NAD biosynthesis in human placenta: Purification and characterization of homogeneous NMN adenylyltransferase

Emanuelli, Monica; Natalini, Paolo; Raffaelli, Nadia; Ruggieri, Silverio; Vita, Alberto; Magni, Giulio

doi:10.1016/0003-9861(92)90089-f

Cited by 40 publications

(54 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The native molecular mass of the active recombinant enzyme was determined by gel filtration to be 139 kDa. These data indicate that the recombinant enzyme is an oligomer of four identical subunits, agreeing with previous experiments on the wild-type placental enzyme (13).…”

Section: Figsupporting

confidence: 92%

“…An in-frame translational termination codon (TAG) occurs after nucleotide 925. The open reading frame encodes a 279-amino acid protein with a molecular mass of 31,900 Da, close to that estimated by SDS-polyacrylamide gel electrophoresis (13). When the sequences of both tryptic peptides were aligned with the deduced amino acid sequence, there …”

Section: Resultsmentioning

confidence: 57%

“…Human placental NMNAT was purified (13) and subjected to tryptic digestion (17). The resulting fragments were separated by reverse-* This work was supported in part by Consiglio Nazionale delle Ricerche Target Project "Biotechnology" and by Cofinanziamento Ministero dell'Università e della Ricerca Scientifica e Tecnologica "Nucleotidi e Nucleosidi: Segnali Chimici, Regolatori Metabolici e Potenziali Farmaci."…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Molecular Cloning, Chromosomal Localization, Tissue mRNA Levels, Bacterial Expression, and Enzymatic Properties of Human NMN Adenylyltransferase

Emanuelli

Carnevali²,

Saccucci

et al. 2001

Journal of Biological Chemistry

Self Cite

174

123

View full text Add to dashboard Cite

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.Following genomic damage by various chemical or radiation treatments, the extent of malignant transformation is thought to be limited by enhanced DNA repair. Such repair is accomplished by activation of different metabolic processes, some involving NAD ϩ . This indicates that the pyridine coenzyme NAD ϩ plays significant roles beyond its participation in redox metabolism and in various types of ADP-ribosylation. The cellular NAD ϩ concentration likewise appears to modulate expression of stress response proteins, including the tumor suppressor protein p53. In fact, high levels of pyridine dinucleotide reduce the time required for cellular rescue from DNA damage. Skin biopsy specimens from actinic keratoses and squamous cell carcinomas also exhibit an inverse relationship between skin NAD ϩ content and the severity of the malignant phenotype (1). In view of this role of NAD ϩ in cellular rescue from DNA damage, a better understanding of how human NAD ϩ biosynthesis is regulated may be of vital importance in developing effective approaches for preventing and treating cancer.For many years, we have studied NMN adenylyltransferases (NMNATs), 1 which play central roles in both de novo biosynthetic and salvage pathways for nicotinamide nucleotides. These enzymes convert NMN (or nicotinic acid mononucleotide) and ATP to NAD ϩ (or nicotinic acid adenine dinucleotide) and inorganic pyrophosphate (2), and their activity has been correlated with crucial cellular events such as mitosis and DNA synthesis (3, 4). In prokaryotes, cell survival and viability appear to require NMNAT activity (5). Because tumor cell NMNAT concentrations are very low, the enzyme represents a potential chemotherapeutic target (6, 7). This adenylyltransferase catalyzes the essential last step in the metabolic conversion of the potent antitumor agent tiazofurin from its prodrug form to tiazofurin adenine dinucleotide. Low tumor levels of this enzyme activity are associated with the development of drug resistance (8, 9).Our laboratory has purified and characterized NMN adenylyltransferases from yeast, bull testis, thermophilic bacteria, and human placenta (10 -13). We have also identified, cloned, and expressed the gene for this enzyme from Methanoc...

show abstract

Section: Figsupporting

confidence: 92%

Section: Resultsmentioning

confidence: 57%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Molecular Cloning, Chromosomal Localization, Tissue mRNA Levels, Bacterial Expression, and Enzymatic Properties of Human NMN Adenylyltransferase

Emanuelli

Carnevali²,

Saccucci

et al. 2001

Journal of Biological Chemistry

Self Cite

174

123

View full text Add to dashboard Cite

show abstract

“…Comparison of the Active Site of the Human Enzyme with Those of Other NMN Adenylyltransferases-The catalytic mechanism of this key enzyme has been investigated in depth, showing a kinetic behavior fully compatible with an ordered sequential Bi-Bi mechanism (36). According to the proposed mechanism, NMN binds first followed by ATP, and in the reversed reaction, PP i precedes NAD.…”

Section: Resultsmentioning

confidence: 99%

Structure of Human NMN Adenylyltransferase

Garavaglia¹,

D’Angelo²,

Emanuelli³

et al. 2002

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Nicotinamide mononucleotide adenylyltransferase (NMNAT), a member of the nucleotidyltransferase ␣/␤-phosphodiesterases superfamily, catalyzes a universal step (NMN ؉ ATP ‫؍‬ NAD ؉ PP i ) in NAD biosynthesis. Localized within the nucleus, the activity of the human enzyme is greatly altered in tumor cells, rendering it a promising target for cancer chemotherapy. By using a combination of single isomorphous replacement and density modification techniques, the human NMNAT structure was solved by x-ray crystallography to a 2.5-Å resolution, revealing a hexamer that is composed of ␣/␤-topology subunits. The active site topology of the enzyme, analyzed through homology modeling and structural comparison with other NMNATs, yielded convincing evidence for a substrate-induced conformational change. We also observed remarkable structural conservation in the ATP-recognition motifs GXXXPX-(T/H)XXH and SXTXXR, which we take to be the universal signature for NMNATs. Structural comparison of human and prokaryotic NMNATs may also lead to the rational design of highly selective antimicrobial drugs.Beyond its pivotal role as a redox cofactor in energy transduction and cellular metabolism, NAD is also intimately involved in signaling pathways. The NAD(P) derivatives nicotinic acid adenine dinucleotide phosphate and cyclic ADP-ribose are likewise among the most potent calcium-mobilizing agents (1, 2). NAD is the substrate for poly(ADP-ribosyl)ation, a vitally important post-translation modification occurring within the nuclei of higher eukaryotes by poly(ADP-ribose) polymerase (3). NAD is also the substrate for mono-ADP-ribosyltransferase, an enzyme that transfers a single ADP-ribose unit from NAD onto target proteins in both mammalian and prokaryotic cells (4). Most recently, high levels of NAD were shown to extend yeast cell life-span, a phenomenon linked to the action of NAD-dependent catalysis of protein deacetylation (5, 6).Like ATP and GTP, NAD performs multiple tasks in both energy and signaling transduction, indicating why NAD homeostasis must be tightly regulated in all living organisms. Any impairment in NAD synthesis seriously impairs cellular metabolism, eventually resulting in cell death (7). The biosynthesis of this key redox coenzyme is accomplished either through a de novo pathway or through NAD-recycling salvage routes with notable differences between prokaryotes and eukaryotes (8). All the known biochemical pathways converge to the reaction catalyzed by NMN adenylyltransferase (EC 2.7.7.1), abbreviated NMNAT 1 (8). This transferase catalyzes the nucleophilic attack by the 5Ј phosphate NMN or nicotinic acid mononucleotide on the ␣-phosphoryl of ATP, releasing PP i and NAD or nicotinic acid adenine dinucleotide (9). Although the eukaryotic enzyme forms NAD and nicotinic acid adenine dinucleotide at similar rates, its prokaryotic counterpart prefers the deamidated substrate (nicotinic acid mononucleotide) (8). Because the reaction is fully reversible, NAD can also be reconverted to ATP (10).Human NMNAT, the only NAD/n...

show abstract

“…by post-translational modification by poly(ADP-ribosylation)] in response to DNA damage. Emanuelli et al (1992) observed that high concentrations of ADP-ribose inhibited NMMAT. However, no evidence for this was found after MNNG treatment, in either the presence or absence of NU1025 (see Table 2).…”

Section: Discussionmentioning

confidence: 99%

Low nicotinamide mononucleotide adenylyltransferase activity in a tiazofurin-resistant cell line: effects on NAD metabolism and DNA repair

Boulton

Kyle²,

Durkacz³

1997

Br J Cancer

View full text Add to dashboard Cite

Summary Poly(ADP-ribose) polymerase (PADPRP), which uses NAD to synthesize ADP-ribose polymers, is activated by DNA strand breaks and mediates cellular responses to DNA damage. The consequences of low cellular NAD levels in a cell line deficient in nicotinamide mononucleotide adenylyltransferase (NMNAT), an enzyme essential for NAD biosynthesis, were investigated by assessing NAD metabolism and DNA repair after treatment with alkylating agents. A tiazofurin-resistant L1210 cell line (TZR) was isolated. NAD levels were approximately 5933 and 3375 pmol mg-1 protein for parental (wild type, WT) and TZR cells respectively, and NMNAT levels were reduced by > 95%. TZR cells were more sensitive to temozolomide (TM) and 1 -methyl-3-nitro-1 -nitroso-guanidine (MNNG), particularly at concentrations that caused > 50% NAD depletion. TM and MNNG treatment decreased NAD levels in both cell lines, but took longer to return to control levels in TZR cells. For example, MNNG (5 gM), depleted NAD levels at 6 h to approximately 4512 (WT) and 1442 (TZR) pmol mg-1 protein; however, NAD levels had returned to control levels by 8 h in WT cells, but were not restored by 16 h in TZR cells. Both cell lines were equisensitive to the growth-inhibitory effects of NU1025 per se (IC50 370 ,UM). Co-exposure of the cell lines to TM (100 gM) with increasing concentrations of NU1025 led to a synergistic enhancement of cytotoxicity, with IC50 values for NU1025 decreasing to 17 ± 4,gM (TZR) and 37 ± 6 gM (WT). A similar enhanced sensitivity to NU1025 (approximately 2.7-fold) was obtained when TZR cells were co-exposed to MNNG + NU1025. TM-induced DNA strand breaks were increased by co-incubation with NU1025, and again the TZR cell line showed increased sensitivity to NU1025. There were no significant changes in NMNAT activity in response to MNNG treatment over 24 h, either in the presence or in the absence of NU1025. These data demonstrate that modest decreases in cellular NAD levels can sensitize cells to alkylating agents and PADPRP inhibitors.

show abstract

NAD biosynthesis in human placenta: Purification and characterization of homogeneous NMN adenylyltransferase

Cited by 40 publications

References 23 publications

Molecular Cloning, Chromosomal Localization, Tissue mRNA Levels, Bacterial Expression, and Enzymatic Properties of Human NMN Adenylyltransferase

Molecular Cloning, Chromosomal Localization, Tissue mRNA Levels, Bacterial Expression, and Enzymatic Properties of Human NMN Adenylyltransferase

Structure of Human NMN Adenylyltransferase

Low nicotinamide mononucleotide adenylyltransferase activity in a tiazofurin-resistant cell line: effects on NAD metabolism and DNA repair

Contact Info

Product

Resources

About