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European Journal of Biochemistry Jul 2004We report here that Escherichia coli pyrophosphatase aggregates in the presence of millimolar Cd(2+). This highly cooperative process was specific to both the metal ion...
We report here that Escherichia coli pyrophosphatase aggregates in the presence of millimolar Cd(2+). This highly cooperative process was specific to both the metal ion and the protein and could be reversed fully by decreasing the Cd(2+) concentration. Aggregation was enhanced by Mg(2+), the natural cofactor of pyrophosphatase, and Mn(2+). Mutations at the intersubunit metal-binding site had no effect, whereas mutation at Glu139, which is part of the peripheral metal-binding site found in pyrophosphatase crystals near the contact region between two enzyme molecules, suppressed aggregation. These findings indicate that aggregation is affected by Cd(2+) binding to the peripheral metal-binding site, probably by strengthening intermolecular Trp149-Trp149' stacking interactions.
Topics: Binding Sites; Cadmium; Escherichia coli; Escherichia coli Proteins; Magnesium; Manganese; Models, Molecular; Protein Structure, Secondary; Pyrophosphatases
PubMed: 15233803
DOI: 10.1111/j.1432-1033.2004.04239.x -
International Journal of Molecular... Sep 2019All living things have pyrophosphatases that hydrolyze pyrophosphate and release energy. This energetically favorable reaction drives many energetically unfavorable...
All living things have pyrophosphatases that hydrolyze pyrophosphate and release energy. This energetically favorable reaction drives many energetically unfavorable reactions. An accepted catalytic model of pyrophosphatase shows that a water molecule activated by two divalent cations (M1 and M2) within the catalytic center can attack pyrophosphate in an S2 mechanism and thus hydrolyze the molecule. However, our co-crystal structure of pyrophosphatase with pyrophosphate shows that a water molecule from the solvent may, in fact, be the actual catalytic water. In the co-crystal structure of the wild-type pyrophosphatase with pyrophosphate, the electron density of the catalytic centers of each monomer are different from one another. This indicates that pyrophosphates in the catalytic center are dynamic. Our mass spectroscopy results have identified a highly conserved lysine residue (Lys30) in the catalytic center that is phosphorylated, indicating that the enzyme could form a phosphoryl enzyme intermediate during hydrolysis. Mutation of Lys30 to Arg abolished the activity of the enzyme. In the structure of the apo wild type enzyme, we observed that a Na ion is coordinated by residues within a loop proximal to the catalytic center. Therefore, we mutated three key residues within the loop (K143R, P147G, and K149R) and determined Na and K-induced inhibition on their activities. Compared to the wild type enzyme, P147G is most sensitive to these cations, whereas K143R was inactive and K149R showed no change in activity. These data indicate that monovalent cations could play a role in down-regulating pyrophosphatase activity in vivo. Overall, our results reveal new aspects of pyrophosphatase catalysis and could assist in the design of specific inhibitors of growth.
Topics: Acinetobacter baumannii; Amino Acid Sequence; Binding Sites; Catalysis; Catalytic Domain; Diphosphates; Enzyme Activation; Hydrolysis; Models, Molecular; Peptides; Phosphorylation; Protein Binding; Protein Conformation; Pyrophosphatases; Structure-Activity Relationship
PubMed: 31500178
DOI: 10.3390/ijms20184394 -
International Journal of Molecular... May 2024Regulatory cystathionine β-synthase (CBS) domains are widespread in proteins; however, difficulty in structure determination prevents a comprehensive understanding of...
Regulatory cystathionine β-synthase (CBS) domains are widespread in proteins; however, difficulty in structure determination prevents a comprehensive understanding of the underlying regulation mechanism. Tetrameric microbial inorganic pyrophosphatase containing such domains (CBS-PPase) is allosterically inhibited by AMP and ADP and activated by ATP and cell alarmones diadenosine polyphosphates. Each CBS-PPase subunit contains a pair of CBS domains but binds cooperatively to only one molecule of the mono-adenosine derivatives. We used site-directed mutagenesis of CBS-PPase to identify the key elements determining the direction of the effect (activation or inhibition) and the "half-of-the-sites" ligand binding stoichiometry. Seven amino acid residues were selected in the CBS1 domain, based on the available X-ray structure of the regulatory domains, and substituted by alanine and other residues. The interaction of 11 CBS-PPase variants with the regulating ligands was characterized by activity measurements and isothermal titration calorimetry. Lys100 replacement reversed the effect of ADP from inhibition to activation, whereas Lys95 and Gly118 replacements made ADP an activator at low concentrations but an inhibitor at high concentrations. Replacement of these residues for alanine increased the stoichiometry of mono-adenosine phosphate binding by twofold. These findings identified several key protein residues and suggested a "two non-interacting pairs of interacting regulatory sites" concept in CBS-PPase regulation.
Topics: Cystathionine beta-Synthase; Mutation; Protein Binding; Mutagenesis, Site-Directed; Adenine Nucleotides; Protein Domains; Pyrophosphatases; Adenosine Diphosphate; Adenosine Triphosphate; Bacterial Proteins; Inorganic Pyrophosphatase; Models, Molecular; Binding Sites
PubMed: 38891956
DOI: 10.3390/ijms25115768 -
Bone Jan 2010The physiologic selectivity of calcification in bone tissue reflects selective co-expression by osteoblasts of fibrillar collagen I and of tissue nonspecific alkaline...
INTRODUCTION
The physiologic selectivity of calcification in bone tissue reflects selective co-expression by osteoblasts of fibrillar collagen I and of tissue nonspecific alkaline phosphatase (TNAP), which hydrolyzes the calcification inhibitor pyrophosphate (PP(i)) and generates phosphate (P(i)). Humans and mice deficient in the PP(i)-generating ecto-enzyme NPP1 demonstrate soft tissue calcification, occurring at sites of extracellular matrix expansion. Significantly, the function in osteoblasts of cytosolic inorganic pyrophosphatase (abbreviated iPP(i)ase), which generates P(i) via PP(i) hydrolysis with neutral pH optimum, remains unknown. We assessed iPP(i)ase in Enpp1(-/-) and wild type (WT) mouse osteoblasts and we tested the hypothesis that iPP(i)ase regulates collagen I expression.
METHODS
We treated mouse calvarial osteoblasts with ascorbate and beta-glycerol phosphate to promote calcification, and we assessed cytosolic P(i) and PP(i) levels, sodium-dependent P(i) uptake, Pit-1 P(i) co-transporter expression, and iPP(i)ase and TNAP activity and expression. We also assessed the function of transfected Ppa1 in osteoblasts.
RESULTS
Inorganic pyrophosphatase but not TNAP was elevated in Enpp1(-/-) calvariae in situ. Cultured primary Enpp1(-/-) calvarial osteoblasts demonstrated increased calcification despite flat TNAP activity rather than physiologic TNAP up-regulation seen in WT osteoblasts. Despite decreased cytosolic PP(i) in early culture, Enpp1(-/-) osteoblasts maintained cytosolic P(i) levels comparable to WT osteoblasts, in association with increased iPP(i)ase, enhanced sodium-dependent P(i) uptake and expression of Pit-1, and markedly increased collagen I synthesis. Suppression of collagen synthesis in Enpp1(-/-) osteoblasts using 3,4-dehydroproline markedly suppressed calcification. Last, transfection of Ppa1 in WT osteoblasts increased cytosolic P(i) and decreased cytosolic but not extracellular PP(i), and induced both collagen I synthesis and calcification.
CONCLUSIONS
Increased iPP(i)ase is associated with "P(i) hunger" and increased calcification by NPP1-deficient osteoblasts. Furthermore, iPP(i)ase induces collagen I at the levels of mRNA expression and synthesis and, unlike TNAP, stimulates calcification by osteoblasts without reducing the extracellular concentration of the hydroxyapatite crystal inhibitor PP(i).
Topics: Activating Transcription Factor 4; Animals; Cells, Cultured; Collagen Type I; Core Binding Factor Alpha 1 Subunit; Immunohistochemistry; Inorganic Pyrophosphatase; Mice; Mice, Inbred C57BL; Osteoblasts; Phosphoric Diester Hydrolases; Pyrophosphatases; Reverse Transcriptase Polymerase Chain Reaction; Transfection
PubMed: 19733704
DOI: 10.1016/j.bone.2009.08.055 -
Proceedings of the National Academy of... Jan 2021Macrodomains are proteins that recognize and hydrolyze ADP ribose (ADPR) modifications of intracellular proteins. Macrodomains are implicated in viral genome replication...
Macrodomains are proteins that recognize and hydrolyze ADP ribose (ADPR) modifications of intracellular proteins. Macrodomains are implicated in viral genome replication and interference with host cell immune responses. They are important to the infectious cycle of and viruses. We describe crystal structures of the conserved macrodomain from the bat coronavirus (CoV) HKU4 in complex with ligands. The structures reveal a binding cavity that accommodates ADPR and analogs via local structural changes within the pocket. Using a radioactive assay, we present evidence of mono-ADPR (MAR) hydrolase activity. In silico analysis presents further evidence on recognition of the ADPR modification for hydrolysis. Mutational analysis of residues within the binding pocket resulted in diminished enzymatic activity and binding affinity. We conclude that the common structural features observed in the macrodomain in a bat CoV contribute to a conserved function that can be extended to other known macrodomains.
Topics: Adenosine Diphosphate Ribose; Animals; Binding Sites; Chiroptera; Coronavirus; Crystallography, X-Ray; Hydrolysis; Pyrophosphatases; Viral Nonstructural Proteins
PubMed: 33397718
DOI: 10.1073/pnas.2004500118 -
European Biophysics Journal : EBJ Oct 2023The Nudt15 enzyme of the NUDIX protein family is the subject of extensive study due to its action on thiopurine drugs used in the treatment of cancer and inflammatory...
The Nudt15 enzyme of the NUDIX protein family is the subject of extensive study due to its action on thiopurine drugs used in the treatment of cancer and inflammatory diseases. In addition to thiopurines, Nudt15 is enzymatically active in vitro on several nucleotide substrates. It has also been suggested that this enzyme may play a role in 5'RNA turnover by hydrolyzing mGDP, a product of mRNA decapping. However, no detailed studies on this substrate with Nudt15 are available. Here, we analyzed the enzymatic activity of Nudt15 with mGDP, its triphosphate form mGTP, and the trimethylated counterparts (mGDP and mGTP). Kinetic data revealed a moderate activity of Nudt15 toward these methylated mononucleotides compared to the dGTP substrate. However mGDP and mGDP showed a distinct stabilization of Nudt15 upon ligand binding, in the same range as dGTP, and thus these two mononucleotides may be used as leading structures in the design of small molecule binders of Nudt15.
Topics: Animals; Guanosine; Pyrophosphatases; RNA, Messenger; Mammals
PubMed: 37644211
DOI: 10.1007/s00249-023-01678-5 -
The Journal of Biological Chemistry Jan 1995An Escherichia coli open reading frame containing significant homology to the active site of the MutT enzyme codes for a novel dinucleotide pyrophosphatase. The motif... (Comparative Study)
Comparative Study
An Escherichia coli open reading frame containing significant homology to the active site of the MutT enzyme codes for a novel dinucleotide pyrophosphatase. The motif shared by these two proteins and several others is conserved throughout nature and may designate a nucleotide-binding or pyrophosphatase domain. The E. coli NADH pyrophosphatase has been cloned, overexpressed, and purified to near homogeneity. The protein contains 257 amino acids (M(r) = 29,774) and migrates on gel filtration columns as an apparent dimer. The enzyme catalyzes the hydrolysis of a broad range of dinucleotide pyrophosphates, but uniquely prefers the reduced form of NADH. The Vmax/Km for NADH (69 mumol min-1 mg-1 mM-1) is an order of magnitude higher than for any other dinucleotide pyrophosphate tested. In addition, the Km for NADH (0.1 mM) is 50-fold lower than the Km for NAD+. The hydrolysis of dinucleotide pyrophosphates requires divalent metal ions and yields two mononucleoside 5'-phosphates. The metals that most efficiently stimulate activity are Mg2+ and Mn2+. Although these metals support similar Vmax values at optimal metal concentration, the apparent Km for Mg2+ is 3.7 mM (at 1 mM NADH), whereas the apparent Km for Mn2+ at the same NADH concentration is 30 microM.
Topics: Amino Acid Sequence; Bacterial Proteins; Binding Sites; Cations, Divalent; Chlorides; Chromatography, Gel; Chromatography, Ion Exchange; Cloning, Molecular; DNA Primers; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Escherichia coli; Escherichia coli Proteins; Kinetics; Magnesium Chloride; Manganese Compounds; Molecular Sequence Data; Molecular Weight; Open Reading Frames; Phosphoric Monoester Hydrolases; Pyrophosphatases; Recombinant Proteins; Sequence Homology, Amino Acid; Substrate Specificity
PubMed: 7829480
DOI: 10.1074/jbc.270.4.1529 -
European Journal of Biochemistry Jul 2003Inorganic pyrophosphatase (PPase) is an important enzyme that catalyzes the hydrolysis of inorganic pyrophosphate (PPi) into ortho-phosphate (Pi). We report here the...
Inorganic pyrophosphatase (PPase) is an important enzyme that catalyzes the hydrolysis of inorganic pyrophosphate (PPi) into ortho-phosphate (Pi). We report here the molecular cloning and characterization of a gene encoding the soluble PPase of the roundworm Ascaris suum. The predicted A. suum PPase consists of 360 amino acids with a molecular mass of 40.6 kDa and a pI of 7.1. Amino acid sequence alignment and phylogenetic analysis indicates that the gene encodes a functional Family I soluble PPase containing features identical to those of prokaryotic, plant and animal/fungal soluble PPases. The Escherichia coli-expressed recombinant enzyme has a specific activity of 937 micro mol Pi.min-1.mg-1 protein corresponding to a kcat value of 638 s-1 at 55 degrees C. Its activity was strongly dependent on Mg2+ and was inhibited by Ca2+. Native PPases were expressed in all developmental stages of A. suum. A homolog was also detected in the most closely related human and dog roundworms A. lumbricoides and Toxocara canis, respectively. The enzyme was intensely localized in the body wall, gut epithelium, ovary and uterus of adult female worms. We observed that native PPase activity together with development and molting in vitro of A. suum L3 to L4 were efficiently inhibited in a dose-dependent manner by imidodiphosphate and sodium fluoride, which are potent inhibitor of both soluble- and membrane-bound H+-PPases. The studies provide evidence that the PPases are novel enzymes in the roundworm Ascaris, and may have crucial role in the development and molting process.
Topics: Amino Acid Sequence; Animals; Ascaris suum; Child; Enzyme Inhibitors; Female; Humans; Immunohistochemistry; Molecular Sequence Data; Molting; Phylogeny; Pyrophosphatases; Recombinant Proteins; Sequence Alignment; Sodium Fluoride
PubMed: 12823552
DOI: 10.1046/j.1432-1033.2003.03658.x -
The Biochemical Journal Nov 19661. The disappearance of pyridine nucleotides during incubation with mosquito homogenates proceeds through the hydrolysis of the pyrophosphate linkage of these compounds...
1. The disappearance of pyridine nucleotides during incubation with mosquito homogenates proceeds through the hydrolysis of the pyrophosphate linkage of these compounds as demonstrated by the formation of NMN and AMP from NAD(+). This reaction was also demonstrated by the loss in the coenzyme functioning property of NAD(+) (yeast alcohol dehydrogenase reaction) without a concomitant loss in reactivity towards cyanide. Transglycosidase activity was not observed in the mosquito homogenates, and low concentrations of nicotinamide did not inhibit the NAD(+) splitting activity of these homogenates. These observations are all in accord with the presence in these homogenates of a NAD(+) pyrophosphatase rather than a NADase. 2. The NAD(+) pyrophosphatase is destroyed by boiling, is not heat-activated, and has a pH optimum at pH8.75. In addition to NAD(+), other dinucleotides such as NADP(+), the 3-acetylpyridine and thionicotinamide analogues of NAD(+) and the thionicotinamide analogue of NADP(+), function as substrates in the hydrolysis catalysed by the pyrophosphatase. 3. A decrease in the specific activity of NAD(+) pyrophosphatase was observed during larval development, and a barely detectable activity was found in the pupa and adult. 4. Enzyme activity per organism increased in the larva but decreased to a very low value in the pupa and adult. These results indicate that the decrease in specific activity was due to a decrease in enzyme concentration rather than an increase in amounts of protein.
Topics: Adenine Nucleotides; Aedes; Animals; Hydrogen-Ion Concentration; In Vitro Techniques; NAD; NADP; Pyrophosphatases; Spectrophotometry
PubMed: 4381708
DOI: 10.1042/bj1010392 -
Acta Crystallographica. Section D,... Feb 2013The hyperthermophilic bacterium Thermotoga maritima has a noncanonical nucleoside triphosphatase that catalyzes the conversion of inosine triphosphate (ITP),...
The hyperthermophilic bacterium Thermotoga maritima has a noncanonical nucleoside triphosphatase that catalyzes the conversion of inosine triphosphate (ITP), deoxyinosine triphosphate (dITP) and xanthosine triphosphate (XTP) into inosine monophosphate (IMP), deoxyinosine monophosphate (IMP) and xanthosine monophosphate (XMP), respectively. The k(cat)/K(m) values determined at 323 and 353 K fall between 1.31 × 10(4) and 7.80 × 10(4) M(-1) s(-1). ITP and dITP are slightly preferred over XTP. Activity towards canonical nucleoside triphosphates (ATP and GTP) was not detected. The enzyme has an absolute requirement for Mg(2+) as a cofactor and has a preference for alkaline conditions. A protein X-ray structure of the enzyme with bound IMP was obtained at 2.15 Å resolution. The active site houses a well conserved network of residues that are critical for substrate recognition and catalysis. The crystal structure shows a tetramer with two possible dimer interfaces. One of these interfaces strongly resembles the dimer interface that is found in the structures of other noncanonical nucleoside pyrophosphatases from human (human ITPase) and archaea (Mj0226 and PhNTPase).
Topics: Bacterial Proteins; Crystallography, X-Ray; Humans; Protein Biosynthesis; Protein Conformation; Pyrophosphatases; Random Allocation; Thermotoga maritima
PubMed: 23385455
DOI: 10.1107/S0907444912044630