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Protein Science : a Publication of the... Sep 2022Membrane-bound pyrophosphatase (mPPase) found in microbes and plants is a membrane H pump that transports the H ion generated in coupled pyrophosphate hydrolysis out of...
Membrane-bound pyrophosphatase (mPPase) found in microbes and plants is a membrane H pump that transports the H ion generated in coupled pyrophosphate hydrolysis out of the cytoplasm. Certain bacterial and archaeal mPPases can in parallel transport Na via a hypothetical "billiard-type" mechanism, also involving the hydrolysis-generated proton. Here, we present the functional evidence supporting this coupling mechanism. Rapid-quench and pulse-chase measurements with [ P]pyrophosphate indicated that the chemical step (pyrophosphate hydrolysis) is rate-limiting in mPPase catalysis and is preceded by a fast isomerization of the enzyme-substrate complex. Na , whose binding is a prerequisite for the hydrolysis step, is not required for substrate binding. Replacement of H O with D O decreased the rates of pyrophosphate hydrolysis by both Na - and H -transporting bacterial mPPases, the effect being more significant than with a non-transporting soluble pyrophosphatase. We also show that the Na -pumping mPPase of Thermotoga maritima resembles other dimeric mPPases in demonstrating negative kinetic cooperativity and the requirement for general acid catalysis. The findings point to a crucial role for the hydrolysis-generated proton both in H -pumping and Na -pumping by mPPases.
Topics: Diphosphates; Hydrolysis; Isotopes; Kinetics; Protons; Pyrophosphatases; Sodium; Solvents
PubMed: 36040263
DOI: 10.1002/pro.4394 -
International Journal of Molecular... Oct 2022Pyrophosphate (PP) is a byproduct of over 120 biosynthetic reactions, and an overabundance of PP can inhibit industrial synthesis. Pyrophosphatases (PPases) can...
Pyrophosphate (PP) is a byproduct of over 120 biosynthetic reactions, and an overabundance of PP can inhibit industrial synthesis. Pyrophosphatases (PPases) can effectively hydrolyze pyrophosphate to remove the inhibitory effect of pyrophosphate. In the present work, a thermophilic alkaline inorganic pyrophosphatase from NA1 was studied. The optimum pH and temperature of Ton1914 were 9.0 and 80 °C, respectively, and the half-life was 52 h at 70 °C and 2.5 h at 90 °C. Ton1914 showed excellent thermal stability, and its relative enzyme activity, when incubated in Tris-HCl 9.0 containing 1.6 mM Mg at 90 °C for 5 h, was still 100%, which was much higher than the control, whose relative activity was only 37%. Real-time quantitative PCR (qPCR) results showed that the promotion of Ton1914 on long-chain DNA was more efficient than that on short-chain DNA when the same concentration of templates was supplemented. The yield of long-chain products was increased by 32-41%, while that of short-chain DNA was only improved by 9.5-15%. Ton1914 also increased the yields of UDP-glucose and UDP-galactose enzymatic synthesis from 40.1% to 84.8% and 20.9% to 35.4%, respectively. These findings suggested that Ton1914 has considerable potential for industrial applications.
Topics: Thermococcus; Inorganic Pyrophosphatase; Diphosphates; Archaeal Proteins; Pyrophosphatases; Uridine Diphosphate
PubMed: 36361526
DOI: 10.3390/ijms232112735 -
Biochemistry Jul 2020Farnesyl diphosphate synthase (FPPS) is an isoprenoid chain elongation enzyme that catalyzes the sequential condensation of dimethylallyl diphosphate (C) with...
Farnesyl diphosphate synthase (FPPS) is an isoprenoid chain elongation enzyme that catalyzes the sequential condensation of dimethylallyl diphosphate (C) with isopentenyl diphosphate (IPP; C) and the resulting geranyl diphosphate (GPP; C) with another molecule of IPP, eventually producing farnesyl diphosphate (FPP; C), which is a precursor for the biosynthesis of a vast majority of isoprenoids. Previous studies of FPPS have highlighted the importance of the structure around the hydrophobic chain elongation path in determining product specificity. To investigate what structural features define the final chain length of the product in FPPS from , we designed and expressed six mutants of FPPS by replacing small amino acids around the binding pocket with bulky residues. Using enzymatic assays, binding kinetics, and crystallographic studies, we analyzed the effects of these mutations on the activity and product specificity of FPPS. Our results revealed that replacement of Thr-164 with tryptophan and phenylalanine completely abolished the activity of FPPS. Intriguingly, the T164Y substitution displayed dual product specificity and produced a mixture GPP and FPP as final products, with an activity for FPP synthesis that was lower than that of the wild-type enzyme. These data indicate that Thr-164 is a potential regulator of product specificity.
Topics: Binding Sites; Crystallography, X-Ray; Diphosphates; Diterpenes; Geranyltranstransferase; Hemiterpenes; Humans; Leishmania major; Leishmaniasis, Cutaneous; Models, Molecular; Organophosphorus Compounds; Polyisoprenyl Phosphates; Protein Conformation; Sesquiterpenes; Substrate Specificity
PubMed: 32584028
DOI: 10.1021/acs.biochem.0c00432 -
Biomolecules Jan 2024The inositol pyrophosphate pathway, a complex cell signaling network, plays a pivotal role in orchestrating vital cellular processes in the budding yeast, where it... (Review)
Review
The inositol pyrophosphate pathway, a complex cell signaling network, plays a pivotal role in orchestrating vital cellular processes in the budding yeast, where it regulates cell cycle progression, growth, endocytosis, exocytosis, apoptosis, telomere elongation, ribosome biogenesis, and stress responses. This pathway has gained significant attention in pharmacology and medicine due to its role in generating inositol pyrophosphates, which serve as crucial signaling molecules not only in yeast, but also in higher eukaryotes. As targets for therapeutic development, genetic modifications within this pathway hold promise for disease treatment strategies, offering practical applications in biotechnology. The model organism , renowned for its genetic tractability, has been instrumental in various studies related to the inositol pyrophosphate pathway. This review is focused on the Kcs1 and Vip1, the two enzymes involved in the biosynthesis of inositol pyrophosphate in , highlighting their roles in various cell processes, and providing an up-to-date overview of their relationship with phosphate homeostasis. Moreover, the review underscores the potential applications of these findings in the realms of medicine and biotechnology, highlighting the profound implications of comprehending this intricate signaling network.
Topics: Diphosphates; Inositol Phosphates; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Signal Transduction
PubMed: 38397389
DOI: 10.3390/biom14020152 -
International Journal of Molecular... Dec 2022Novel sulfur and selenium substituted 5',5'-linked dinucleoside pyrophate analogues were prepared in a vibration ball mill from the corresponding persilylated...
Novel sulfur and selenium substituted 5',5'-linked dinucleoside pyrophate analogues were prepared in a vibration ball mill from the corresponding persilylated monophosphate. The chemical hydrolysis of pyrophosphorochalcogenolate-linked dimers was studied over a wide pH-range. The effect of the chalcogeno-substitution on the reactivity of dinucleoside pyrophosphates was surprisingly modest, and the chemical stability is promising considering the potential therapeutic or diagnostic applications. The chemical stability of the precursor phosphorochalcogenolate monoesters was also investigated. Hydrolytic desilylation of these materials was effected in aqueous buffer at pH 3, 7 or 11 and resulted in phosphorus-chalcogen bond scission which was monitored using P NMR. The rate of dephosphorylation was dependent upon both the nature of the chalcogen and the pH. The integrity of the P-S bond in the corresponding phosphorothiolate was maintained at high pH but rapidly degraded at pH 3. In contrast, P-Se bond cleavage of the phosphoroselenolate monoester was rapid and the rate increased with alkalinity. The results obtained in kinetic experiments provide insight on the reactivity of the novel pyrophosphates studied as well as of other types of thiosubstituted biological phosphates. At the same time, these results also provide evidence for possible formation of unexpectedly reactive intermediates as the chalcogen-substituted analogues are metabolised.
Topics: Nucleosides; Phosphates; Hydrolysis; Diphosphates; Chalcogens
PubMed: 36555224
DOI: 10.3390/ijms232415582 -
Biological Chemistry Sep 2012Diphosphoinositol phosphates are a subclass of inositol phosphates possessing one or two high energy diphosphate groups instead of phosphoester substituents of the... (Review)
Review
Diphosphoinositol phosphates are a subclass of inositol phosphates possessing one or two high energy diphosphate groups instead of phosphoester substituents of the myo-inositol. Here we describe the enzymes responsible for their synthesis and degradation and how these may be regulated. Formation of diphosphoinositol phosphates in yeast and mammals is driven by an increase of the cellular energy charge, a lack of inorganic phosphate, and in mammals by osmotic or heat stress and in some cases by receptor mediated signaling. Known cellular actions are an improvement of the cell homeostasis by a reduction of the energy charge, increased phosphate uptake, improvement of mitochondrial performance, and an increase of insulin secretion in mammals. The underlying molecular mechanisms of action are far from being clarified but an increasing body of knowledge about molecular details has highlighted their complex participation in many cellular systems and metabolic processes.
Topics: Animals; Diphosphates; Homeostasis; Humans; Inositol Phosphates
PubMed: 22944697
DOI: 10.1515/hsz-2012-0133 -
Journal of Visualized Experiments : JoVE Aug 2013Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and...
Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and detect genetic mutations that confer resistance to anti-microbial agents. The advantages of pyrosequencing for microbiology applications include rapid and reliable high-throughput screening and accurate identification of microbes and microbial genome mutations. Pyrosequencing involves sequencing of DNA by synthesizing the complementary strand a single base at a time, while determining the specific nucleotide being incorporated during the synthesis reaction. The reaction occurs on immobilized single stranded template DNA where the four deoxyribonucleotides (dNTP) are added sequentially and the unincorporated dNTPs are enzymatically degraded before addition of the next dNTP to the synthesis reaction. Detection of the specific base incorporated into the template is monitored by generation of chemiluminescent signals. The order of dNTPs that produce the chemiluminescent signals determines the DNA sequence of the template. The real-time sequencing capability of pyrosequencing technology enables rapid microbial identification in a single assay. In addition, the pyrosequencing instrument, can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may occur in some anti-microbial resistance patterns.
Topics: Bacteria; Base Sequence; DNA, Bacterial; DNA-Directed DNA Polymerase; Diphosphates; Molecular Sequence Data; Nucleotides; Sequence Analysis, DNA; Sulfate Adenylyltransferase; Templates, Genetic
PubMed: 23995536
DOI: 10.3791/50405 -
Angewandte Chemie (International Ed. in... Oct 2022Two analogues of the diterpene precursor geranylgeranyl diphosphate with shifted double bonds, named iso-GGPP I and iso-GGPP II, were enzymatically converted with twelve...
Two analogues of the diterpene precursor geranylgeranyl diphosphate with shifted double bonds, named iso-GGPP I and iso-GGPP II, were enzymatically converted with twelve diterpene synthases from bacteria, fungi and protists. The changed reactivity in the substrate analogues resulted in the formation of 28 new diterpenes, many of which exhibit novel skeletons.
Topics: Polyisoprenyl Phosphates; Diterpenes; Diphosphates; Fungi; Alkyl and Aryl Transferases
PubMed: 36066489
DOI: 10.1002/anie.202211054 -
Molecules (Basel, Switzerland) Apr 2020A greenhouse pot trial was conducted to investigate the effect of organic amendments combined with triple superphosphate on the bioavailability of heavy metals (HMs...
A greenhouse pot trial was conducted to investigate the effect of organic amendments combined with triple superphosphate on the bioavailability of heavy metals (HMs growth and metal uptake from Pb-Zn mine tailings. Cattle manure compost (CMC), spent mushroom compost (SMC) and agricultural field soil (AFS) were applied to tailings at 5%, 10%, 20% and 30% / ratio, whereas sewage sludge (SS) and wood biochar (WB) were mixed at 2.5%, 5%, 10% and 20% / ratio. Triple superphosphate (TSP) was added to all the treatments at 4:1 (molar ratio). Amendments efficiently decreased DTPA-extracted Pb, Zn, Cd and Cu in treatments. Chlorophyll contents and shoot and root dry biomass significantly (< 0.05) increased in the treatments of CMC (except T4 for chlorophyll b) and SMC, whereas treatments of SS (except T1 for chlorophyll a and b), WB and AFS (except T4 for chlorophyll a and b) did not show positive effects as compared to CK1. Bioconcentration factor (BCF) and translocation factor (TF) values in plant tissues were below 1 for most treatments. In amended treatments, soluble protein content increased, phenylalanine ammonialyase (PAL) and polyphenol oxidase (PPO) decreased, and catalase (CAT) activity showed varied results as compared to CK1 and CK2. Results suggested that can be a potential metal phytostabilizer and use of CMC or SMC in combination with TSP are more effective than other combinations for the in situ stabilization of Pb-Zn mine tailings.
Topics: Biodegradation, Environmental; Biomass; Chemical Phenomena; Chlorophyll; Diphosphates; Fabaceae; Hydrogen-Ion Concentration; Lead; Metals, Heavy; Photochemical Processes; Zinc
PubMed: 32244753
DOI: 10.3390/molecules25071617 -
Journal of Nuclear Cardiology :... Oct 2022
Review
Radiopharmaceutical supply disruptions and the use of Tc-hydroxymethylene diphosphonate as an alternative to Tc-pyrophosphate for the diagnosis of transthyretin cardiac amyloidosis: An ASNC Information Statement.
Topics: Amyloidosis; Cardiomyopathies; Diphosphates; Diphosphonates; Humans; Prealbumin; Radiopharmaceuticals; Technetium Tc 99m Pyrophosphate
PubMed: 35838892
DOI: 10.1007/s12350-022-03059-5