Identification, Separation and Characterisation of Two Forms of Cytosolic 5′-Nucleotidase/Nucleoside Phosphotransferase in Calf Thymus

Pesi, Rossana; Baiocchi, Cristina; Allegrini, Simone; Moretti, Elisa; Sgarrella, Francesco; Camici, Marcella; Tozzi, Maria Grazia

doi:10.1515/bchm.1998.379.6.699

Cited by 7 publications

(14 citation statements)

References 12 publications

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“…Two active forms of cN-II were isolated from calf thymus (51). The 59-kDa form A appeared to contain two effector sites, one for ATP and ADP and one for 2,3-BPG, while the 54-kDa form B contained three separate effector sites for ATP, ADP, and 2,3-BPG (51).…”

Section: Discussionmentioning

confidence: 99%

Crystal Structure of Human Cytosolic 5′-Nucleotidase II

Wallden

Stenmark

Nyman

et al. 2007

Journal of Biological Chemistry

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Cytosolic 5-nucleotidase II catalyzes the dephosphorylation of 6-hydroxypurine nucleoside 5-monophosphates and regulates the IMP and GMP pools within the cell. It possesses phosphotransferase activity and thereby also catalyzes the reverse reaction. Both reactions are allosterically activated by adeninebased nucleotides and 2,3-bisphosphoglycerate. We have solved structures of cytosolic 5-nucleotidase II as native protein (2.2 Å ) and in complex with adenosine (1.5 Å ) and beryllium trifluoride (2.15 Å ). The tetrameric enzyme is structurally similar to enzymes of the haloacid dehalogenase (HAD) superfamily, including mitochondrial 5(3)-deoxyribonucleotidase and cytosolic 5-nucleotidase III but possesses additional regulatory regions that contain two allosteric effector sites. At effector site 1 located near a subunit interface we modeled diadenosine tetraphosphate with one adenosine moiety in each subunit. This efficiently glues the tetramer subunits together in pairs. The model shows why diadenosine tetraphosphate but not diadenosine triphosphate activates the enzyme and supports a role for cN-II during apoptosis when the level of diadenosine tetraphosphate increases. We have also modeled 2,3-bisphosphoglycerate in effector site 1 using one phosphate site from each subunit. By comparing the structure of cytosolic 5-nucleotidase II with that of mitochondrial 5(3)-deoxyribonucleotidase in complex with dGMP, we identified residues involved in substrate recognition.Cytosolic 5Ј-nucleotidase II (cN-II), 2 also called purine 5Ј-nucleotidase, IMP-GMP specific nucleotidase or high K m 5Ј-nucleotidase, is one of the seven known mammalian 5Ј-nucleotidases that catalyze the dephosphorylation of ribo-and deoxyribonucleoside monophosphates to nucleoside and inorganic phosphate (1, 2). The seven enzymes differ in substrate specificity, subcellular location, and tissue-specific expression. Intracellular 5Ј-nucleotidases regulate nucleotide levels by counteracting the action of nucleoside kinases and competing with other enzymes that use the nucleoside monophosphates. This complex network of interactions contributes to maintain nucleotide pools in tune with the metabolic needs of the cell (1, 2).The structures of two of the six human intracellular 5Ј-nucleotidases, i.e. mitochondrial 5Ј(3Ј)-deoxyribonucleotidase (mdN) (3) and cytosolic 5Ј-nucleotidase III (cN-III) (PDB entry 2CN1) are known. The latter is almost identical to the published structure of mouse cN-III (4). Both mdN and cN-III belong to the haloacid dehalogenase (HAD) superfamily, which is defined by an ␣/␤-Rossmann-like domain and a smaller 4-helix bundle domain, which, however, is not present in all HAD superfamily enzymes. The intracellular 5Ј-nucleotidases share three conserved motifs that have been found in HAD superfamily enzymes such as phosphoserine phosphatase (5) , are located in the ␣/␤-Rossmann-like domain and build up the catalytic phosphate-binding site in these enzymes. Motif I is directly involved in the reaction mechanism of 5Ј-nucleotidases (3),...

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Section: Discussionmentioning

confidence: 99%

Crystal Structure of Human Cytosolic 5′-Nucleotidase II

Wallden

Stenmark

Nyman

et al. 2007

Journal of Biological Chemistry

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“…In a previous work, we observed the simultaneous presence of two cN-II forms in preparations from calf thymus, distinguishable for electrophoretic, chromatographic and regulatory properties [35]. In fact, the cN-II species (form B) with faster electrophoretic mobility (54 kDa) was activated by ADP and BPG, and a synergistic stimulatory effect of these compounds was also observed.…”

Section: Proteolytic Generation Of Two Cn-ii Formsmentioning

confidence: 80%

“…[8][9][10][11][12][13][14] C]Inosine was purchased from Sigma Chemical Co. (St Louis, MO, USA). Thrombin was from Amersham Pharmacia Biotech (Uppsala, Sweden).…”

Section: Methodsmentioning

confidence: 99%

“…Unless stated otherwise, the nucleotidase activity of cN-II and its mutants was measured as the rate of [8][9][10][11][12][13][14] C]inosine formation from 2 mM [8][9][10][11][12][13][14] C]IMP in the presence of 1.4 mM inosine, 20 mM MgCl 2 , 4.5 mM ATP and 5 mM dithiothreitol, as previously described [11]. Phosphotransferase activity was measured as the rate of [8][9][10][11][12][13][14] C]IMP formation from 1.4 mM [8][9][10][11][12][13][14] C]inosine, in the presence of 2 mM IMP, 20 mM MgCl 2 , 4.5 mM ATP and 5 mM dithiothreitol, as previously described [11].…”

Section: Enzyme Assaysmentioning

confidence: 99%

“…Phosphotransferase activity was measured as the rate of [8][9][10][11][12][13][14] C]IMP formation from 1.4 mM [8][9][10][11][12][13][14] C]inosine, in the presence of 2 mM IMP, 20 mM MgCl 2 , 4.5 mM ATP and 5 mM dithiothreitol, as previously described [11]. For the determination of kinetic parameters (K m and k cat ) the concentration of the labelled substrates ranged from 0.02 to 4 mM.…”

Section: Enzyme Assaysmentioning

confidence: 99%

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Mechanistic studies on bovine cytosolic 5′‐nucleotidase II, an enzyme belonging to the HAD superfamily

Allegrini

Scaloni

Careddu

et al. 2004

European Journal of Biochemistry

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Cytosolic 5¢-nucleotidase/phosphotransferase specific for 6-hydroxypurine monophosphate derivatives (cN-II), belongs to a class of phosphohydrolases that act through the formation of an enzyme-phosphate intermediate. Sequence alignment with members of the P-type ATPases/L-2-haloacid dehalogenase superfamily identified three highly conserved motifs in cN-II and other cytosolic nucleotidases. Mutagenesis studies at specific amino acids occurring in cN-II conserved motifs were performed. The modification of the measured kinetic parameters, caused by conservative and nonconservative substitutions, suggested that motif I is involved in the formation and stabilization of the covalent enzyme-phosphate intermediate. Similarly, T249 in motif II as well as K292 in motif III also contribute to stabilize the phospho-enzyme adduct. Finally, D351 and D356 in motif III coordinate magnesium ion, which is required for catalysis. These findings were consistent with data already determined for P-type ATPases, haloacid dehalogenases and phosphotransferases, thus suggesting that cN-II and other mammalian 5¢-nucleotidases are characterized by a 3D arrangement related to the 2-haloacid dehalogenase superfold. Structural determinants involved in differential regulation by nonprotein ligands and redox reagents of the two naturally occurring cN-II forms generated by proteolysis were ascertained by combined biochemical and mass spectrometric investigations. These experiments indicated that the C-terminal region of cN-II contains a cysteine prone to form a disulfide bond, thereby inactivating the enzyme. Proteolysis events that generate the observed cN-II forms, eliminating this C-terminal portion, may prevent loss of enzymic activity and can be regarded as regulatory phenomena.Keywords: catalytic residues; HAD; nucleotidase; regulation; site-directed mutagenesis.Mammalian 5¢-nucleotidases (eN, cN-Ia, cN-Ib, cN-II, cN-III, cdN and mdN) make up a family of proteins with different subcellular locations and remarkably low sequence similarities [1]. Besides ectosolic 5¢-nucleotidase, one mitochondrial and five cytosolic enzymes have been described to date. According to its substrate specificity and tissue distribution, each protein seems to play a specific role within the cell. In fact, cN-Is, which is highly expressed in skeletal muscle, heart and testis, is specific for AMP and seems to be involved in adenosine production during hypoxia or ischemia, because it mediates the cell response to low energy charges [2]. On the other hand, cN-II is more specific for inosine monophosphate (IMP) and GMP, and is a ubiquitous enzyme involved in the regulation of intracellular IMP and GMP concentrations [3]. Furthermore, cN-III, which is expressed in red blood cells and is specific for pyrimidines, seems to participate in RNA degradation during erythrocyte maturation [4]. Likewise, cytosolic and mitochondrial deoxynucleotidases (cdN and mdN) regulate nucleotide pools in their respective compartments [1].cN-II was the first member of the cytosolic 5¢-nucl...

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Structural Basis for the Allosteric Regulation and Substrate Recognition of Human Cytosolic 5′-Nucleotidase II

Wallden

Nordlund

2011

Journal of Molecular Biology

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Identification, Separation and Characterisation of Two Forms of Cytosolic 5′-Nucleotidase/Nucleoside Phosphotransferase in Calf Thymus

Cited by 7 publications

References 12 publications

Crystal Structure of Human Cytosolic 5′-Nucleotidase II

Crystal Structure of Human Cytosolic 5′-Nucleotidase II

Mechanistic studies on bovine cytosolic 5′‐nucleotidase II, an enzyme belonging to the HAD superfamily

Structural Basis for the Allosteric Regulation and Substrate Recognition of Human Cytosolic 5′-Nucleotidase II

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