasic patterns of [Ca 2+] c oscillations. Slow oscillations are initiated by including a Ca 2+ leak current through highly uncoupled SERCA3 pumps. Both types of oscillations may superimpose yielding compound bursting and mixed oscillations of [Ca 2+] c.

Development of models of active ion transport for whole-cell modelling: cardiac sodium–potassium pump as a case study

This study presents a method for the reduction of biophysically-based kinetic models for the active transport of ions. A lumping scheme is presented which exploits the differences in timescales associated with fast and slow transitions between model states, while maintaining the thermodynamic properties of the model. The goal of this approach is to contribute to modelling of the effects of disturbances to metabolism, associated with ischaemic heart disease, on cardiac cell function. The approach is illustrated for the sodium-potassium pump in the myocyte. The lumping scheme is applied to produce a 4-state representation from the detailed 15-state model of Läuger and Apell, Eur. Biophys. J. 13 (1986) 309, for which the principles of free energy transduction are used to link the free energy released from ATP hydrolysis (Δ G ATP) to the transition rates between states of the model. An iterative minimisation algorithm is implemented to determine the transition rate parameters based on the model fit to experimental data. Finally, the relationship between Δ G ATP and pump cycling direction is investigated and compared with recent experimental findings.

Kinetics of a self-amplifying substrate cycle: ADP‒ATP cycling assay

A kinetic study of an ATP–ADP amplification cyclic system involving the enzymes adenylate kinase, pyruvate kinase and l-lactate dehydrogenase has been made. The stoichiometry of the cycle is 2:1, because two molecules of ADP are synthesized from one each of ATP and AMP, and one molecule of ADP is converted back into one of ATP at each turn of the cycle. This results in a continuous exponential increase in the concentrations of ATP and ADP in the reaction medium, according to the equations obtained. This is therefore a substrate cycle that amplifies itself, the cycling rate increasing continuously with time. The background signal of the reagent was reduced by using apyrase to degrade ATP and ADP in the reagent, permitting detection limits as low as 16pmol of ATP and/or ADP in a continuous spectrophotometric assay.

Kinetics of a self-amplifying substrate cycle: ADP–ATP cycling assay

A kinetic study of an ATP-ADP amplification cyclic system involving the enzymes adenylate kinase, pyruvate kinase and L-lactate dehydrogenase has been made. The stoichiometry of the cycle is 2:1, because two molecules of ADP are synthesized from one each of ATP and AMP, and one molecule of ADP is converted back into one of ATP at each turn of the cycle. This results in a continuous exponential increase in the concentrations of ATP and ADP in the reaction medium, according to the equations obtained. This is therefore a substrate cycle that amplifies itself, the cycling rate increasing continuously with time. The background signal of the reagent was reduced by using apyrase to degrade ATP and ADP in the reagent, permitting detection limits as low as 16 pmol of ATP and/or ADP in a continuous spectrophotometric assay.

Interaction of Calcium with Bordetella pertussis Adenylate Cyclase Toxin: CHARACTERIZATION OF MULTIPLE CALCIUM-BINDING SITES AND CALCIUM-INDUCED CONFORMATIONAL CHANGES

The adenylate cyclase (CyaA) secreted by Bordetella pertussis is a toxin that is able to enter eukaryotic cells and cause a dramatic increase in cAMP level. In addition, the toxin also exhibits an intrinsic hemolytic activity that is independent from the ATP cycling catalytic activity of the toxin. Both the cytotoxic and hemolytic activities are calcium-dependent. In this work, we have analyzed the calcium interacting properties of CyaA. We have shown that CyaA exposed to CaCl2 could retain membrane binding capability and hemolytic activity when it was further assayed in the presence of an excess of EGTA. Determination of the calcium content of CyaA exposed first to calcium and subsequently to EGTA indicated that some (3-5) calcium ions remained bound to the protein, suggesting the existence of Ca2+ binding sites of high affinity. Binding of Ca2+ to these sites might be necessary for both the membrane binding capability and the hemolytic activity of the toxin. In addition, CyaA possesses a large number (about 45) of low affinity (KD = 0.5-0.8 mM) Ca2+ binding sites that are located in the C terminus of the toxin, between amino acids 1007 and 1706. This region mainly consists of about 45 repeated sequences of the type GGXGXDXLX (where X represents any amino acid) that are characteristic of the RTX (Repeat in ToXin) bacterial protein family. Our data suggest that each one can bind one calcium ion. Circular dichroism spectroscopy analysis showed that calcium binding to the low affinity sites induces a large conformational change of CyaA, as revealed by an important increase in the content of alpha-helical structures. This conformational change might be directly involved in the Ca(2+)-dependent translocation of the catalytic domain of CyaA through the plasma membrane of target cells.

Effects of hypocaloric feeding and refeeding on myocardial Ca and ATP cycling in the rat.

Hypocaloric feeding (HCF) depresses heart function causing cardiac atrophy, bradycardia, and decreased cardiac output. We tested the hypothesis that HCF results in decreased myocardial Ca- and ATP cycling. We reduced protein-calorie intake of adult rats by 20% for 7 days and then allowed them to recover for 3 days. Changes in ionized Ca concentration (nM/s) of 2.5% myocardial homogenates that were attributable to the Ca-ATPase pump and Ca-release channel (CRC), respectively, of the sarcoplasmic reticulum (SR) were depressed 41 and 85% by HCF from 61.6 +/- 9.4 and 24.7 +/- 3.3, to 36.1 +/- 2.8 and 3.6 +/- 2.9. Activity of the Ca-pump was restored after 3 days of refeeding, whereas the CRC remained 23% depressed (all p < 0.05). Additionally, the CRC activity was inhibited to a 3-fold greater extent than controls by HCF, but was disinhibited within one day of refeeding. The greater effect on CRC than Ca-pump activity resulted in net Ca-uptake being unaffected by HCF. In addition to depression of Ca-cycling, ATP sythetase and total ATPase activities (IU/g), respectively, were depressed 20 and 15% by HCF from 174 +/- 19 and 51.3 +/- 3.8 to 140 +/- 15 and 43.7 +/- 4.7, but were restored to control values within one day of refeeding. We conclude that HCF produces a compensatory, reversible, and asymmetric downregulation and inhibition of Ca-cycling, with the CRC being preferentially affected.