Mammalian 5′-Nucleotidases

Abstract

Nucleoside monophosphate phosphohydrolases or 5′-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 5′-nucleotidases with different subcellular localization have been cloned (Table I). Sequence comparisons show high homology only between cytosolic 5′-nucleotidase IA (cN-IA) 1The abbreviations used are: cNcytosolic 5′-nucleotidasecdNcytosolic 5′(3′)-deoxynucleotidaseeNecto-5′-nucleotidasemdNmitochondrial 5′(3′)-deoxynucleotidaseNTnucleotidase. and B and between cytosolic 5′(3′)-deoxynucleotidase (cdN) and mitochondrial 5′(3′)-deoxynucleotidase (mdN). However, the existence of common motifs suggests a common catalytic mechanism for all intracellular 5′-nucleotidases. Some 5′-nucleotidases are ubiquitous (ecto-5′-nucleotidase (eN), cN-II, cdN, and mdN); others display tissue-specific expression (cN-I and cN-III).Table IClassification of 5′-nucleotidasesRevised protein nomenclatureFull name and gene symbolUniGene cluster no.AliasesRefs.eNEcto-5′-nucleotidase, NT5EHs.153952Ecto-5′-NT; low Km 5′-NT; eNT; CD731Misumi Y. Ogata S. Ohkubo K. Hirose S. Ikehara Y. Eur. J. Biochem. 1990; 191: 563-569Crossref PubMed Scopus (122) Google ScholarcN-IACytosolic 5′-nucleotidase IA, NT5C1AHs.307006AMP-specific 5′-NT; cN-I2Sala-Newby G.B. Skladanowski A.C. Newby A.C. J. Biol. Chem. 1999; 274: 17789-17793Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 3Hunsucker S.A. Spychala J. Mitchell B.S. J. Biol. Chem. 2001; 276: 10498-10504Abstract Full Text Full Text PDF PubMed Scopus (96) Google ScholarcN-IBCytosolic 5′-nucleotidase IB, NT5C1BHs.120319cN-IA homolog; AIRP4Sala-Newby G.B. Newby A.C. Biochim. Biophys. Acta. 2001; 1521: 12-18Crossref PubMed Scopus (28) Google ScholarcN-IICytosolic 5′-nucleotidase II, NT5C2Hs.138593High Km 5′-NT; purine 5′-NT; GMP,IMP-specific 5′-NT5Oka J. Matsumoto A. Hosokawa Y. Inoue S. Biochem. Biophys. Res. Commun. 1994; 205: 917-922Crossref PubMed Scopus (60) Google ScholarcN-IIICytosolic 5′-nucleotidase III, NT5C3Hs.55189PN-I; P5′N-1; UMPH6Amici A. Emanuelli M. Raffaelli N. Ruggieri S. Saccucci F. Magni G. Blood. 2000; 96: 1596-1598Crossref PubMed Google ScholarcdNCytosolic 5′(3′)-deoxynucleotidase, NT5CHs.67201dNT-1; PN-II7Rampazzo C. Johansson M. Gallinaro L. Ferraro P. Hellman U. Karlsson A. Reichard P. Bianchi V. J. Biol. Chem. 2000; 275: 5409-5415Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 8Mazzon C. Rampazzo C. Scaini M.C. Gallinaro L. Karlsson A. Meier C. Balzarini J. Reichard P. Bianchi V. Biochem. Pharmacol. 2003; 66: 471-479Crossref PubMed Scopus (90) Google ScholarmdNMitochondrial 5′(3′)- deoxynucleotidase, NT5MHs.16614dNT-29Rampazzo C. Gallinaro L. Milanesi E. Frigimelica E. Reichard P. Bianchi V. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8239-8244Crossref PubMed Scopus (84) Google Scholar Open table in a new tab cytosolic 5′-nucleotidase cytosolic 5′(3′)-deoxynucleotidase ecto-5′-nucleotidase mitochondrial 5′(3′)-deoxynucleotidase nucleotidase. Here we summarize recent advances on the structure and cellular functions of the cloned 5′-nucleotidases. We also propose a revised nomenclature, agreed upon with other colleagues active in the nucleotidase field. Crystal structures are known for human mdN (10Rinaldo-Matthis A. Rampazzo C. Reichard P. Bianchi V. Nordlund P. Nat. Struct. Biol. 2002; 9: 779-787Crossref PubMed Scopus (71) Google Scholar) and cdN 2A. Rinaldo-Matthis and P. Nordlund, manuscript in preparation. and for Escherichia coli periplasmic 5′-nucleotidase (11Knöfel T. Sträter N. Nat. Struct. Biol. 1999; 6: 448-453Crossref PubMed Scopus (103) Google Scholar), 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 (10Rinaldo-Matthis A. Rampazzo C. Reichard P. Bianchi V. Nordlund P. Nat. Struct. Biol. 2002; 9: 779-787Crossref PubMed Scopus (71) Google Scholar). eN belongs to a separate family that includes also 2′,3′-cyclic phosphodiesterases and apyrases (11Knöfel T. Sträter N. Nat. Struct. Biol. 1999; 6: 448-453Crossref PubMed Scopus (103) Google Scholar). The crystal structure of mdN and work on the active site of cN-II form the basis for a reaction mechanism of intracellular 5′-nucleotidases (10Rinaldo-Matthis A. Rampazzo C. Reichard P. Bianchi V. Nordlund P. Nat. Struct. Biol. 2002; 9: 779-787Crossref PubMed Scopus (71) Google Scholar, 12Allegrini S. Scaloni A. Ferrara L. Pesi R. Pinna P. Sgarrella F. Camici M. Eriksson S. Tozzi M.G. J. Biol. Chem. 2001; 276: 33526-33532Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The reaction creates a phosphoenzyme intermediate involving the first aspartate in the DX- DX(V/T) motif (12Allegrini S. Scaloni A. Ferrara L. Pesi R. Pinna P. Sgarrella F. Camici M. Eriksson S. Tozzi M.G. J. Biol. Chem. 2001; 276: 33526-33532Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). A detailed scheme of the catalytic process derived from the crystal structure of mdN (10Rinaldo-Matthis A. Rampazzo C. Reichard P. Bianchi V. Nordlund P. Nat. Struct. Biol. 2002; 9: 779-787Crossref PubMed Scopus (71) Google Scholar) 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 β-phosphoglucomutase (13Lahiri S.D. Zhang G. Dunaway-Mariano D. Allen K.N. Science. 2003; 299: 2067-2071Crossref PubMed Scopus (291) Google Scholar). 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 (10Rinaldo-Matthis A. Rampazzo C. Reichard P. Bianchi V. Nordlund P. Nat. Struct. Biol. 2002; 9: 779-787Crossref PubMed Scopus (71) Google Scholar). 2A. Rinaldo-Matthis and P. Nordlund, manuscript in preparation. Using the structurally important residues the best alignment was between the two deoxynucleotidases and cN-III (10Rinaldo-Matthis A. Rampazzo C. Reichard P. Bianchi V. Nordlund P. Nat. Struct. Biol. 2002; 9: 779-787Crossref PubMed Scopus (71) Google Scholar). Two 5′-nucleotidases, cN-II and cN-III, exhibit phosphotransferase activity (for reviews see Refs. 14Itoh R. Comp. Biochem. Physiol. [B]. 1993; 105: 13-19Crossref PubMed Scopus (57) Google Scholar and 15Rees D.C. Duley J.A. Marinaki A.M. Br. J. Haematol. 2003; 120: 375-383Crossref PubMed Scopus (47) Google Scholar) 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 5′-nucleotidase, the paradigm for eN, contains two zinc ions and the catalytic dyad Asp-His (11Knöfel T. Sträter N. Nat. Struct. Biol. 1999; 6: 448-453Crossref PubMed Scopus (103) Google Scholar). No phosphoenzyme intermediate is formed during catalysis, but a water molecule performs the nucleophilic attack on the phosphate (16Knöfel T. Sträter N. J. Mol. Biol. 2001; 309: 239-254Crossref PubMed Scopus (87) Google Scholar). All 5′-nucleotidases have relatively broad substrate specificities. In agreement with the structural information on the active sites (10Rinaldo-Matthis A. Rampazzo C. Reichard P. Bianchi V. Nordlund P. Nat. Struct. Biol. 2002; 9: 779-787Crossref PubMed Scopus (71) Google Scholar, 11Knöfel T. Sträter N. Nat. Struct. Biol. 1999; 6: 448-453Crossref PubMed Scopus (103) Google Scholar), all family members except eN are absolutely dependent on magnesium for activity. Table II summarizes some distinctive properties of 5′-nucleotidases. Detection of individual nucleotidases by enzymatic assays in cell lysates is problematic because different nucleotidases are co-expressed in the same tissue or cell type. The problem was earlier addressed by immunotitration (for review, see Ref. 14Itoh R. Comp. Biochem. Physiol. [B]. 1993; 105: 13-19Crossref PubMed Scopus (57) Google Scholar) and more recently by a strategy that exploits differences in optimal conditions for the ubiquitous nucleotidases (17Rampazzo C. Mazzon C. Reichard P. Bianchi V. Biochem. Biophys. Res. Commun. 2002; 293: 258-263Crossref PubMed Scopus (23) Google Scholar). Differential assays can take advantage of inhibitors of individual nucleotidases (8Mazzon C. Rampazzo C. Scaini M.C. Gallinaro L. Karlsson A. Meier C. Balzarini J. Reichard P. Bianchi V. Biochem. Pharmacol. 2003; 66: 471-479Crossref PubMed Scopus (90) Google Scholar, 17Rampazzo C. Mazzon C. Reichard P. Bianchi V. Biochem. Biophys. Res. Commun. 2002; 293: 258-263Crossref PubMed Scopus (23) Google Scholar, 18Garvey E.P. Lowen G.T. Almond M.R. Biochemistry. 1998; 37: 9043-9051Crossref PubMed Scopus (32) Google Scholar, 19Skladanowski A.C. Sala G.B. Newby A.C. Biochem. J. 1989; 262: 203-208Crossref PubMed Scopus (15) Google Scholar, 20Zimmermann H. Biochem. J. 1992; 285: 345-365Crossref PubMed Scopus (760) Google Scholar). The most active inhibitors described so far are pyrimidine nucleotide and nucleoside analogs inhibiting cN-I at nanomolar or low micromolar concentrations with up to 1000-fold selectivity for cN-I relative to cN-II or eN (18Garvey E.P. Lowen G.T. Almond M.R. Biochemistry. 1998; 37: 9043-9051Crossref PubMed Scopus (32) Google Scholar). Two pyrimidine phosphonates inhibit cdN and mdN (8Mazzon C. Rampazzo C. Scaini M.C. Gallinaro L. Karlsson A. Meier C. Balzarini J. Reichard P. Bianchi V. Biochem. Pharmacol. 2003; 66: 471-479Crossref PubMed Scopus (90) Google Scholar, 17Rampazzo C. Mazzon C. Reichard P. Bianchi V. Biochem. Biophys. Res. Commun. 2002; 293: 258-263Crossref PubMed Scopus (23) Google Scholar) with weaker inhibition of cN-I (17Rampazzo C. Mazzon C. Reichard P. Bianchi V. Biochem. Biophys. Res. Commun. 2002; 293: 258-263Crossref PubMed Scopus (23) Google Scholar). Specific properties of individual 5′-nucleotidases are discussed below.Table IIDistinctive features of 5′-nucleotidasesEnzymeProtein structure (monomer kDaaPredicted from cDNA sequence and not including posttranslational modifications.)Substrate affinity (Km)Effect of ATPpH optimumeNDimer (63)μm-7.5cN-IAbcN-IB not yet characterized.Tetramer (41)μm-mmcMicromolar Km values for pyrimidine deoxynucleotides; millimolar or submillimolar for purine substrates (3, 18).+ (ADP ++)dADP and dADP are best activators (S. A. Hunsucker and Y. Spychala, manuscript in preparation).7cN-IITetramer (65)sub mm++6.5cN-IIIMonomer (33)sub mmnone7.5cdNDimer (23)mmnone6-6.5mdNDimer (26)sub mmnone5.5a Predicted from cDNA sequence and not including posttranslational modifications.b cN-IB not yet characterized.c Micromolar Km values for pyrimidine deoxynucleotides; millimolar or submillimolar for purine substrates (3Hunsucker S.A. Spychala J. Mitchell B.S. J. Biol. Chem. 2001; 276: 10498-10504Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 18Garvey E.P. Lowen G.T. Almond M.R. Biochemistry. 1998; 37: 9043-9051Crossref PubMed Scopus (32) Google Scholar).d ADP and dADP are best activators (S. A. Hunsucker and Y. Spychala, manuscript in preparation). Open table in a new tab Ecto-5′-nucleotidase—eN, also known as CD73, is a glycosylated protein bound to the outer surface of the plasma membrane by a glycosylphosphatidylinositol anchor (1Misumi Y. Ogata S. Ohkubo K. Hirose S. Ikehara Y. Eur. J. Biochem. 1990; 191: 563-569Crossref PubMed Scopus (122) Google Scholar) and co-localizes with detergent-resistant and glycolipid-rich membrane subdomains called lipid rafts. Up to 50% of the enzyme may be associated to intracellular membranes (for review, see Ref. 20Zimmermann H. Biochem. J. 1992; 285: 345-365Crossref PubMed Scopus (760) Google Scholar) and be released during homogenization. Early reports of a soluble low Km nucleotidase (for review, see Ref. 20Zimmermann H. Biochem. J. 1992; 285: 345-365Crossref PubMed Scopus (760) Google Scholar) were because of this phenomenon (21Piec G. Le Hir M. Biochem. J. 1991; 273: 409-413Crossref PubMed Scopus (23) Google Scholar). Although eN has broad substrate specificity, AMP is considered to be the major physiological substrate (for review, see Ref. 20Zimmermann H. Biochem. J. 1992; 285: 345-365Crossref PubMed Scopus (760) Google Scholar) (22Yegutkin G.G. Henttinen T. Samburski S.S. Spychala J. Jalkanen S. Biochem. J. 2002; 367: 121-128Crossref PubMed Scopus (122) Google Scholar, 23Resta R. Yamashita Y. Thompson L.F. Immunol. Rev. 1998; 161: 95-109Crossref PubMed Scopus (271) Google Scholar, 24Spychala J. Pharmacol. Ther. 2000; 87: 161-173Crossref PubMed Scopus (239) Google Scholar). Independently of the enzymatic function, the protein acts as co-receptor in T cell activation (for review, see Ref. 23Resta R. Yamashita Y. Thompson L.F. Immunol. Rev. 1998; 161: 95-109Crossref PubMed Scopus (271) Google Scholar) and as cell adhesion molecule (for review, see Ref. 24Spychala J. Pharmacol. Ther. 2000; 87: 161-173Crossref PubMed Scopus (239) Google Scholar). eN is variably expressed in a wide number of cell types under physiological and pathological conditions (for review, see Refs. 20Zimmermann H. Biochem. J. 1992; 285: 345-365Crossref PubMed Scopus (760) Google Scholar, 23Resta R. Yamashita Y. Thompson L.F. Immunol. Rev. 1998; 161: 95-109Crossref PubMed Scopus (271) Google Scholar, and 24Spychala J. Pharmacol. Ther. 2000; 87: 161-173Crossref PubMed Scopus (239) Google Scholar). In neuronal cells eN expression is linked to developing or plastic states (for review, see Ref. 24Spychala J. Pharmacol. Ther. 2000; 87: 161-173Crossref PubMed Scopus (239) Google Scholar). The proximal promoter region of the gene contains a number of tissue-specific elements (25Hansen K.R. Resta R. Webb C.F. Thompson L.F. Gene (Amst.). 1995; 167: 307-312Crossref PubMed Scopus (43) Google Scholar, 26Spychala J. Zimmermann A.G. Mitchell B.S. J. Biol. Chem. 1999; 274: 22705-22712Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Cytosolic 5′-Nucleotidase IA—cN-IA was named AMP-specific 5′-nucleotidase for its high specific activity with AMP at millimolar concentrations. Subsequent detailed kinetic studies revealed high affinities toward deoxypyrimidine monophosphates (18Garvey E.P. Lowen G.T. Almond M.R. Biochemistry. 1998; 37: 9043-9051Crossref PubMed Scopus (32) Google Scholar). It is highly expressed in skeletal and heart muscle where it has a physiological function in the generation of signaling adenosine during ischemia (2Sala-Newby G.B. Skladanowski A.C. Newby A.C. J. Biol. Chem. 1999; 274: 17789-17793Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 27Sala-Newby G.B. Freeman N.V. Skladanowski A.C. Newby A.C. J. Biol. Chem. 2000; 275: 11666-11671Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The high affinity for deoxynucleoside monophosphates suggests a role in the regulation of deoxyribonucleotide pools. A homologous sequence related to human autoimmune infertility gene (AIRP) and with highest expression in testis has been cloned and designated cN-IB (4Sala-Newby G.B. Newby A.C. Biochim. Biophys. Acta. 2001; 1521: 12-18Crossref PubMed Scopus (28) Google Scholar). Cytosolic 5′-Nucleotidase II—cN-II is a 6-hydroxypurine-specific nucleotidase, most active with (d)IMP (for review, see Ref. 14Itoh R. Comp. Biochem. Physiol. [B]. 1993; 105: 13-19Crossref PubMed Scopus (57) Google Scholar) and critically positioned to regulate ATP and GTP pools. This tetrameric protein is stimulated by (d)ATP and GTP and regulated by substrate and phosphate in a complex manner (for review, see Ref. 14Itoh R. Comp. Biochem. Physiol. [B]. 1993; 105: 13-19Crossref PubMed Scopus (57) Google Scholar) (27Sala-Newby G.B. Freeman N.V. Skladanowski A.C. Newby A.C. J. Biol. Chem. 2000; 275: 11666-11671Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 28Spychala J. Madrid-Marina V. Fox I.H. J. Biol. Chem. 1988; 263: 18759-18765Abstract Full Text PDF PubMed Google Scholar, 29Gazziola C. Moras M. Ferraro P. Gallinaro L. Verin R. Rampazzo C. Reichard P. Bianchi V. Exp. Cell Res. 1999; 253: 474-482Crossref PubMed Scopus (31) Google Scholar), possibly involving subunit association and dissociation (30Spychala J. Chen V. Oka J. Mitchell B.S. Eur. J. Biochem. 1999; 259: 851-858Crossref PubMed Scopus (29) Google Scholar). Under physiological conditions cN-II can catalyze phosphotransfer from a purine nucleotide donor to inosine or guanosine (31Worku Y. Newby A.C. Biochem. J. 1982; 205: 503-510Crossref PubMed Scopus (60) Google Scholar, 32Pesi R. Turriani M. Allegrini S. Scolozzi C. Camici M. Ipata P.L. Tozzi M.G. Arch. Biochem. Biophys. 1994; 312: 75-80Crossref PubMed Scopus (76) Google Scholar). This reaction is responsible for the activation of several anti-viral and anti-cancer nucleoside analogs that are not substrates for cellular nucleoside kinases (33Johnson M.A. Fridland A. Mol. Pharmacol. 1989; 36: 291-295PubMed Google Scholar, 34Keller P.M. McKee S.A. Fyfe J.A. J. Biol. Chem. 1985; 260: 8664-8667Abstract Full Text PDF PubMed Google Scholar). Cytosolic 5′-Nucleotidase III—cN-III is highly expressed in red blood cells where it participates in the degradation of RNA during erythrocyte maturation (for review, see Ref. 15Rees D.C. Duley J.A. Marinaki A.M. Br. J. Haematol. 2003; 120: 375-383Crossref PubMed Scopus (47) Google Scholar). It prefers pyrimidine riboover deoxyribonucleotides with CMP being the best substrate. It is inactive with purine nucleotides. The enzyme has a phosphotransferase activity (35Amici A. Emanuelli M. Magni G. Raffaelli N. Ruggieri S. FEBS Lett. 1997; 419: 263-267Crossref PubMed Scopus (51) Google Scholar) less efficient than cN-II (32Pesi R. Turriani M. Allegrini S. Scolozzi C. Camici M. Ipata P.L. Tozzi M.G. Arch. Biochem. Biophys. 1994; 312: 75-80Crossref PubMed Scopus (76) Google Scholar). The sequence of cN-III coincides with that of p36, an interferon α-induced protein of unknown function (6Amici A. Emanuelli M. Raffaelli N. Ruggieri S. Saccucci F. Magni G. Blood. 2000; 96: 1596-1598Crossref PubMed Google Scholar). Alternative splicing of exon 2 gives rise to two proteins that are 286 and 297 amino acids long (36Marinaki A.M. Escuredo E. Dudley J.A. Simmonds H.A. Amici A. Naponelli V. Magni G. Seip M. Ben-Bassat I. Harley E.H. Lay Thein S. Rees D.C. Blood. 2001; 97: 3327-3332Crossref PubMed Scopus (53) Google Scholar), with the shorter form corresponding to cN-III. Cytosolic 5′(3′)-Deoxynucleotidase—cdN is a ubiquitous enzyme, first purified to homogeneity from human placenta (37Höglund L. Reichard P. J. Biol. Chem. 1990; 265: 6589-6595Abstract Full Text PDF PubMed Google Scholar). It is the major deoxynucleotidase activity in cultured human cells (17Rampazzo C. Mazzon C. Reichard P. Bianchi V. Biochem. Biophys. Res. Commun. 2002; 293: 258-263Crossref PubMed Scopus (23) Google Scholar, 38Gallinaro L. Crovatto K. Rampazzo C. Pontarin G. Ferraro P. Milanesi E. Reichard P. Bianchi V. J. Biol. Chem. 2002; 277: 35080-35087Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). In contrast to cN-III, human cdN is not strictly pyrimidine-specific and works efficiently with dIMP and dGMP. dAMP is a poor substrate and dCMP is inactive (8Mazzon C. Rampazzo C. Scaini M.C. Gallinaro L. Karlsson A. Meier C. Balzarini J. Reichard P. Bianchi V. Biochem. Pharmacol. 2003; 66: 471-479Crossref PubMed Scopus (90) Google Scholar, 37Höglund L. Reichard P. J. Biol. Chem. 1990; 265: 6589-6595Abstract Full Text PDF PubMed Google Scholar). The enzyme is very active on 2′- and 3′-phosphates (7Rampazzo C. Johansson M. Gallinaro L. Ferraro P. Hellman U. Karlsson A. Reichard P. Bianchi V. J. Biol. Chem. 2000; 275: 5409-5415Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 37Höglund L. Reichard P. J. Biol. Chem. 1990; 265: 6589-6595Abstract Full Text PDF PubMed Google Scholar). Neither the highly purified human placental cdN nor the recombinant mouse and human enzymes showed phosphotransferase activity (7Rampazzo C. Johansson M. Gallinaro L. Ferraro P. Hellman U. Karlsson A. Reichard P. Bianchi V. J. Biol. Chem. 2000; 275: 5409-5415Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 37Höglund L. Reichard P. J. Biol. Chem. 1990; 265: 6589-6595Abstract Full Text PDF PubMed Google Scholar), in contrast to what was reported for cdN purified from human red blood cells (35Amici A. Emanuelli M. Magni G. Raffaelli N. Ruggieri S. FEBS Lett. 1997; 419: 263-267Crossref PubMed Scopus (51) Google Scholar). Mitochondrial 5′(3′)-Deoxynucleotidase—mdN is highly homologous to cytosolic cdN (52% amino acid identity). The two enzymes are coded by nuclear genes with identical structure, probably derived by a gene duplication event (9Rampazzo C. Gallinaro L. Milanesi E. Frigimelica E. Reichard P. Bianchi V. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8239-8244Crossref PubMed Scopus (84) Google Scholar). With its high preference for dUMP and dTMP, mdN shows remarkably narrow substrate specificity. Similarly to cdN, mdN prefers deoxyover ribonucleotides and accepts 2′- and 3′-nucleoside monophosphates (9Rampazzo C. Gallinaro L. Milanesi E. Frigimelica E. Reichard P. Bianchi V. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8239-8244Crossref PubMed Scopus (84) Google Scholar, 38Gallinaro L. Crovatto K. Rampazzo C. Pontarin G. Ferraro P. Milanesi E. Reichard P. Bianchi V. J. Biol. Chem. 2002; 277: 35080-35087Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Its enzymatic features suggest that mdN regulates mitochondrial dTTP and prevents accumulation of mutagenic dUTP within mitochondria. By opposing the phosphorylation of nucleosides by kinases, intracellular 5′-nucleotidases participate in substrate cycles that regulate the cellular levels of ribo- and deoxyribonucleoside monophosphates and thereby all ribo- and deoxyribonucleotide pools (for review, see Ref. 39Reichard P. Annu. Rev. Biochem. 1988; 57: 349-374Crossref PubMed Scopus (634) Google Scholar) (40Gazziola C. Ferraro P. Moras M. Reichard P. Bianchi V. J. Biol. Chem. 2001; 276: 6185-6190Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Intracellular 5′-nucleotidases have relatively high Km values and operate on substrates generally present at (very) low concentrations. Thus they are exquisitely sensitive to oscillations of substrate concentration. Given their overlapping substrate specificities, it is difficult to tie a given enzyme to the maintenance of a specific nucleotide pool. Important information has been obtained with cell lines engineered to overexpress individual nucleotidases. Involvement of a 5′-nucleotidase in a specific substrate cycle is signaled by the increased excretion of the nucleoside produced by that cycle (40Gazziola C. Ferraro P. Moras M. Reichard P. Bianchi V. J. Biol. Chem. 2001; 276: 6185-6190Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). In such experiments it is important to analyze the turnover of individual pools during brief time windows. Changes in nucleotide pool sizes only show the final end point of complex metabolic adaptations and may be a consequence of reduced ATP availability (29Gazziola C. Moras M. Ferraro P. Gallinaro L. Verin R. Rampazzo C. Reichard P. Bianchi V. Exp. Cell Res. 1999; 253: 474-482Crossref PubMed Scopus (31) Google Scholar, 41Rampazzo C. Gazziola C. Ferraro P. Gallinaro L. Johansson M. Reichard P. Bianchi V. Eur. J. Biochem. 1999; 261: 689-697Crossref PubMed Scopus (42) Google Scholar). By this strategy cN-IA was shown to operate on AMP (27Sala-Newby G.B. Freeman N.V. Skladanowski A.C. Newby A.C. J. Biol. Chem. 2000; 275: 11666-11671Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 42Sala-Newby G.B. Freeman N.V. Curto M.A. Newby A.C. Am. J. Physiol. 2003; 285: H991-H998Crossref PubMed Scopus (15) Google Scholar) and cN-II on IMP and GMP (27Sala-Newby G.B. Freeman N.V. Skladanowski A.C. Newby A.C. J. Biol. Chem. 2000; 275: 11666-11671Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 40Gazziola C. Ferraro P. Moras M. Reichard P. Bianchi V. J. Biol. Chem. 2001; 276: 6185-6190Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), and murine cdN was shown to regulate all pyrimidine deoxyribonucleotide pools (40Gazziola C. Ferraro P. Moras M. Reichard P. Bianchi V. J. Biol. Chem. 2001; 276: 6185-6190Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). In human cells dCMP should be dephosphorylated by a different enzyme, as human cdN is inactive on dCMP. A potential candidate is cN-IA that has high affinity for all deoxyribonucleotides (3Hunsucker S.A. Spychala J. Mitchell B.S. J. Biol. Chem. 2001; 276: 10498-10504Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 18Garvey E.P. Lowen G.T. Almond M.R. Biochemistry. 1998; 37: 9043-9051Crossref PubMed Scopus (32) Google Scholar), although it is still not clear whether the expression of this enzyme outside skeletal and heart muscle is sufficient to perform this function (3Hunsucker S.A. Spychala J. Mitchell B.S. J. Biol. Chem. 2001; 276: 10498-10504Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Strategies such as knockout mice and small interfering RNA may help solve these issues. Indeed, down-regulation of mdN in cultured human cells by small interfering RNA showed that mdN participates in a mitochondrial substrate cycle with the mitochondrial thymidine kinase. 3C. Rampazzo and V. Bianchi, unpublished data. The only known genetic syndrome to 5′-nucleotidase is the by of cN-III (for review, see Ref. 15Rees D.C. Duley J.A. Marinaki A.M. Br. J. Haematol. 2003; 120: 375-383Crossref PubMed Scopus (47) Google Scholar). of pyrimidine in of the important role of cN-III during maturation of red blood and nucleoside analogs be by phosphorylation to their Intracellular 5′-nucleotidases the of the analogs by the activation and thereby their of the in of nucleoside were linked to high expression of cN-II and cN-IA (3Hunsucker S.A. Spychala J. Mitchell B.S. J. Biol. Chem. 2001; 276: 10498-10504Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, T. Biochim. Biophys. Acta. 1991; PubMed Scopus Google Scholar) (for review, see Ref. C. 2001; PubMed Scopus Google Scholar). with the substrate cycle the relative of nucleoside to 5′-nucleotidase may have with 5′-nucleotidases to K. E. L. C. Br. J. Haematol. 2003; PubMed Scopus (84) Google Scholar). of that inhibit 5′-nucleotidase activity may and the of nucleoside analogs that are poor substrates for 5′-nucleotidases may to more anti-viral show mitochondrial that a on their M.C. Nat. 1995; PubMed Scopus Google Scholar). to this problem is to that are by cytosolic and mitochondrial 5′-nucleotidases, for accumulation of active of cdN and mdN inhibitors that these enzymes suggests that such strategy is (17Rampazzo C. Mazzon C. Reichard P. Bianchi V. Biochem. Biophys. Res. Commun. 2002; 293: 258-263Crossref PubMed Scopus (23) Google Scholar). The in the human of at genes for 5′-nucleotidases suggests that these enzymes perform important metabolic With the enzymes in recombinant form it be to their Gene regulation an field. We not is the tissue-specific expression of cN-III, the expression levels of the ubiquitous nucleotidases revealed by are expression of individual enzymes can be or in specific and of expression are in specific