Characterization of recombinant human nicotinamide mononucleotide adenylyl transferase (NMNAT), a nuclear enzyme essential for NAD synthesis

Schweiger, Manfred; Hennig, K.; Lerner, Felicitas; Niere, Marc; Hirsch‐Kauffmann, Monica; Specht, Thomas; Weise, Christoph; Oei, Shiao Li; Ziegler, Mathias

doi:10.1016/s0014-5793(01)02180-9

Cited by 118 publications

(124 citation statements)

References 28 publications

Supporting

Mentioning

122

Contrasting

Order By: Relevance

“…Sections were pretreated by incubating overnight at 451C in 1 mM sodium citrate, pH 6.0, followed by 10 min in 0.1% Trypsin in PBS. They were then immunostained for Nmnat1 using two different affinity-purified rabbit polyclonal antibodies (each diluted 1 : 50) that detect only Nmnat1 on Western blots: one kindly supplied by Professor Mathias Ziegler (Figure 2), 34 Nerve lesion. Mice were anaesthetised with a mixture of ketamine (100 mg/kg; Fort Dodge Animal Health, Southampton) and xylazine (5 mg/kg; Parke Davis/ Pfizer, Karlsruhe, Germany).…”

Section: Methodsmentioning

confidence: 99%

NAD+ and axon degeneration revisited: Nmnat1 cannot substitute for WldS to delay Wallerian degeneration

et al. 2006

View full text Add to dashboard Cite

The slow Wallerian degeneration protein (Wld S ), a fusion protein incorporating full-length nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1), delays axon degeneration caused by injury, toxins and genetic mutation. Nmnat1 overexpression is reported to protect axons in vitro, but its effect in vivo and its potency remain unclear. We generated Nmnat1-overexpressing transgenic mice whose Nmnat activities closely match that of Wld S mice. Nmnat1 overexpression in five lines of transgenic mice failed to delay Wallerian degeneration in transected sciatic nerves in contrast to Wld S mice where nearly all axons were protected. Transected neurites in Nmnat1 transgenic dorsal root ganglion explant cultures also degenerated rapidly. The delay in vincristine-induced neurite degeneration following lentiviral overexpression of Nmnat1 was significantly less potent than for Wld S , and lentiviral overexpressed enzyme-dead Wld S still displayed residual neurite protection. Thus, Nmnat1 is significantly weaker than Wld S at protecting axons against traumatic or toxic injury in vitro, and has no detectable effect in vivo. The full protective effect of Wld S requires more N-terminal sequences of the protein.

show abstract

Section: Methodsmentioning

confidence: 99%

NAD+ and axon degeneration revisited: Nmnat1 cannot substitute for WldS to delay Wallerian degeneration

et al. 2006

View full text Add to dashboard Cite

show abstract

“…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).…”

Section: The Coenzymes Nadmentioning

confidence: 99%

“…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). Crystal structures of hsPNAT-1 have been solved in its apo form (20) and in complex with NMN (21), NAD, NaAD, or TAD (19).…”

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

Journal of Biological Chemistry

125

148

View full text Add to dashboard Cite

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...

show abstract

“…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

Journal of Biological Chemistry

869

792

View full text Add to dashboard Cite

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.

show abstract

Characterization of recombinant human nicotinamide mononucleotide adenylyl transferase (NMNAT), a nuclear enzyme essential for NAD synthesis

Cited by 118 publications

References 28 publications

NAD+ and axon degeneration revisited: Nmnat1 cannot substitute for WldS to delay Wallerian degeneration

NAD+ and axon degeneration revisited: Nmnat1 cannot substitute for WldS to delay Wallerian degeneration

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

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

Contact Info

Product

Resources

About