The Bifunctional Cytosolic 5′-Nucleotidase: Regulation of the Phosphotransferase and Nucleotidase Activities

Pesi, Rossana; Turriani, Maura; Allegrini, Simone; Scolozzi, C.; Camici, Marcella; Ipata, Piero Luigi; Tozzi, Maria Grazia

doi:10.1006/abbi.1994.1282

Cited by 72 publications

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“…The cN-II was first purified from chicken liver (10) and has been intensively investigated in vertebrates (11). As substrates it prefers the 6-hydroxypurine nucleoside monophosphates IMP, dIMP, GMP, dGMP, and XMP (10,(12)(13)(14)(15) and functions as a tetramer (12, 16 -18). It is ubiquitously expressed and is likely to play an important role in the regulation of purine nucleotide interconversions and in the regulation of IMP and GMP pools within the cell (1).…”

Section: The Three Motifs Motif I (Dxdx[t/v]l) Motif Ii ([T/s]) mentioning

confidence: 99%

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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: The Three Motifs Motif I (Dxdx[t/v]l) Motif Ii ([T/s]) mentioning

confidence: 99%

“…Phosphate donors include any 6-hydroxypurine monophosphate substrate of the nucleotidase reaction (13)(14)(15)(22)(23)(24).…”

Section: The Three Motifs Motif I (Dxdx[t/v]l) Motif Ii ([T/s]) mentioning

confidence: 99%

Crystal Structure of Human Cytosolic 5′-Nucleotidase II

Wallden

Stenmark

Nyman

et al. 2007

Journal of Biological Chemistry

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“…It is inactive with purine nucleotides. The enzyme has a phosphotransferase activity (35) less efficient than cN-II (32). The sequence of cN-III coincides with that of p36, an interferon ␣-induced protein of unknown function (6).…”

Section: Properties Detection and Inhibition Of 5-nucleotidasesmentioning

confidence: 99%

“…(27)(28)(29), possibly involving subunit association and dissociation (30). Under physiological conditions cN-II can catalyze phosphotransfer from a purine nucleotide donor to inosine or guanosine (31,32). This reaction is responsible for the activation of several anti-viral and anti-cancer nucleoside analogs that are not substrates for cellular nucleoside kinases (33,34).…”

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confidence: 99%

Mammalian 5′-Nucleotidases

Bianchi

Spychała

2003

Journal of Biological Chemistry

192

184

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Nucleoside monophosphate phosphohydrolases or 5Ј-nucleotidases (members of EC 3.1.3.5 and EC 3.1.3.6) dephosphorylate non-cyclic nucleoside monophosphates to nucleosides and inorganic phosphate. Seven human 5Ј-nucleotidases with different subcellular localization have been cloned (Table I). Sequence comparisons show high homology only between cytosolic 5Ј-nucleotidase IA (cN-IA) 1 and B and between cytosolic 5Ј(3Ј)-deoxynucleotidase (cdN) and mitochondrial 5Ј(3Ј)-deoxynucleotidase (mdN). However, the existence of common motifs suggests a common catalytic mechanism for all intracellular 5Ј-nucleotidases. Some 5Ј-nucleotidases are ubiquitous (ecto-5Ј-nucleotidase (eN), cN-II, cdN, and mdN); others display tissue-specific expression (cN-I and cN-III).Here we summarize recent advances on the structure and cellular functions of the cloned 5Ј-nucleotidases. We also propose a revised nomenclature, agreed upon with other colleagues active in the nucleotidase field. Catalytic MechanismCrystal structures are known for human mdN (10) and cdN 2 and for Escherichia coli periplasmic 5Ј-nucleotidase (11), a homologue of eN. All intracellular nucleotidases share a DX-DX(V/T) motif critical for catalysis and show structural similarity to the haloacid dehalogenase superfamily of enzymes (10). eN belongs to a separate family that includes also 2Ј,3Ј-cyclic phosphodiesterases and apyrases (11).The crystal structure of mdN and work on the active site of cN-II form the basis for a reaction mechanism of intracellular 5Ј-nucleotidases (10, 12). The reaction creates a phosphoenzyme intermediate involving the first aspartate in the DX-DX(V/T) motif (12). A detailed scheme of the catalytic process derived from the crystal structure of mdN (10) involved both aspartates in the above motif (Fig. 1). The first (Asp-41) generates a pentacovalent phosphorus intermediate with similar basic organization as the intermediate detected in the structure of ␤-phosphoglucomutase (13). The x-ray structure of cdN suggests a catalytic mechanism identical with that of mdN. Differences within the active sites account for differences in substrate specificity (10).2 Using the structurally important residues the best alignment was between the two deoxynucleotidases and cN-III (10). Two 5Ј-nucleotidases, cN-II and cN-III, exhibit phosphotransferase activity (for reviews see Refs. 14 and 15) possibly because of higher stability of the phosphoenzyme intermediate or faster exchange of the nucleoside product with the nucleoside acceptor. The active site of E. coli 5Ј-nucleotidase, the paradigm for eN, contains two zinc ions and the catalytic dyad . No phosphoenzyme intermediate is formed during catalysis, but a water molecule performs the nucleophilic attack on the phosphate (16). Properties, Detection, and Inhibition of 5-NucleotidasesAll 5Ј-nucleotidases have relatively broad substrate specificities. In agreement with the structural information on the active sites (10, 11), all family members except eN are absolutely dependent on magnesium for activity. Table II s...

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“…Intracellular nucleotidases such as cytosolic 5Ј-nucleotidase II (NT5C2; also known as cN-II and high K m 5Ј-nucleotidase) and cytoplasmic 5Ј-nucleotidase III (NT5C3) are involved in the final step of dephosphorylation before export of nucleosides out of the cell. Although NT5C2 has a preference for 6-hydroxypurine nucleotide monophosphates (such as IMP, dIMP, GMP, dGMP, and XMP) as substrates, NT5C3 only hydrolyzes pyrimidine monophosphates (Pesi et al, 1994;Amici and Magni, 2002).…”

Section: Introductionmentioning

confidence: 99%

Genetic Variants in Cytosolic 5′-Nucleotidase II Are Associated with Its Expression and Cytarabine Sensitivity in HapMap Cell Lines and in Patients with Acute Myeloid Leukemia

Mitra

Crews

Pounds

et al. 2011

J Pharmacol Exp Ther

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Cytosolic 5Ј-nucleotidase II (NT5C2) is involved in the development of 1-␤-D-arabinofuranosylcytosine (ara-C) resistance and has been associated with clinical outcome in patients receiving ara-C-based chemotherapy. NT5C2 inactivates ara-C by dephosphorylating ara-C monophosphate to ara-C. In this study, we sequenced NT5C2 in genomic DNA samples from International HapMap project panels with European [Centre d'Etude du Polymorphisme Humain (CEU); n ϭ 90] or African [Yoruba people in Ibadan, Nigeria (YRI); n ϭ 90] ancestry. We identified 41 genetic variants [one insertion-deletion and 40 single nucleotide polymorphisms (SNPs)], including three nonsynonymous SNPs (Y3A, K47R, and Q136R). Twenty-five SNPs were novel and 16 overlapped with the HapMap data. Subjects with African ancestry had NT5C2 mRNA expression levels that was significantly higher than those with European ancestry (p ϭ 0.005). Furthermore, there was a correlation between NT5C2 mRNA expression and ara-C sensitivity in CEU but not in YRI cell lines. None of the nonsynonymous SNPs demonstrated any effect on NT5C2 activity. The genotypes of several SNPs were significantly associated with NT5C2 mRNA expression and/or ara-C sensitivity in CEU cell lines, but very few were significant in YRI cell lines. Of most interest, SNPs (linkage disequilibrium group CEU.12) in the 5Ј-untranslated region were associated with NT5C2 expression and ara-C sensitivity in HapMap cell lines and with NT5C2 mRNA expression and ara-C sensitivity in diagnostic leukemic blasts from pediatric patients with acute myeloid leukemia. Functional genomics analysis demonstrated that the promoter SNP rs11191612 was associated with altered luciferase activation in reporter assays and altered DNA-protein binding in gel shift assays. These results suggest that genetic variations in NT5C2 influence its expression and, potentially, cellular responses to nucleoside analogs.

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The Bifunctional Cytosolic 5′-Nucleotidase: Regulation of the Phosphotransferase and Nucleotidase Activities

Cited by 72 publications

References 0 publications

Crystal Structure of Human Cytosolic 5′-Nucleotidase II

Crystal Structure of Human Cytosolic 5′-Nucleotidase II

Mammalian 5′-Nucleotidases

Genetic Variants in Cytosolic 5′-Nucleotidase II Are Associated with Its Expression and Cytarabine Sensitivity in HapMap Cell Lines and in Patients with Acute Myeloid Leukemia

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