Pyrimidine nucleotidases from human erythrocyte possess phosphotransferase activities specific for pyrimidine nucleotides

Amici, Adolfo; Emanuelli, Monica; Magni, Giulio; Raffaelli, Nadia; Ruggieri, Silverio

doi:10.1016/s0014-5793(97)01464-6

Cited by 52 publications

(67 citation statements)

References 21 publications

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“…Mg(II) Coordination-hP5N-1 requires Mg(II) ion for activity (3,48). Our structures established that Mg(II) binds in the active site of mP5N-1 and is octahedrally coordinated by 1) the carboxyl oxygens of Asp 49 and Asp 238 , 2) the main-chain carbonyl oxygen of Asp 51 , 3) two water molecules, and 4) either an additional water (state II) or an oxygen of the phosphate group (or fluorine of phosphate analogs) (states V-VII) (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…phosphoenzyme intermediate analog was obtained by using BeF 3 Ϫ , which acts as a phosphate analog in proteins that are phosphorylated on aspartate residues (46,50). The intermediate with the covalently modified Asp 49 is shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…These enzymes catalyze dephosphorylation of pyrimidine mononucleotides to their respective nucleosides. In addition, pyrimidine nucleotidases also possess phosphotransferase activity between pyrimidine mononucleotides and pyrimidine nucleosides (3,4). Two different subtypes, hP5N-1 2 (UniProt code Q9H0P0) and hPN-2, which differ in their substrate specificities, were identified in human erythrocytes (5)(6)(7).…”

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confidence: 99%

“…Two different subtypes, hP5N-1 2 (UniProt code Q9H0P0) and hPN-2, which differ in their substrate specificities, were identified in human erythrocytes (5)(6)(7). Subtype hP5N-1 preferentially dephosphorylates 5Ј-UMP, 5Ј-CMP, and at a lower rate also 5Ј-dCMP, 5Ј-dTMP, and 5Ј-dUMP, whereas hPN-2 preferentially dephosphorylates 3Ј-dTMP, 3Ј-dUMP, and 3Ј-UMP but shows no activity against 5Ј-CMP and 5Ј-dCMP (3,4,8). Both enzymes are essentially inactive against purine nucleotides.…”

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Structure of Pyrimidine 5′-Nucleotidase Type 1

Bitto

Bingman

Wesenberg

et al. 2006

Journal of Biological Chemistry

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Eukaryotic pyrimidine 5-nucleotidase type 1 (P5N-1) catalyzes dephosphorylation of pyrimidine 5-mononucleotides. Deficiency of P5N-1 activity in red blood cells results in nonspherocytic hemolytic anemia. The enzyme deficiency is either familial or can be acquired through lead poisoning. We present the crystal structure of mouse P5N-1 refined to 2.35 Å resolution. The mouse P5N-1 has a 92% sequence identity to its human counterpart. The structure revealed that P5N-1 adopts a fold similar to enzymes of the haloacid dehydrogenase superfamily. The active site of this enzyme is structurally highly similar to those of phosphoserine phosphatases. We propose a catalytic mechanism for P5N-1 that is also similar to that of phosphoserine phosphatases and provide experimental evidence for the mechanism in the form of structures of several reaction cycle states, including: 1) P5N-1 with bound Mg(II) at 2.25 Å , 2) phosphoenzyme intermediate analog at 2.30 Å , 3) product-transition complex analog at 2.35 Å , and 4) product complex at 2.1 Å resolution with phosphate bound in the active site. Furthermore the structure of Pb(II)-inhibited P5N-1 (at 2.35 Å ) revealed that Pb(II) binds within the active site in a way that compromises function of the cationic cavity, which is required for the recognition and binding of the phosphate group of nucleotides.Eukaryotic pyrimidine nucleotidases are enzymes involved in catabolism of RNA and in the pyrimidine salvage pathway (1, 2). These enzymes catalyze dephosphorylation of pyrimidine mononucleotides to their respective nucleosides. In addition, pyrimidine nucleotidases also possess phosphotransferase activity between pyrimidine mononucleotides and pyrimidine nucleosides (3, 4). Two different subtypes, hP5N-1 2 (UniProt code Q9H0P0) and hPN-2, which differ in their substrate specificities, were identified in human erythrocytes (5-7). Subtype hP5N-1 preferentially dephosphorylates 5Ј-UMP, 5Ј-CMP, and at a lower rate also 5Ј-dCMP, 5Ј-dTMP, and 5Ј-dUMP, whereas hPN-2 preferentially dephosphorylates 3Ј-dTMP, 3Ј-dUMP, and 3Ј-UMP but shows no activity against 5Ј-CMP and 5Ј-dCMP (3,4,8). Both enzymes are essentially inactive against purine nucleotides. This specificity of hPNs is unique among 5Ј-nucleotidases (EC 3.1.3.5), which are typically not discriminatory to the identity of the nucleotide base (9). The specificity of hPNs is crucial in erythrocytes for preservation of the internal pool of ATP/GTP (6). Two types of hP5N-1 deficiency are known: hereditary and acquired. Hereditary deficiency is associated with nonspherocytic hemolytic anemia (NHA) (2, 7, 10). Acquired deficiency results from inhibition of hP5N-1 by Pb(II) during lead poisoning, and its severity is related to the blood levels of Pb(II) (11)(12)(13)(14). Both NHA and severe lead poisoning (Ͼ80 g/dl Pb(II) in blood) have the same clinical manifestation: erythrocytes in blood smears of affected patients show a distinct basophilic stippling upon Wright's staining (7,14,15). At the molecular level, NHA is characterized by signifi...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Structure of Pyrimidine 5′-Nucleotidase Type 1

Bitto

Bingman

Wesenberg

et al. 2006

Journal of Biological Chemistry

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“…This suggests that Thr of Motif 1 may be important for the phosphotransferase reaction. In apparent contradiction, it was claimed that cdN isolated from human erythrocytes that lacks the strategic Thr has phosphotransferase activity (44). However, this appears unlikely as cdN isolated from human placenta and recombinant murine cdN did not show this activity (45,46).…”

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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