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Biochemistry Feb 2021DNA polymerases play vital roles in the maintenance and replication of genomic DNA by synthesizing new nucleotide polymers using nucleoside triphosphates as substrates....
DNA polymerases play vital roles in the maintenance and replication of genomic DNA by synthesizing new nucleotide polymers using nucleoside triphosphates as substrates. Deoxynucleoside triphosphates (dNTPs) are the canonical substrates for DNA polymerases; however, some bacterial polymerases have been demonstrated to insert deoxynucleoside diphosphates (dNDPs), which lack a third phosphate group, the γ-phosphate. Whether eukaryotic polymerases can efficiently incorporate dNDPs has not been investigated, and much about the chemical or structural role played by the γ-phosphate of dNTPs remains unknown. Using the model mammalian polymerase (Pol) β, we examine how Pol β incorporates a substrate lacking a γ-phosphate [deoxyguanosine diphosphate (dGDP)] utilizing kinetic and crystallographic approaches. Using single-turnover kinetics, we determined dGDP insertion across a templating dC by Pol β to be drastically impaired when compared to dGTP insertion. We found the most significant impairment in the apparent insertion rate (), which was reduced 32000-fold compared to that of dGTP insertion. X-ray crystal structures revealed similar enzyme-substrate contacts for both dGDP and dGTP. These findings suggest the insertion efficiency of dGDP is greatly decreased due to impairments in polymerase chemistry. This work is the first instance of a mammalian polymerase inserting a diphosphate nucleotide and provides insight into the nature of polymerase mechanisms by highlighting how these enzymes have evolved to use triphosphate nucleotide substrates.
Topics: DNA; DNA Polymerase beta; DNA-Directed DNA Polymerase; Deoxyguanine Nucleotides; Deoxyguanosine; Diphosphates; Humans; Kinetics; Substrate Specificity
PubMed: 33475337
DOI: 10.1021/acs.biochem.0c00847 -
Molecules (Basel, Switzerland) May 2018Betulin-3,28-diphosphate (BDP) obtained by phosphorylation of betulin using POCl₃ has two main structural forms-BDP-1 and BDP-2-which differ in ethanol solubility,...
Betulin-3,28-diphosphate (BDP) obtained by phosphorylation of betulin using POCl₃ has two main structural forms-BDP-1 and BDP-2-which differ in ethanol solubility, melting point, FTIR spectra, thermoanalytical characteristics and biological activity. Betulin-3,28-diphosphate and its sodium salt (Na-BDP) were characterized using C and P-NMR spectra, powder XRD experiments, as well as differential scanning calorimetry (DSC) and thermogravimetric analysis (TG) methods. The exo-effects at 193 ± 8 °C for ethanol soluble BDP-1 samples (-19.7 ± 0.2 kJ∙mol) were about three times less than for ethanol insoluble BDP-2 samples f (-70.5 ± 0.7 kJ∙mol). The DSC curves of Na-BDP-1 and Na-BDP-2 characterized the endo-effects having a maximum at 95⁻112 °C. Water-soluble Na-BDP-1 was obtained as needle-like crystals, unlike poorly crystalline Na-BDP-2, whereas BDP-1 and BDP-2 aged with time and were isolated as amorphous substances. In vitro experiments on rats showed that compared to the control, Na-BDP-1 increased catalase and SOD activity and improved energy metabolism more effectively than Na-BDP-2.
Topics: Calorimetry, Differential Scanning; Diphosphates; Magnetic Resonance Spectroscopy; Molecular Structure; Solubility; Spectroscopy, Fourier Transform Infrared; Triterpenes; Water; X-Ray Diffraction
PubMed: 29757999
DOI: 10.3390/molecules23051175 -
Chemical Senses Jun 2017Rodents consume solutions of phosphates and pyrophosphates in preference to water. Recently, we found that the preference for trisodium pyrophosphate (Na3HP2O7) was...
Rodents consume solutions of phosphates and pyrophosphates in preference to water. Recently, we found that the preference for trisodium pyrophosphate (Na3HP2O7) was greater in T1R3 knockout (KO) mice than wild-type (WT) controls, suggesting that T1R3 is a pyrophosphate detector. We now show that this heightened Na3HP2O7 preference of T1R3 KO mice extends to disodium phosphate (Na2HPO4), disodium and tetrasodium pyrophosphate (Na2H2PO4 and Na4H2PO4), a tripolyphosphate (Na5P3O10), a non-sodium phosphate [(NH4)2HPO4], and a non-sodium pyrophosphate (K4P2O7) but not to non-P salts with large anions (sodium gluconate, acetate, or propionate). Licking rates for Na3HP2O7 are higher in T1R2 KO mice than WT controls; Na3HP2O7 preference scores are increased even more in T1R2 KO mice and T1R2+T1R3 double KO mice than in T1R3 KO mice; preference scores for Na3HP2O7 are normal in T1R1 KO mice. These results implicate each subunit of the T1R2+T1R3 dimer in the behavioral response to P-containing taste compounds.
Topics: Animals; Diphosphates; Food Preferences; Mice; Mice, Inbred C57BL; Mice, Knockout; Receptors, G-Protein-Coupled; Taste
PubMed: 28383662
DOI: 10.1093/chemse/bjx019 -
PLoS Computational Biology Oct 2022Membrane-integral pyrophosphatases (mPPases) are membrane-bound enzymes responsible for hydrolysing inorganic pyrophosphate and translocating a cation across the...
Membrane-integral pyrophosphatases (mPPases) are membrane-bound enzymes responsible for hydrolysing inorganic pyrophosphate and translocating a cation across the membrane. Their function is essential for the infectivity of clinically relevant protozoan parasites and plant maturation. Recent developments have indicated that their mechanism is more complicated than previously thought and that the membrane environment may be important for their function. In this work, we use multiscale molecular dynamics simulations to demonstrate for the first time that mPPases form specific anionic lipid interactions at 4 sites at the distal and interfacial regions of the protein. These interactions are conserved in simulations of the mPPases from Thermotoga maritima, Vigna radiata and Clostridium leptum and characterised by interactions with positive residues on helices 1, 2, 3 and 4 for the distal site, or 9, 10, 13 and 14 for the interfacial site. Due to the importance of these helices in protein stability and function, these lipid interactions may play a crucial role in the mPPase mechanism and enable future structural and functional studies.
Topics: Cations; Cell Membrane; Diphosphates; Lipids; Pyrophosphatases
PubMed: 36191052
DOI: 10.1371/journal.pcbi.1010578 -
Nature Communications Apr 2024Ribose-5-phosphate (R5P) is a precursor for nucleic acid biogenesis; however, the importance and homeostasis of R5P in the intracellular parasite Toxoplasma gondii...
Ribose-5-phosphate (R5P) is a precursor for nucleic acid biogenesis; however, the importance and homeostasis of R5P in the intracellular parasite Toxoplasma gondii remain enigmatic. Here, we show that the cytoplasmic sedoheptulose-1,7-bisphosphatase (SBPase) is dispensable. Still, its co-deletion with transaldolase (TAL) impairs the double mutant's growth and increases C-glucose-derived flux into pentose sugars via the transketolase (TKT) enzyme. Deletion of the latter protein affects the parasite's fitness but is not lethal and is correlated with an increased carbon flux via the oxidative pentose phosphate pathway. Further, loss of TKT leads to a decline in C incorporation into glycolysis and the TCA cycle, resulting in a decrease in ATP levels and the inability of phosphoribosyl-pyrophosphate synthetase (PRPS) to convert R5P into 5'-phosphoribosyl-pyrophosphate and thereby contribute to the production of AMP and IMP. Likewise, PRPS is essential for the lytic cycle. Not least, we show that RuPE-mediated metabolic compensation is imperative for the survival of the ΔsbpaseΔtal strain. In conclusion, we demonstrate that multiple routes can flexibly supply R5P to enable parasite growth and identify catalysis by TKT and PRPS as critical enzymatic steps. Our work provides novel biological and therapeutic insights into the network design principles of intracellular parasitism in a clinically-relevant pathogen.
Topics: Toxoplasma; Diphosphates; Ribosemonophosphates; Glycolysis; Pentose Phosphate Pathway
PubMed: 38589375
DOI: 10.1038/s41467-024-47097-8 -
Biometals : An International Journal on... Dec 2018Iron deficiency is a significant health problem across the world. While many patients benefit from oral iron supplements, some, including those on hemodialysis require...
Iron deficiency is a significant health problem across the world. While many patients benefit from oral iron supplements, some, including those on hemodialysis require intravenous iron therapy to maintain adequate iron levels. Until recently, all iron compounds suitable for parenteral administration were colloidal iron-carbohydrate conjugates that require uptake and processing by macrophages. These compounds are associated with variable risk of anaphylaxis, oxidative stress, and inflammation, depending on their physicochemical characteristics. Ferric pyrophosphate citrate (FPC) is a novel iron compound that was approved for parenteral administration by US Food and Drug Administration in 2015. Here we report the physicochemical characteristics of FPC. FPC is a noncolloidal, highly water soluble, complex iron salt that does not contain a carbohydrate moiety. X-ray absorption spectroscopy data indicate that FPC consists of iron (III) complexed with one pyrophosphate and two citrate molecules in the solid state. This structure is preserved in solution and stable for several months, rendering it suitable for pharmaceutical applications in solid or solution state.
Topics: Chemistry, Physical; Citric Acid; Diphosphates; Iron; Molecular Structure
PubMed: 30324285
DOI: 10.1007/s10534-018-0151-1 -
Microbial Cell Factories Oct 2022Linalool is a monoterpenoid, also a vital silvichemical with commercial applications in cosmetics, flavoring ingredients, and medicines. Regulation of mevalonate (MVA)...
BACKGROUND
Linalool is a monoterpenoid, also a vital silvichemical with commercial applications in cosmetics, flavoring ingredients, and medicines. Regulation of mevalonate (MVA) pathway metabolic flux is a common strategy to engineer Saccharomyces cerevisiae for efficient linalool production. However, metabolic regulation of the MVA pathway is complex and involves competition for central carbon metabolism, resulting in limited contents of target metabolites.
RESULTS
In this study, first, a truncated linalool synthase (t26AaLS1) from Actinidia arguta was selected for the production of linalool in S. cerevisiae. To simplify the complexity of the metabolic regulation of the MVA pathway and increase the flux of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), we introduced the two-step isopentenyl utilization pathway (IUP) into S. cerevisiae, which could produce large amounts of IPP/DMAPP. Further, the S. cerevisiae IDI1 (ecoding isopentenyl diphosphate delta-isomerase) and ERG20 (encoding farnesyl diphosphate synthase) genes were integrated into the yeast genome, combined with the strategies of copy number variation of the t26AaLS1 and ERG20 genes to increase the metabolic flux of the downstream IPP, as well as optimization of isoprenol and prenol concentrations, resulting in a 4.8-fold increase in the linalool titer. Eventually, under the optimization of carbon sources and Mg addition, a maximum linalool titer of 142.88 mg/L was obtained in the two-phase extractive shake flask fermentation.
CONCLUSIONS
The results show that the efficient synthesis of linalool in S. cerevisiae could be achieved through a two-step pathway, gene expression adjustment, and optimization of culture conditions. The study may provide a valuable reference for the other monoterpenoid production in S. cerevisiae.
Topics: Acyclic Monoterpenes; Carbon; DNA Copy Number Variations; Diphosphates; Geranyltranstransferase; Hemiterpenes; Metabolic Engineering; Mevalonic Acid; Monoterpenes; Organophosphorus Compounds; Pentanols; Saccharomyces cerevisiae
PubMed: 36243714
DOI: 10.1186/s12934-022-01934-x -
Proceedings of the National Academy of... Apr 2020Inositol diphosphates (PP-IPs), also known as inositol pyrophosphates, are high-energy cellular signaling codes involved in nutrient and regulatory responses. We report...
Inositol diphosphates (PP-IPs), also known as inositol pyrophosphates, are high-energy cellular signaling codes involved in nutrient and regulatory responses. We report that the evolutionarily conserved gene product, Vip1, possesses autonomous kinase and pyrophosphatase domains capable of synthesis and destruction of D-1 PP-IPs. Our studies provide atomic-resolution structures of the PP-IP products and unequivocally define that the Vip1 gene product is a highly selective 1-kinase and 1-pyrophosphatase enzyme whose activities arise through distinct active sites. Kinetic analyses of kinase and pyrophosphatase parameters are consistent with Vip1 evolving to modulate levels of 1-IP and 1,5-IP Individual perturbations in kinase and pyrophosphatase activities in cells result in differential effects on vacuolar morphology and osmotic responses. Analogous to the dual-functional key energy metabolism regulator, phosphofructokinase 2, Vip1 is a kinase and pyrophosphatase switch whose 1-PP-IP products play an important role in a cellular adaptation.
Topics: Diphosphates; Inositol Phosphates; Kinetics; Phosphorylation; Phosphotransferases (Phosphate Group Acceptor); Pyrophosphatases; Saccharomyces cerevisiae; Signal Transduction
PubMed: 32303658
DOI: 10.1073/pnas.1908875117 -
Molecules (Basel, Switzerland) Oct 2020Inositol pyrophosphates (PP-InsPs) comprise an important group of intracellular, diffusible cellular signals that a wide range of biological processes throughout the... (Review)
Review
Inositol pyrophosphates (PP-InsPs) comprise an important group of intracellular, diffusible cellular signals that a wide range of biological processes throughout the yeast, plant, and animal kingdoms. It has been difficult to gain a molecular-level mechanistic understanding of the actions of these molecules, due to their highly phosphorylated nature, their low levels, and their rapid metabolic turnover. More recently, these obstacles to success are being surmounted by the chemical synthesis of a number of insightful PP-InsP analogs. This review will describe these analogs and will indicate the important chemical and biological information gained by using them.
Topics: Animals; Diphosphates; Humans; Inositol; Metabolic Networks and Pathways; Phosphorylation; Protein Domains; Protein Stability
PubMed: 33023101
DOI: 10.3390/molecules25194515 -
Nucleic Acids Research Jan 2024Enzymatic methods to quantify deoxyribonucleoside triphosphates have existed for decades. In contrast, no general enzymatic method to quantify ribonucleoside...
Enzymatic methods to quantify deoxyribonucleoside triphosphates have existed for decades. In contrast, no general enzymatic method to quantify ribonucleoside triphosphates (rNTPs), which drive almost all cellular processes and serve as precursors of RNA, exists to date. ATP can be measured with an enzymatic luminometric method employing firefly luciferase, but the quantification of other ribonucleoside mono-, di-, and triphosphates is still a challenge for a non-specialized laboratory and practically impossible without chromatography equipment. To allow feasible quantification of ribonucleoside phosphates in any laboratory with typical molecular biology and biochemistry tools, we developed a robust microplate assay based on real-time detection of the Broccoli RNA aptamer during in vitro transcription. The assay employs the bacteriophage T7 and SP6 RNA polymerases, two oligonucleotide templates encoding the 49-nucleotide Broccoli aptamer, and a high-affinity fluorogenic aptamer-binding dye to quantify each of the four canonical rNTPs. The inclusion of nucleoside mono- and diphosphate kinases in the assay reactions enabled the quantification of the mono- and diphosphate counterparts. The assay is inherently specific and tolerates concentrated tissue and cell extracts. In summary, we describe the first chromatography-free method to quantify ATP, ADP, AMP, GTP, GDP, GMP, UTP, UDP, UMP, CTP, CDP and CMP in biological samples.
Topics: Diphosphates; Nucleotides; Ribonucleotides; Biochemistry
PubMed: 38008466
DOI: 10.1093/nar/gkad1091