Publications

• Nicotinamide mononucleotide adenylyltransferase. Molecular and enzymatic properties of the homogeneous enzyme from baker's yeast.

  Natalini P., Ruggieri S., Raffaelli N., Magni G.

  Nicotinamide mononucleotide (NMN) adenylyltransferase has been purified to homogeneity from baker's yeast crude extract. The purification procedure is relatively simple and consists of high-salt extraction of enzyme activity and precipitation with poly(ethylenimine), followed by ion-exchange and d ... >> More

  Nicotinamide mononucleotide (NMN) adenylyltransferase has been purified to homogeneity from baker's yeast crude extract. The purification procedure is relatively simple and consists of high-salt extraction of enzyme activity and precipitation with poly(ethylenimine), followed by ion-exchange and dye ligand chromatography separations. The final enzyme preparation is homogeneous as judged by a single Coomassie blue stainable band when run on nondenaturating and denaturating polyacrylamide gels. The native enzyme shows a molecular weight of about 200 000, calculated by gel filtration and sucrose gradient centrifugation. The protein possesses quaternary structure and is composed of four apparently identical Mr 50 000 subunits. The absorption spectrum shows a maximum at 280 nm and a minimum at 253 nm. The isoelectric point is 6.2. Amino acid composition analysis shows the presence of 28 half-cystine residues. The same result has been obtained by titrating the enzyme in denaturating conditions with Ellman's reagent after incubation with sodium borohydride. NMN adenylyltransferase is a glycoprotein containing 2% sugar, 2 mol of alkali-labile phosphate per mole of enzyme, and 1 mol of adenine moiety per mole of enzyme. Therefore, the possibility that the enzyme is ADP-ribosylated exists. The Km values for ATP, NMN, and nicotinate mononucleotide are 0.11 mM, 0.19 nM, and 5 mM, respectively. Kinetic analysis reveals a behavior that is consistent with an ordered sequential Bi-Bi mechanism. The pH optimum is in the range 7.2-8.4. << Less

  Biochemistry 25:3725-3729(1986) [PubMed] [EuropePMC]

• Initial-rate kinetics of human NMN-adenylyltransferases: substrate and metal ion specificity, inhibition by products and multisubstrate analogues, and isozyme contributions to NAD+ biosynthesis.

  Sorci L., Cimadamore F., Scotti S., Petrelli R., Cappellacci L., Franchetti P., Orsomando G., Magni G.

  Initial-rate and product inhibition studies revealed distinctive ordered ternary complex kinetic mechanisms, substrate specificities, and metal ion preferences for the three isozymes of human nicotinamide mononucleotide adenylyl-transferase (NMNAT, EC 2.7.7.1). ATP binds before NMN with nuclear is ... >> More

  Initial-rate and product inhibition studies revealed distinctive ordered ternary complex kinetic mechanisms, substrate specificities, and metal ion preferences for the three isozymes of human nicotinamide mononucleotide adenylyl-transferase (NMNAT, EC 2.7.7.1). ATP binds before NMN with nuclear isozyme NMNAT1 and Golgi apparatus NMNAT2, but the opposite order is observed with the mitochondrial isozyme NMNAT3. Only the latter utilizes ITP efficiently in place of ATP, and while NMNH conversion to NADH by NMNAT1 and NMNAT3 occurs at similar rates, conversion by NMNAT2 is much slower. These isozymes can also be discriminated by their action on tiazofurin monophosphate (TrMP), a metabolite of the antineoplastic prodrug tiazofurin. Our finding that TrMP is only a substrate with NMNAT1 and NMNAT3 reveals for the first time an organelle selectivity in the metabolism of this important drug. In search of additional ways to discriminate these isozymes, we synthesized and tested the P1-(nicotinamide/nicotinate-riboside-5')-Pn-(adenosine-5') dinucleotides Np3AD, Np4AD, and Nap4AD. In addition to being highly effective inhibitors, these multisubstrate geometric inhibitors gave inhibition patterns that are consistent with the aforementioned isozyme differences in substrate binding order. Distinctive differences in their substrate specificity and metal ion selectivity also permitted us to quantify individual isozyme contributions to NAD+ formation in human cell extracts. << Less

  Biochemistry 46:4912-4922(2007) [PubMed] [EuropePMC]

• [Concurrent nonspecific pathology in patients with osteoarticular tuberculosis].

  Chenskikh E.P., Tian A.D.

  Probl Tuberk 1987:10-12(1987) [PubMed] [EuropePMC]

• Molecular cloning, chromosomal localization, tissue mRNA levels, bacterial expression, and enzymatic properties of human NMN adenylyltransferase.

  Emanuelli M., Carnevali F., Saccucci F., Pierella F., Amici A., Raffaelli N., Magni G.

  A 1329-base pair clone isolated from a human placenta cDNA library contains a full-length 837-base pair coding region for a 31.9-kDa protein whose deduced primary structure exhibits high homology to consensus sequences found in other NMN adenylyltransferases. Northern blotting detected a major 3.1 ... >> More

  A 1329-base pair clone isolated from a human placenta cDNA library contains a full-length 837-base pair coding region for a 31.9-kDa protein whose deduced primary structure exhibits high homology to consensus sequences found in other NMN adenylyltransferases. Northern blotting detected a major 3.1-kilobase mRNA transcript as well as a minor 4.1-kilobase transcript in all human tissues examined. In several cancer cell lines, lower levels of mRNA expression were clearly evident. The gene encoding the human enzyme was mapped to chromosome band 1p32-35. High efficiency bacterial expression yielded 1.5 mg of recombinant enzyme/liter of culture medium. The molecular and kinetic properties of recombinant human NMN adenylyltransferase provide new directions for investigating metabolic pathways involving this enzyme. << Less

  J. Biol. Chem. 276:406-412(2001) [PubMed] [EuropePMC]

• YCL047C/POF1 is a novel nicotinamide mononucleotide adenylyltransferase (NMNAT) in Saccharomyces cerevisiae.

  Kato M., Lin S.J.

  NAD(+) is an essential metabolic cofactor involved in various cellular biochemical processes. Nicotinamide riboside (NR) is an endogenously produced key pyridine metabolite that plays important roles in the maintenance of NAD(+) pool. Using a NR-specific cell-based screen, we identified mutants th ... >> More

  NAD(+) is an essential metabolic cofactor involved in various cellular biochemical processes. Nicotinamide riboside (NR) is an endogenously produced key pyridine metabolite that plays important roles in the maintenance of NAD(+) pool. Using a NR-specific cell-based screen, we identified mutants that exhibit altered NR release phenotype. Yeast cells lacking the ORF YCL047C/POF1 release considerably more NR compared with wild type, suggesting that POF1 plays an important role in NR/NAD(+) metabolism. The amino acid sequence of Pof1 indicates that it is a putative nicotinamide mononucleotide adenylyltransferase (NMNAT). Unlike other yeast NMNATs, Pof1 exhibits NMN-specific adenylyltransferase activity. Deletion of POF1 significantly lowers NAD(+) levels and decreases the efficiency of NR utilization, resistance to oxidative stress, and NR-induced life span extension. We also show that NR is constantly produced by multiple nucleotidases and that the intracellular NR pools are likely to be compartmentalized, which contributes to the regulation of NAD(+) homeostasis. Our findings may contribute to the understanding of the molecular basis and regulation of NAD(+) metabolism in higher eukaryotes. << Less

  J. Biol. Chem. 289:15577-15587(2014) [PubMed] [EuropePMC]

• The deamido-diphosphopyridine nucleotide and diphosphopyridine nucleotide pyrophosphorylases of Escherichia coli and yeast.

  Dahmen W., Webb B., Preiss J.

  Arch. Biochem. Biophys. 120:440-450(1967) [PubMed] [EuropePMC]

• The NAD biosynthesis pathway mediated by nicotinamide phosphoribosyltransferase regulates Sir2 activity in mammalian cells.

  Revollo J.R., Grimm A.A., Imai S.

  Recent studies have revealed new roles for NAD and its derivatives in transcriptional regulation. The evolutionarily conserved Sir2 protein family requires NAD for its deacetylase activity and regulates a variety of biological processes, such as stress response, differentiation, metabolism, and ag ... >> More

  Recent studies have revealed new roles for NAD and its derivatives in transcriptional regulation. The evolutionarily conserved Sir2 protein family requires NAD for its deacetylase activity and regulates a variety of biological processes, such as stress response, differentiation, metabolism, and aging. Despite its absolute requirement for NAD, the regulation of Sir2 function by NAD biosynthesis pathways is poorly understood in mammals. In this study, we determined the kinetics of the NAD biosynthesis mediated by nicotinamide phosphoribosyltransferase (Nampt) and nicotinamide/nicotinic acid mononucleotide adenylyltransferase (Nmnat), and we examined its effects on the transcriptional regulatory function of the mouse Sir2 ortholog, Sir2alpha, in mouse fibroblasts. We found that Nampt was the rate-limiting component in this mammalian NAD biosynthesis pathway. Increased dosage of Nampt, but not Nmnat, increased the total cellular NAD level and enhanced the transcriptional regulatory activity of the catalytic domain of Sir2alpha recruited onto a reporter gene in mouse fibroblasts. Gene expression profiling with oligonucleotide microarrays also demonstrated a significant correlation between the expression profiles of Nampt- and Sir2alpha-overexpressing cells. These findings suggest that NAD biosynthesis mediated by Nampt regulates the function of Sir2alpha and thereby plays an important role in controlling various biological events in mammals. << Less

  J. Biol. Chem. 279:50754-50763(2004) [PubMed] [EuropePMC]

• Enzymatic cleavage of diphosphopyridine nucleotide with radioactive pyrophosphate.

  KORNBERG A., PRICER W.E. Jr.

  J Biol Chem 191:535-541(1951) [PubMed] [EuropePMC]

• Identification and characterization of YLR328W, the Saccharomyces cerevisiae structural gene encoding NMN adenylyltransferase. Expression and characterization of the recombinant enzyme.

  Emanuelli M., Carnevali F., Lorenzi M., Raffaelli N., Amici A., Ruggieri S., Magni G.

  The enzyme nicotinamide mononucleotide (NMN) adenylyltransferase (EC 2.7.7.1) catalyzes the transfer of the adenylyl moiety of ATP to NMN to form NAD. A new purification procedure for NMN adenylyltransferase from Saccharomyces cerevisiae provided sufficient amounts of enzyme for tryptic fragmentat ... >> More

  The enzyme nicotinamide mononucleotide (NMN) adenylyltransferase (EC 2.7.7.1) catalyzes the transfer of the adenylyl moiety of ATP to NMN to form NAD. A new purification procedure for NMN adenylyltransferase from Saccharomyces cerevisiae provided sufficient amounts of enzyme for tryptic fragmentation. Through data-base search a full matching was found between the sequence of tryptic fragments and the sequence of a hypothetical protein encoded by the S. cerevisiae YLR328W open reading frame (GenBank accession number U20618). The YLR328W gene was isolated, cloned into a T7-based vector and successfully expressed in Escherichia coli BL21 cells, yielding a high level of NMN adenylyltransferase activity. The purification of recombinant protein, by a two-step chromatographic procedure, resulted in a single polypeptide of 48 kDa under SDS-PAGE, in agreement with the molecular mass of the hypothetical protein encoded by YLR328W ORF. The N-terminal sequence of the purified recombinant NMN adenylyltransferase exactly corresponds to the predicted sequence. Molecular and kinetic properties of recombinant NMN adenylyltransferase are reported and compared with those already known for the enzyme obtained from different sources. << Less

  FEBS Lett. 455:13-17(1999) [PubMed] [EuropePMC]

• Regulation of NAD synthesis by the trifunctional NadR protein of Salmonella enterica.

  Grose J.H., Bergthorsson U., Roth J.R.

  The three activities of NadR were demonstrated in purified protein and assigned to separate domains by missense mutations. The N-terminal domain represses transcription of genes for NAD synthesis and salvage. The C-terminal domain has nicotinamide ribose kinase (NmR-K; EC 2.7.1.22) activity, which ... >> More

  The three activities of NadR were demonstrated in purified protein and assigned to separate domains by missense mutations. The N-terminal domain represses transcription of genes for NAD synthesis and salvage. The C-terminal domain has nicotinamide ribose kinase (NmR-K; EC 2.7.1.22) activity, which is essential for assimilation of NmR, converting it internally to nicotinamide mononucleotide (NMN). The central domain has a weak adenylyltransferase (NMN-AT; EC 2.7.7.1) activity that converts NMN directly to NAD but is physiologically irrelevant. This central domain mediates regulatory effects of NAD on all NadR activities. In the absence of effectors, pure NadR protein binds operator DNA (the default state) and is released by ATP (expected to be present in vivo). NAD allows NadR to bind DNA in the presence of ATP and causes repression in vivo. A superrepressor mutation alters an ATP-binding residue in the central (NMN-AT) domain. This eliminates NMN-AT activity and places the enzyme in its default (DNA binding) state. The mutant protein shows full NmR kinase activity that is 10-fold more sensitive to NAD inhibition than the wild type. It is proposed that NAD and the superrepressor mutation exert their effects by preventing ATP from binding to the central domain. << Less

  J. Bacteriol. 187:2774-2782(2005) [PubMed] [EuropePMC]

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