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Structure (London, England : 1993) Dec 1996Soluble inorganic pyrophosphatase (PPase), an essential enzyme central to phosphorus metabolism, catalyzes the hydrolysis of the phosphoanhydride bond in inorganic...
BACKGROUND
Soluble inorganic pyrophosphatase (PPase), an essential enzyme central to phosphorus metabolism, catalyzes the hydrolysis of the phosphoanhydride bond in inorganic pyrophosphate. Catalysis requires divalent metal ions which affect the apparent pKas of the essential general acid and base on the enzyme, and the pKa of the substrate. Three to five metal ions are required for maximal activity, depending on pH and enzyme source. A detailed understanding of catalysis would aid both in understanding the nature of biological mechanisms of phosphoryl transfer, and in understanding the role of divalent cations. Without a high-resolution complex structure such a model has previously been unobtainable.
RESULTS
We report the first two high-resolution structures of yeast PPase, at 2.2 and 2.0 A resolution with R factors of around 17%. One structure contains the two activating metal ions; the other, the product (MnPi)2 as well. The latter structure shows an extensive network of hydrogen bond and metal ion interactions that account for virtually every lone pair on the product phosphates. It also contains a water molecule/hydroxide ion bridging two metal ions and, uniquely, a phosphate bound to four Mn2+ ions.
CONCLUSIONS
Our structure-based model of the PPase mechanism posits that the nucleophile is the hydroxide ion mentioned above. This aspect of the mechanism is formally analogous to the "two-metal ion' mechanism of alkaline phosphatase, exonucleases and polymerases. A third metal ion coordinates another water molecule that is probably the required general acid. Extensive Lewis acid coordination and hydrogen bonds provide charge shielding of the electrophile and lower the pKa of the leaving group. This "three-metal ion' mechanism is in detail different from that of other phosphoryl transfer enzymes, presumably reflecting how ancient the reaction is.
Topics: Alkaline Phosphatase; Binding Sites; Crystallography, X-Ray; DNA-Directed DNA Polymerase; Dimerization; Diphosphates; Hydrogen Bonding; Hydroxides; Inorganic Pyrophosphatase; Manganese; Models, Chemical; Models, Molecular; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Pyrophosphatases; Saccharomyces cerevisiae
PubMed: 8994974
DOI: 10.1016/s0969-2126(96)00155-4 -
The Journal of Biological Chemistry Jan 2003Here we report the isolation and characterization of a type I vacuolar-type H(+)-pyrophosphatase (V-PPase), TgVP1, from an apicomplexan, Toxoplasma gondii, a parasitic...
Isolation and characterization of TgVP1, a type I vacuolar H+-translocating pyrophosphatase from Toxoplasma gondii. The dynamics of its subcellular localization and the cellular effects of a diphosphonate inhibitor.
Here we report the isolation and characterization of a type I vacuolar-type H(+)-pyrophosphatase (V-PPase), TgVP1, from an apicomplexan, Toxoplasma gondii, a parasitic protist that is particularly amenable to molecular and genetic manipulation. The 816-amino acid TgVP1 polypeptide is 50% sequence-identical (65% similar) to the prototypical type I V-PPase from Arabidopsis thaliana, AVP1, and contains all the sequence motifs characteristic of this pump category. Unlike AVP1 and other known type I enzymes, however, TgVP1 contains a 74-residue N-terminal extension encompassing a 42-residue N-terminal signal peptide sequence, sufficient for targeting proteins to the secretory pathway of T. gondii. Providing that the coding sequence for the entire N-terminal extension is omitted from the plasmid, transformation of Saccharomyces cerevisiae with plasmid-borne TgVP1 yields a stable and functional translation product that is competent in aminomethylenediphosphonate (AMDP)-inhibitable K(+)-activated pyrophosphate (PP(i)) hydrolysis and PP(i)-energized H(+) translocation. Immunofluorescence microscopy of both free and intracellular T. gondii tachyzoites using purified universal V-PPase polyclonal antibodies reveals a punctate apical distribution for the enzyme. Equivalent studies of the tachyzoites during host cell invasion, by contrast, disclose a transverse radial distribution in which the V-PPase is associated with a collar-like structure that migrates along the length of the parasite in synchrony with and in close apposition to the penetration furrow. Although treatment of T. gondii with AMDP concentrations as high as 100 microm had no discernible effect on the efficiency of host cell invasion and integration, concentrations commensurate with the I(50) for the inhibition of TgVP1 activity in vitro (0.9 microm) do inhibit cell division and elicit nuclear enlargement concomitant with the inflation and eventual disintegration of acidocalcisome-like vesicular structures. A dynamic association of TgVP1 with the host cell invasion apparatus is invoked, one in which the effects of inhibitory V-PPase substrate analogs are exerted after rather than during host cell invasion.
Topics: Amino Acid Sequence; Animals; Calcium; Diphosphonates; Dose-Response Relationship, Drug; Enzyme Inhibitors; Ion Transport; Molecular Sequence Data; Protons; Protozoan Proteins; Pyrophosphatases; Saccharomyces cerevisiae; Toxoplasma; Vacuoles
PubMed: 12411435
DOI: 10.1074/jbc.M209436200 -
Fukuoka Igaku Zasshi = Hukuoka Acta... Nov 2011
Review
Topics: Adenosine Diphosphate Ribose; Guanine; Humans; Hydrolysis; Mutation; Pyrophosphatases; Substrate Specificity
PubMed: 22351996
DOI: No ID Found -
Clinical and Translational Science Jan 2021Enzyme replacement with ectonucleotide pyrophosphatase phospodiesterase-1 (ENPP1) eliminates mortality in a murine model of the lethal calcification disorder generalized...
Enzyme replacement with ectonucleotide pyrophosphatase phospodiesterase-1 (ENPP1) eliminates mortality in a murine model of the lethal calcification disorder generalized arterial calcification of infancy. We used protein engineering, glycan optimization, and a novel biomanufacturing platform to enhance potency by using a three-prong strategy. First, we added new N-glycans to ENPP1; second, we optimized pH-dependent cellular recycling by protein engineering of the Fc neonatal receptor; finally, we used a two-step process to improve sialylation by first producing ENPP1-Fc in cells stably transfected with human α-2,6-sialyltransferase (ST6) and further enhanced terminal sialylation by supplementing production with 1,3,4-O-Bu ManNAc. These steps sequentially increased the half-life of the parent compound in rodents from 37 hours to ~ 67 hours with an added N-glycan, to ~ 96 hours with optimized pH-dependent Fc recycling, to ~ 204 hours when the therapeutic was produced in ST6-overexpressing cells with 1,3,4-O-Bu ManNAc supplementation. The alterations were demonstrated to increase drug potency by maintaining efficacious levels of plasma phosphoanhydride pyrophosphate in ENPP1-deficient mice when the optimized biologic was administered at a 10-fold lower mass dose less frequently than the parent compound-once every 10 days vs. 3 times a week. We believe these improvements represent a general strategy to rationally optimize protein therapeutics.
Topics: Animals; Area Under Curve; Disease Models, Animal; Enzyme Replacement Therapy; Glycosylation; Half-Life; Histocompatibility Antigens Class I; Humans; Male; Mice, Transgenic; Phosphoric Diester Hydrolases; Protein Engineering; Protein Structure, Tertiary; Pyrophosphatases; Receptors, Fc; Recombinant Fusion Proteins; Vascular Calcification
PubMed: 33064927
DOI: 10.1111/cts.12887 -
Acta Biochimica Polonica 2015Bacteria living in marine environment encounter various challenges and limitations, thus in order to survive, they need to employ efficient stress-response mechanisms....
Bacteria living in marine environment encounter various challenges and limitations, thus in order to survive, they need to employ efficient stress-response mechanisms. One of these mechanisms is the stringent response, where unusual nucleotides, guanosine tetra- and pentaphosphates, herald starvation and physico-chemical stresses. All so far sequenced free-living bacteria contain the gene(s) responsible for (p)ppGpp synthesis - rsh (named after Escherichia coli genes, relA and spoT). Two similar genes were identified mostly in β- and γ-proteobacteria while other bacteria have only one gene coding the dual function of (p)ppGpp synthesis and degradation. Although the presence of (p)ppGpp-mediated response to the stress conditions has been shown for a few, and predicted for some other marine microorganisms, the (p)ppGpp effects may vary among different organisms. Thus, in this work we asked whether marine bacteria could have evolved a genetic adaptation specifically suited to adapt to environment with limited resources. The phylogenetic analyses of SpoT, RelA and RSH proteins from organisms associated with marine environment showed, however, that the evolutionary correlations obtained for these proteins are congruent with those constructed for 16S rRNA sequences and reflect taxonomical relationships of these organisms. Likewise, the similarity of specific amino acid residues indispensable for catalytic activity of these enzymes is very high, and any observed changes parallel with the taxonomical and evolutionary relationships. However, potential homologs of Mesh1 enzyme (metazoan SpoT homologs) that occur in both eukaryotic and prokaryotic organisms and contain the hydrolytic domain orthologous to SpoT were identified in Cellulophaga, Erythrobacter and Flavobacterium genera for the first time, as well as in soil bacterium Cytophaga hutchinsonii and freshwater Rhodothermus marinus.
Topics: Adaptation, Physiological; Amino Acid Sequence; Bacteria; Genes, Bacterial; Marine Biology; Molecular Sequence Data; Phylogeny; Pyrophosphatases; Sequence Homology, Amino Acid; Water Microbiology
PubMed: 26641635
DOI: 10.18388/abp.2015_1132 -
Nature Structural & Molecular Biology Feb 2011Autotaxin (ATX, also known as ectonucleotide pyrophosphatase/phosphodiesterase-2, ENPP2) is a secreted lysophospholipase D that generates the lipid mediator...
Autotaxin (ATX, also known as ectonucleotide pyrophosphatase/phosphodiesterase-2, ENPP2) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA), a mitogen and chemoattractant for many cell types. ATX-LPA signaling is involved in various pathologies including tumor progression and inflammation. However, the molecular basis of substrate recognition and catalysis by ATX and the mechanism by which it interacts with target cells are unclear. Here, we present the crystal structure of ATX, alone and in complex with a small-molecule inhibitor. We have identified a hydrophobic lipid-binding pocket and mapped key residues for catalysis and selection between nucleotide and phospholipid substrates. We have shown that ATX interacts with cell-surface integrins through its N-terminal somatomedin B-like domains, using an atypical mechanism. Our results define determinants of substrate discrimination by the ENPP family, suggest how ATX promotes localized LPA signaling and suggest new approaches for targeting ATX with small-molecule therapeutic agents.
Topics: Amino Acid Sequence; Animals; Binding Sites; Catalytic Domain; Cell Line; Crystallography, X-Ray; Humans; Integrins; Lysophospholipids; Molecular Sequence Data; Mutation; Phosphoric Diester Hydrolases; Protein Binding; Protein Structure, Tertiary; Pyrophosphatases; Rats; Substrate Specificity
PubMed: 21240271
DOI: 10.1038/nsmb.1980 -
Nature Communications Nov 2017The NUDIX enzymes are involved in cellular metabolism and homeostasis, as well as mRNA processing. Although highly conserved throughout all organisms, their biological...
The NUDIX enzymes are involved in cellular metabolism and homeostasis, as well as mRNA processing. Although highly conserved throughout all organisms, their biological roles and biochemical redundancies remain largely unclear. To address this, we globally resolve their individual properties and inter-relationships. We purify 18 of the human NUDIX proteins and screen 52 substrates, providing a substrate redundancy map. Using crystal structures, we generate sequence alignment analyses revealing four major structural classes. To a certain extent, their substrate preference redundancies correlate with structural classes, thus linking structure and activity relationships. To elucidate interdependence among the NUDIX hydrolases, we pairwise deplete them generating an epistatic interaction map, evaluate cell cycle perturbations upon knockdown in normal and cancer cells, and analyse their protein and mRNA expression in normal and cancer tissues. Using a novel FUSION algorithm, we integrate all data creating a comprehensive NUDIX enzyme profile map, which will prove fundamental to understanding their biological functionality.
Topics: A549 Cells; Cell Line; Cell Line, Tumor; Gene Expression Profiling; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Humans; MCF-7 Cells; Multigene Family; Phylogeny; Pyrophosphatases; RNA Interference; Substrate Specificity; Nudix Hydrolases
PubMed: 29142246
DOI: 10.1038/s41467-017-01642-w -
Analytical Biochemistry Apr 2020Nudix proteins are members of a large family of homologous enzymes that hydrolyze nucleoside diphosphates linked to other compounds. The substrates for a subset of Nudix...
Nudix proteins are members of a large family of homologous enzymes that hydrolyze nucleoside diphosphates linked to other compounds. The substrates for a subset of Nudix enzymes are all nucleotides linked to RNA, like the mG mRNA caps and the more recently discovered NAD(H) RNA caps. However, the RNA affinity and nucleic acid specificity of Nudix proteins has not yet been explored in depth. In this study we designed new fluorescence-based assays to examine the interaction of purified recombinant E. coli NudC and human Nudt1 (aka MTH1) Nudt3, Nudt12, Nudt16, and Nudt20 (aka Dcp2). All Nudix proteins except Nudt1 and Nudt12 bound both RNA and DNA stoichiometrically with high affinity (dissociation constants in the nanomolar range) and no clear sequence specificity. In stark contrast, Nudt12 binds RNA but not similar DNA oligonucleotides. Nudt12 also bound RNAs with 5' NAD caps more tightly than those with NADH or mG cap. NudC was similarly selective against mG caps but did not differentiate between NAD and NADH capped RNA. Nudt3, Nudt16, and Nudt20 bound mG capped RNA more tightly than RNA with NADH caps.
Topics: Binding Sites; DNA; DNA Repair Enzymes; Escherichia coli; Fluorescent Dyes; Humans; Phosphoric Monoester Hydrolases; Pyrophosphatases; RNA; Recombinant Proteins; Nudix Hydrolases
PubMed: 32059949
DOI: 10.1016/j.ab.2020.113622 -
The Biochemical Journal Jan 19671. The inorganic-pyrophosphatase activity of alkaline phosphatases prepared from human liver and small intestine was investigated at different stages of purification. 2....
1. The inorganic-pyrophosphatase activity of alkaline phosphatases prepared from human liver and small intestine was investigated at different stages of purification. 2. Both liver and intestinal preparations possessed pyrophosphatase activity at all stages of purification, and the two types of activity were not separated by gel filtration or by anion-exchange or cation-exchange chromatography. 3. After starch-gel electrophoresis of the tissue extracts, the zones of pyrophosphatase activity coincided exactly with alkaline-phosphatase zones. 4. Hydrolysis of each type of substrate was inhibited by the presence of the other, and a constant ratio of alkaline-phosphatase activity to pyrophosphatase activity was maintained during inactivation of the enzymes by incubation at 55 degrees . 5. These results are consistent with the view that alkaline phosphatases are also inorganic pyrophosphatases.
Topics: Alkaline Phosphatase; Chromatography, Gel; Chromatography, Ion Exchange; Electrophoresis; Humans; Hydrogen-Ion Concentration; In Vitro Techniques; Intestine, Small; Liver; Pyrophosphatases
PubMed: 6030299
DOI: 10.1042/bj1020053 -
Science Advances May 2019Membrane-bound pyrophosphatases are homodimeric integral membrane proteins that hydrolyze pyrophosphate into orthophosphates, coupled to the active transport of protons...
Membrane-bound pyrophosphatases are homodimeric integral membrane proteins that hydrolyze pyrophosphate into orthophosphates, coupled to the active transport of protons or sodium ions across membranes. They are important in the life cycle of bacteria, archaea, plants, and parasitic protists, but no homologous proteins exist in vertebrates, making them a promising drug target. Here, we report the first nonphosphorus allosteric inhibitor of the thermophilic bacterium membrane-bound pyrophosphatase and its bound structure together with the substrate analog imidodiphosphate. The unit cell contains two protein homodimers, each binding a single inhibitor dimer near the exit channel, creating a hydrophobic clamp that inhibits the movement of β-strand 1-2 during pumping, and thus prevents the hydrophobic gate from opening. This asymmetry of inhibitor binding with respect to each homodimer provides the first clear structural demonstration of asymmetry in the catalytic cycle of membrane-bound pyrophosphatases.
Topics: Algorithms; Allosteric Site; Anti-Bacterial Agents; Bacterial Proteins; Binding Sites; Catalysis; Cell Membrane; Enzyme Inhibitors; Hydrolysis; Ions; Kinetics; Membrane Proteins; Models, Molecular; Protein Conformation; Protein Multimerization; Pyrophosphatases; Saccharomyces cerevisiae; Sodium; Thermotoga maritima
PubMed: 31131322
DOI: 10.1126/sciadv.aav7574