title: A deoxyribonucleotidase in mitochondria: involvement in regulation of dNTP pools and possible link to genetic disease.
abstract: Three cytosolic and one plasma membrane-bound 5'-nucleotidases have been cloned and characterized. Their various substrate specificities suggest widely different functions in nucleotide metabolism. We now describe a 5'-nucleotidase in mitochondria. The enzyme, named dNT-2, dephosphorylates specifically the 5'- and 2'(3')-phosphates of uracil and thymine deoxyribonucleotides. The cDNA of human dNT-2 codes for a 25.9-kDa polypeptide with a typical mitochondrial leader peptide, providing the structural basis for two-step processing during import into the mitochondrial matrix. The deduced amino acid sequence is 52% identical to that of a recently described cytosolic deoxyribonucleotidase (dNT-1). The two enzymes share many catalytic properties, but dNT-2 shows a narrower substrate specificity. Mitochondrial localization of dNT-2 was demonstrated by the mitochondrial fluorescence of 293 cells expressing a dNT-2-green fluorescent protein (GFP) fusion protein. 293 cells expressing fusion proteins without leader peptide or with dNT-1 showed a cytosolic fluorescence. During in vitro import into mitochondria, the preprotein lost the leader peptide. We suggest that dNT-2 protects mitochondrial DNA replication from overproduction of dTTP, in particular in resting cells. Mitochondrial toxicity of dTTP can be inferred from a severe inborn error of metabolism in which the loss of thymidine phosphorylase led to dTTP accumulation and aberrant mitochondrial DNA replication. We localized the gene for dNT-2 on chromosome 17p11.2 in the Smith-Magenis syndrome-critical region, raising the possibility that dNT-2 is involved in the etiology of this genetic disease.
authors.affiliation: Departments of Biology and Biomedical Sciences, University of Padua, I-35131 Padua, Italy.
authors.forename: C
authors.initials: C
authors.lastname: Rampazzo
authors.forename: L
authors.initials: L
authors.lastname: Gallinaro
authors.forename: E
authors.initials: E
authors.lastname: Milanesi
authors.forename: E
authors.initials: E
authors.lastname: Frigimelica
authors.forename: P
authors.initials: P
authors.lastname: Reichard
authors.forename: V
authors.initials: V
authors.lastname: Bianchi
mesh_terms: D015720:5'-Nucleotidase / Q000235:genetics / Q000378:metabolism*; D000015:Abnormalities, Multiple / Q000201:enzymology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology; D002460:Cell Line; D018076:DNA, Complementary; D003856:Deoxyuracil Nucleotides / Q000378:metabolism*; D030342:Genetic Diseases, Inborn / Q000201:enzymology*; D006801:Humans; D051379:Mice; D008931:Mitochondria, Muscle / Q000201:enzymology*; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology; D009711:Nucleotides / Q000378:metabolism; D011686:Purine-Pyrimidine Metabolism, Inborn Errors / Q000201:enzymology; D051381:Rats; D013379:Substrate Specificity; D013938:Thymidine Monophosphate / Q000378:metabolism*; D014018:Tissue Distribution
chemical_list: D018076:DNA, Complementary; D003856:Deoxyuracil Nucleotides; D009711:Nucleotides; D013938:Thymidine Monophosphate; C007267:2'-deoxyuridylic acid; C063766:5'(3')-nucleotidase; D015720:5'-Nucleotidase