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

NAD synthetase catalyzes the final step in the biosynthesis of NAD. In the present study, we obtained cDNAs for two types of human NAD synthetase (referred as NADsyn1 and NADsyn2). Structural analysis revealed in both NADsyn1 and NADsyn2 a domain required for NAD synthesis from ammonia and in only NADsyn1 an additional carbon-nitrogen hydrolase domain shared with enzymes of the nitrilase family that cleave nitriles as well as amides to produce the corresponding acids and ammonia. Consistent with the domain structures, biochemical assays indicated (i) that both NADsyn1 and NADsyn2 have NAD synthetase activity, (ii) that NADsyn1 uses glutamine as well as ammonia as an amide donor, whereas NADsyn2 catalyzes only ammoniadependent NAD synthesis, and (iii) that mutant NADsyn1 in which Cys-175 corresponding to the catalytic cysteine residue in nitrilases was replaced with Ser does not use glutamine. Kinetic studies suggested that glutamine and ammonia serve as physiological amide donors for NADsyn1 and NADsyn2, respectively. Both synthetases exerted catalytic activity in a multimeric form. In the mouse, NADsyn1 was seen to be abundantly expressed in the small intestine, liver, kidney, and testis but very weakly in the skeletal muscle and heart. In contrast, expression of NADsyn2 was observed in all tissues tested. Therefore, we conclude that humans have two types of NAD synthetase exhibiting different amide donor specificity and tissue distributions. The ammoniadependent synthetase has not been found in eucaryotes until this study. Our results also indicate that the carbon-nitrogen hydrolase domain is the functional domain of NAD synthetase to make use of glutamine as an amide donor in NAD synthesis. Thus, glutamine-dependent NAD synthetase may be classified as a possible glutamine amidase in the nitrilase family. Our molecular identification of NAD synthetases may prove useful to learn more of mechanisms regulating cellular NAD metabolism.The coenzyme NAD has a role in the majority of metabolic redox reactions and represents an essential component of metabolic pathways in all living cells. In a number of signaling pathways, NAD also serves as a precursor of potent calciummobilizing agents such as cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate (1) and serves as a substrate for post-translational modifications of protein, mono-(2-4) and poly(ADP-ribosyl)ations (5). Depletion of cellular NAD by poly-(ADP-ribosyl)transferase activation in response to DNA damage results in cell death (6). Increased NAD synthesis has been shown to extend life span in yeast (7) and in Caenorhabditis elegans (8) via activation of an NAD-dependent histone deacetylase, silent information regulator 2 (Sir2) (9). The cellular level of NAD may modulate the sensitivity of cells to apoptotic responses through deacetylation of the p53 tumor suppressor by a human homologue of Sir2 (10). Recent publications have demonstrated that fluctuation of the NAD level in cells seems to have significant impact on their physiology. Despite the...

Section: Discussionmentioning

confidence: 99%

“…1). Although most of the genes involved in both pathways have been identified in procaryotes (13), little is known of those genes, including that of NAD synthetase in eucaryotes, except for nicotinamide mononucleotide adenylyltransferase (14) and quinolinic acid phosphoribosyltransferase (15) genes.…”

mentioning

confidence: 99%

Molecular Identification of Human Glutamine- and Ammonia-dependent NAD Synthetases

Hara

Yamada

Terashima

et al. 2003

Section: The Coenzymes Nadmentioning

confidence: 99%

“…In human, two PNAT isoforms (gi 11245478 and gi 12620200) have been cloned and purified, and their kinetic properties were analyzed (23)(24)(25)(26). Human nuclear PNAT-1 was shown to present exclusively in the nuclei and express at high levels in heart, skeletal muscle and, to a lesser extent, in kidney and liver (23). Human PNAT-1 has also been reported to interact specifically with poly(ADP-ribose) polymerase (24,27) and may be subjected to further regulation by phosphorylation (24).…”

Section: The Coenzymes Nadmentioning

confidence: 99%

Structural Characterization of a Human Cytosolic NMN/NaMN Adenylyltransferase and Implication in Human NAD Biosynthesis

Zhang

Kurnasov

Karthikeyan

et al. 2003

126

148

Pyridine dinucleotides (NAD and NADP) are ubiquitous cofactors involved in hundreds of redox reactions essential for the energy transduction and metabolism in all living cells. In addition, NAD also serves as a substrate for ADP-ribosylation of a number of nuclear proteins, for silent information regulator 2 (Sir2)-like histone deacetylase that is involved in gene silencing regulation, and for cyclic ADP ribose (cADPR)-dependent Ca 2؉ signaling. Pyridine nucleotide adenylyltransferase (PNAT) is an indispensable central enzyme in the NAD biosynthesis pathways catalyzing the condensation of pyridine mononucleotide (NMN or NaMN) with the AMP moiety of ATP to form NAD (or NaAD). Here we report the identification and structural characterization of a novel human PNAT (hsPNAT-3) that is located in the cytoplasm and mitochondria. Its subcellular localization and tissue distribution are distinct from the previously identified human nuclear PNAT-1 and PNAT-2. Detailed structural analysis of PNAT-3 in its apo form and in complex with its substrate(s) or product revealed the catalytic mechanism of the enzyme. The characterization of the cytosolic human PNAT-3 provided compelling evidence that the final steps of NAD biosynthesis pathways may exist in mammalian cytoplasm and mitochondria, potentially contributing to their NAD/NADP pool. The coenzymes NADϩ (H) 1 and NADP ϩ (H) have been known for many decades as the major hydrogen donor or acceptor in hundreds of metabolic redox reactions throughout the cell. Together these nucleotides have a direct impact on virtually every cellular metabolic pathway. Additionally, NAD serves as a substrate for the covalent modification of nuclear proteins by ADP-ribosylation, a process involved in DNA repair and the regulation of genomic instability (1-3). Recently, many new exciting functions have been discovered for this long-known molecule. These include its role as co-substrate in Sir2-mediated histone deacetylation involved in gene silencing regulation and in increasing the lifespan of species ranging from yeast, to worm, to certain mammals (4, 5). Moreover, several derivatives of NAD and NADP were found to be potent intracellular calcium-mobilizing agents and are involved in a variety of Ca 2ϩ -signaling pathways (6 -8). These recent developments brought a significant amount of additional interest to the investigation of cellular NAD biosynthesis and regulation.NMN and/or NaMN adenylyltransferase (NMNAT and/or NaMNAT, collectively named pyridine nucleotide adenylyltransferase, or PNAT) is an indispensable enzyme catalyzing the central step of all NAD biosynthesis pathways (9, 10). It links the AMP moiety of ATP with the nicotinamide mononucleotide (NMN, or its deamidated form NaMN) to form the dinucleotide product NAD (or deamido-NAD, NaAD). A practical aspect of human PNAT function is that it catalyzes the rate-limiting step in the metabolic conversion of the anticancer agent tiazofurin to its active form TAD (tiazofurin adenine dinucleotide, an NAD analog) (11). The development of ti...

“…Instead of being deamidated, nicotinamide is converted into NMN by nicotinamide phosphoribosyltransferase (Nampt). 1 NMN is then directly synthesized into NAD by nicotinamide/nicotinic acid mononucleotide adenylyltransferase (Nmnat) (28,29).…”

mentioning

confidence: 99%

The NAD Biosynthesis Pathway Mediated by Nicotinamide Phosphoribosyltransferase Regulates Sir2 Activity in Mammalian Cells

Revollo¹,

Grimm²,

Imai

2004

875

792

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, Sir2␣, in mouse fibroblasts. We found that Nampt was the ratelimiting 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 Sir2␣ 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 Sir2␣-overexpressing cells. These findings suggest that NAD biosynthesis mediated by Nampt regulates the function of Sir2␣ and thereby plays an important role in controlling various biological events in mammals.