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MBio Oct 2023Although most bacteria are quickly killed after phagocytosis by a eukaryotic cell, some pathogenic bacteria escape death after phagocytosis. Pathogenic species secrete...
Although most bacteria are quickly killed after phagocytosis by a eukaryotic cell, some pathogenic bacteria escape death after phagocytosis. Pathogenic species secrete polyP, and the polyP is necessary for the bacteria to prevent their killing after phagocytosis. Conversely, exogenous polyP prevents the killing of ingested bacteria that are normally killed after phagocytosis by human macrophages and the eukaryotic microbe . This suggests the possibility that in these cells, a signal transduction pathway is used to sense polyP and prevent killing of ingested bacteria. In this report, we identify key components of the polyP signal transduction pathway in . In cells lacking these components, polyP is unable to inhibit killing of ingested bacteria. The pathway components have orthologs in human cells, and an exciting possibility is that pharmacologically blocking this pathway in human macrophages would cause them to kill ingested pathogens such as .
Topics: Humans; Polyphosphates; Diphosphates; Dictyostelium; Bacteria; Phagocytosis; TOR Serine-Threonine Kinases
PubMed: 37754562
DOI: 10.1128/mbio.01939-23 -
The Plant Cell Oct 2015Phytol from chlorophyll degradation can be phosphorylated to phytyl-phosphate and phytyl-diphosphate, the substrate for tocopherol (vitamin E) synthesis. A candidate for...
Phytol from chlorophyll degradation can be phosphorylated to phytyl-phosphate and phytyl-diphosphate, the substrate for tocopherol (vitamin E) synthesis. A candidate for the phytyl-phosphate kinase from Arabidopsis thaliana (At1g78620) was identified via a phylogeny-based approach. This gene was designated VITAMIN E DEFICIENT6 (VTE6) because the leaves of the Arabidopsis vte6 mutants are tocopherol deficient. The vte6 mutant plants are incapable of photoautotrophic growth. Phytol and phytyl-phosphate accumulate, and the phytyl-diphosphate content is strongly decreased in vte6 leaves. Phytol feeding and enzyme assays with Arabidopsis and recombinant Escherichia coli cells demonstrated that VTE6 has phytyl-P kinase activity. Overexpression of VTE6 resulted in increased phytyl-diphosphate and tocopherol contents in seeds, indicating that VTE6 encodes phytyl-phosphate kinase. The severe growth retardation of vte6 mutants was partially rescued by introducing the phytol kinase mutation vte5. Double mutant plants (vte5 vte6) are tocopherol deficient and contain more chlorophyll, but reduced amounts of phytol and phytyl-phosphate compared with vte6 mutants, suggesting that phytol or phytyl-phosphate are detrimental to plant growth. Therefore, VTE6 represents the missing phytyl-phosphate kinase, linking phytol release from chlorophyll with tocopherol synthesis. Moreover, tocopherol synthesis in leaves depends on phytol derived from chlorophyll, not on de novo synthesis of phytyl-diphosphate from geranylgeranyl-diphosphate.
Topics: Arabidopsis; Arabidopsis Proteins; Biological Transport; Biosynthetic Pathways; Chlorophyll; Diphosphates; Mutation; Phosphorylation; Phosphotransferases; Phylogeny; Phytol; Plant Leaves; Seeds; Tocopherols
PubMed: 26452599
DOI: 10.1105/tpc.15.00395 -
PloS One 2016Pyrophosphate is a potent mitogen, capable of stimulating proliferation in multiple cell types, and a critical participant in bone mineralization. Pyrophosphate can also...
Pyrophosphate is a potent mitogen, capable of stimulating proliferation in multiple cell types, and a critical participant in bone mineralization. Pyrophosphate can also affect the resorption rate and bioactivity of orthopedic ceramics. The present study investigated whether calcium pyrophosphate affected proliferation, differentiation and gene expression in early (MC3T3 pre-osteoblast) and late stage (SAOS-2 osteosarcoma) osteoblasts. Pyrophosphate stimulated peak alkaline phosphatase activity by 50% and 150% at 100μM and 0.1μM in MC3T3, and by 40% in SAOS-2. The expression of differentiation markers collagen 1 (COL1), alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCN) were increased by an average of 1.5, 2, 2 and 3 fold, by high concentrations of sodium pyrophosphate (100μM) after 7 days of exposure in MC3T3. COX-2 and ANK expression did not differ significantly from controls in either treatment group. Though both high and low concentrations of pyrophosphate stimulate ALP activity, only high concentrations (100μM) stimulated osteogenic gene expression. Pyrophosphate did not affect proliferation in either cell type. The results of this study confirm that chronic exposure to pyrophosphate exerts a physiological effect upon osteoblast differentiation and ALP activity, specifically by stimulating osteoblast differentiation markers and extracellular matrix gene expression.
Topics: Alkaline Phosphatase; Animals; Biomarkers; Calcification, Physiologic; Cell Differentiation; Cell Line; Cell Proliferation; Diphosphates; Enzyme Activation; Extracellular Matrix Proteins; Gene Expression Regulation; Humans; Mice; Osteoblasts; Osteogenesis
PubMed: 27701417
DOI: 10.1371/journal.pone.0163530 -
Materials Science & Engineering. C,... Jun 2017Tooth hypersensitivity is a growing problem affecting both the young and ageing population worldwide. Since an effective and permanent solution is not yet available, we...
Tooth hypersensitivity is a growing problem affecting both the young and ageing population worldwide. Since an effective and permanent solution is not yet available, we propose a new methodology for the restoration of dental enamel using femtosecond lasers and novel calcium phosphate biomaterials. During this procedure the irradiated mineral transforms into a densified layer of acid resistant iron doped β-pyrophosphate, bonded with the surface of eroded enamel. Our aim therefore is to evaluate this densified mineral as a potential replacement material for dental hard tissue. To this end, we have tested the hardness of β-pyrophosphate pellets (sintered at 1000°C) and its mineral precursor (brushite), the wear rate during simulated tooth-brushing trials and the cytocompatibility of these minerals in powder form. It was found that the hardness of the β-pyrophosphate pellets is comparable with that of dental enamel and significantly higher than dentine while, the brushing trials prove that the wear rate of β-pyrophosphate is much slower than that of natural enamel. Finally, cytotoxicity and genotoxicity tests suggest that iron doped β-pyrophosphate is cytocompatible and therefore could be used in dental applications. Taken together and with the previously reported results on laser irradiation of these materials we conclude that iron doped β-pyrophosphate may be a promising material for restoring acid eroded and worn enamel.
Topics: Animals; Biocompatible Materials; Calcium Phosphates; Cattle; Dental Enamel; Diphosphates; Microscopy, Electron, Scanning; X-Ray Diffraction
PubMed: 28415544
DOI: 10.1016/j.msec.2017.02.116 -
Biochemistry Jul 2010Isopentenyl diphosphate isomerase (IDI) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the basic five-carbon...
Isopentenyl diphosphate isomerase (IDI) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the basic five-carbon building blocks of isoprenoid molecules. Two structurally unrelated classes of IDIs are known. Type I IPP isomerase (IDI-1) utilizes a divalent metal in a protonation-deprotonation reaction. In contrast, the type II enzyme (IDI-2) requires reduced flavin, raising the possibility that the reaction catalyzed by IDI-2 involves the net addition or abstraction of a hydrogen atom. As part of our studies of the mechanism of isomerization for IDI-2, we synthesized allene and alkyne substrate analogues for the enzyme. These molecules are predicted to be substantially less reactive toward proton addition than IPP and DMAPP but have similar reactivities toward hydrogen atom addition. This prediction was verified by calculations of gas-phase heats of reaction for addition of a proton and of a hydrogen atom to 1-butyne (3) and 1,2-butadiene (4) to form the 1-buten-2-yl carbocation and radical, respectively, and related affinities for 2-methyl-1-butene (5) and 2-methyl-2-butene (6) using G3MP2B3 and CBS-QB3 protocols. Alkyne 1-OPP and allene 2-OPP were not substrates for Thermus thermophilus IDI-2 or Escherichia coli IDI-1 but instead were competitive inhibitors. The experimental and computational results are consistent with a protonation-deprotonation mechanism for the enzyme-catalyzed isomerization of IPP and DMAPP.
Topics: Alkadienes; Alkynes; Carbon-Carbon Double Bond Isomerases; Diphosphates; Escherichia coli; Hemiterpenes; Hot Temperature; Hydrogen; Indoles; Isomerism; Organophosphorus Compounds; Substrate Specificity; Thermus thermophilus
PubMed: 20560533
DOI: 10.1021/bi100844e -
Applied and Environmental Microbiology Nov 2020and were grown in cellobiose-limited chemostat cultures at a fixed dilution rate. produced acetate, ethanol, formate, and lactate. Surprisingly, and in contrast to...
and were grown in cellobiose-limited chemostat cultures at a fixed dilution rate. produced acetate, ethanol, formate, and lactate. Surprisingly, and in contrast to batch cultures, in cellobiose-limited chemostat cultures of , ethanol was the main fermentation product. Enzyme assays confirmed that in , glycolysis proceeds via pyrophosphate (PP)-dependent phosphofructokinase (PFK), pyruvate-phosphate dikinase (PPDK), as well as a malate shunt for the conversion of phosphoenolpyruvate (PEP) to pyruvate. Pyruvate kinase activity was not detectable. In , ATP but not PP served as cofactor for the PFK reaction. High activities of both pyruvate kinase and PPDK were present, whereas the activities of a malate shunt enzymes were low in In , glycolysis via PP-PFK and PPDK obeys the equation glucose + 5 NDP + 3 PP → 2 pyruvate + 5 NTP + P (where NDP is nucleoside diphosphate and NTP is nucleoside triphosphate). Metabolic flux analysis of chemostat data with the wild type and a deletion mutant of the proton-pumping pyrophosphatase showed that a PP-generating mechanism must be present that operates according to ATP + P → ADP + PP Both organisms also produced significant amounts of amino acids in cellobiose-limited cultures. It was anticipated that this phenomenon would be suppressed by growth under nitrogen limitation. Surprisingly, nitrogen-limited chemostat cultivation of wild-type revealed a bottleneck in pyruvate oxidation, as large amounts of pyruvate and amino acids, mainly valine, were excreted; up to 50% of the nitrogen consumed was excreted again as amino acids. This study discusses the fate of pyrophosphate in the metabolism of two thermophilic anaerobes that lack a soluble irreversible pyrophosphatase as present in but instead use a reversible membrane-bound proton-pumping enzyme. In such organisms, the charging of tRNA with amino acids may become more reversible. This may contribute to the observed excretion of amino acids during sugar fermentation by and Calculation of the energetic advantage of reversible pyrophosphate-dependent glycolysis, as occurs in , could not be properly evaluated, as currently available genome-scale models neglect the anabolic generation of pyrophosphate in, for example, polymerization of amino acids to protein. This anabolic pyrophosphate replaces ATP and thus saves energy. Its amount is, however, too small to cover the pyrophosphate requirement of sugar catabolism in glycolysis. Consequently, pyrophosphate for catabolism is generated according to ATP + P → ADP + PP.
Topics: Bioreactors; Clostridium thermocellum; Diphosphates; Metabolic Flux Analysis; Nitrogen; Thermoanaerobacterium
PubMed: 32978139
DOI: 10.1128/AEM.01795-20 -
IP8: A quantitatively minor inositol pyrophosphate signaling molecule that punches above its weight.Advances in Biological Regulation Jan 2024The inositol pyrophosphates (PP-IPs) are specialized members of the wider inositol phosphate signaling family that possess functionally significant diphosphate groups.... (Review)
Review
The inositol pyrophosphates (PP-IPs) are specialized members of the wider inositol phosphate signaling family that possess functionally significant diphosphate groups. The PP-IPs exhibit remarkable functionally versatility throughout the eukaryotic kingdoms. However, a quantitatively minor PP-IP - 1,5 bisdiphosphoinositol tetrakisphosphate (1,5-IP) - has received considerably less attention from the cell signalling community. The main purpose of this review is to summarize recently-published data which have now brought 1,5-IP into the spotlight, by expanding insight into the molecular mechanisms by which this polyphosphate regulates many fundamental biological processes.
Topics: Humans; Diphosphates; Inositol Phosphates; Signal Transduction
PubMed: 38064879
DOI: 10.1016/j.jbior.2023.101002 -
Molecules (Basel, Switzerland) Dec 2021X-ray powder diffraction (XRPD) and thermal analysis (differential scanning calorimetry/derivative of thermogravimetry (DSC/DTG)) are solid-state techniques that can be...
X-ray powder diffraction (XRPD) and thermal analysis (differential scanning calorimetry/derivative of thermogravimetry (DSC/DTG)) are solid-state techniques that can be successfully used to identify and quantify various chemical compounds in polycrystalline mixtures, such as dietary supplements or drugs. In this work, 31 dietary supplements available on the Polish market that contain iron compounds, namely iron gluconate, fumarate, bisglycinate, citrate and pyrophosphate, were evaluated. The aim of the work was to identify iron compounds declared by the manufacturer as food supplements and to try to verify compliance with the manufacturer's claims. Studies performed by X-ray and thermal analysis confirmed that crystalline iron compounds (iron (II) gluconate, iron (II) fumarate), declared by the manufacturers, were present in the investigated dietary supplements. Iron (II) bisglycinate proved to be semi-crystalline. However, depending on the composition of the formulation, it was possible to identify this compound in the tested supplements. For amorphous iron compounds (iron (III) citrate and iron (III) pyrophosphate), the diffraction pattern does not have characteristic diffraction lines. Food supplements containing crystalline iron compounds have a melting point close to the melting point of pure iron compounds. The presence of excipients was found to affect the shapes and positions of the endothermic peaks significantly. Widening of endothermic peaks and changes in their position were observed, as well as exothermic peaks indicating crystallization of amorphous compounds. Weight loss was determined for all dietary supplements tested. Analysis of the DTG curves showed that the thermal decomposition of most food supplements takes place in several steps. The results obtained by a combination of both simple, relatively fast and reliable XRPD and DSC/DTG methods are helpful in determining phase composition, pharmaceutical abnormalities or by detecting the presence of the correct polymorphic form.
Topics: Calorimetry, Differential Scanning; Dietary Supplements; Diphosphates; Fumarates; Gluconates; Iron; Thermogravimetry; X-Ray Diffraction
PubMed: 35011434
DOI: 10.3390/molecules27010197 -
Journal of Experimental Botany Feb 2017Salvia divinorum (Lamiaceae) is an annual herb used by indigenous cultures of Mexico for medicinal and ritual purposes. The biosynthesis of salvinorin A, its major...
Salvia divinorum (Lamiaceae) is an annual herb used by indigenous cultures of Mexico for medicinal and ritual purposes. The biosynthesis of salvinorin A, its major bioactive neo-clerodane diterpenoid, remains virtually unknown. This investigation aimed to identify the enzyme that catalyzes the first reaction of salvinorin A biosynthesis, the formation of (-)-kolavenyl diphosphate [(-)-KPP], which is subsequently dephosphorylated to afford (-)-kolavenol. Peltate glandular trichomes were identified as the major and perhaps exclusive site of salvinorin accumulation in S. divinorum. The trichome-specific transcriptome was used to identify candidate diterpene synthases (diTPSs). In vitro and in planta characterization of a class II diTPS designated as SdKPS confirmed its activity as (-)-KPP synthase and its involvement in salvinorin A biosynthesis. Mutation of a phenylalanine into histidine in the active site of SdKPS completely converts the product from (-)-KPP into ent-copalyl diphosphate. Structural elements were identified that mediate the natural formation of the neo-clerodane backbone by this enzyme and suggest how SdKPS and other diTPSs may have evolved from ent-copalyl diphosphate synthase.
Topics: Diphosphates; Diterpenes, Clerodane; Plant Proteins; Salvia; Transcriptome
PubMed: 28204567
DOI: 10.1093/jxb/erw493 -
PeerJ 2024Pyrophosphatases (PPases) are enzymes that catalyze the hydrolysis of pyrophosphate (PPi), a byproduct of the synthesis and degradation of diverse biomolecules. The... (Review)
Review
Pyrophosphatases (PPases) are enzymes that catalyze the hydrolysis of pyrophosphate (PPi), a byproduct of the synthesis and degradation of diverse biomolecules. The accumulation of PPi in the cell can result in cell death. Although the substrate is the same, there are variations in the catalysis and features of these enzymes. Two enzyme forms have been identified in bacteria: cytoplasmic or soluble pyrophosphatases and membrane-bound pyrophosphatases, which play major roles in cell bioenergetics. In eukaryotic cells, cytoplasmic enzymes are the predominant form of PPases (c-PPases), while membrane enzymes (m-PPases) are found only in protists and plants. The study of bacterial cytoplasmic and membrane-bound pyrophosphatases has slowed in recent years. These enzymes are central to cell metabolism and physiology since phospholipid and nucleic acid synthesis release important amounts of PPi that must be removed to allow biosynthesis to continue. In this review, two aims were pursued: first, to provide insight into the structural features of PPases known to date and that are well characterized, and to provide examples of enzymes with novel features. Second, the scientific community should continue studying these enzymes because they have many biotechnological applications. Additionally, in this review, we provide evidence that there are m-PPases present in fungi; to date, no examples have been characterized. Therefore, the diversity of PPase enzymes is still a fruitful field of research. Additionally, we focused on the roles of H/Na pumps and m-PPases in cell bioenergetics. Finally, we provide some examples of the applications of these enzymes in molecular biology and biotechnology, especially in plants. This review is valuable for professionals in the biochemistry field of protein structure-function relationships and experts in other fields, such as chemistry, nanotechnology, and plant sciences.
Topics: Inorganic Pyrophosphatase; Bacteria; Fungi; Diphosphates
PubMed: 38938619
DOI: 10.7717/peerj.17496