Nicotinamide mononucleotide adenylyl transferase (NMNAT) is an essential enzyme in all organisms, because it catalyzes a key step of NAD synthesis. However, little is known about the structure and regulation of this enzyme. In this study we established the primary structure of human NMNAT. The human sequence represents the first report of the primary structure of this enzyme for an organism higher than yeast. The enzyme was purified from human placenta and internal peptide sequences determined. Analysis of human DNA sequence data then permitted the cloning of a cDNA encoding this enzyme. Recombinant NMNAT exhibited catalytic properties similar to the originally purified enzyme. Human NMNAT (molecular weight 31 932) consists of 279 amino acids and exhibits substantial structural differences to the enzymes from lower organisms. A putative nuclear localization signal was confirmed by immunofluorescence studies. NMNAT strongly inhibited recombinant human poly(ADP-ribose) polymerase 1, however, NMNAT was not modified by poly(ADP-ribose). NMNAT appears to be a substrate of nuclear kinases and contains at least three potential phosphorylation sites. Endogenous and recombinant NMNAT were phosphorylated in nuclear extracts in the presence of [Q Q-32 P]ATP. We propose that NMNAT's activity or interaction with nuclear proteins are likely to be modulated by phosphorylation. ß 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.
Contents
Summary
I. Introduction
II. NAD synthesis and breakdown
III. cADPR in plants
IV. NAADP in plants
V. NAD kinases
VI. NAD and gene regulation
VII. Sir2 is an NAD dependant histone deacetylase
VIII. Nicotinamidases
IX. Poly ADP‐ribosylation
X. Poly(ADP‐ribose) glycohydrolase (PARG)
XI. Subcellular compartmentation of NAD and NADP in plants
XII. Conclusions
Acknowledgements
References
Summary
The pyridine nucleotides, NAD+, NADH, NADP+, and NADPH have long‐established and well‐characterised roles as redox factors in processes such as oxidative phosphorylation, the TCA cycle, and as electron acceptors in photosynthesis. Recent years have seen an increase in the number of signalling and gene regulatory processes where NAD+ or NADP+ are metabolised. Cyclic ADP‐ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) are metabolites of NAD+ and NADP+, respectively, and now have widely accepted roles as potent intracellular calcium releasing agents in animals, but are less well characterised in plants. NAD kinases catalyse the transfer of a phosphate group from ATP to NAD to form NADP and are well characterised in plants in their requirement for the calcium binding protein calmodulin, thereby putatively linking their regulation to stress‐induced intracellular calcium release. A second group of proteins unrelated to those above, the sirtuins (Sir2) and poly ADP‐ribose polymerases (PARPs), cleave NAD and transfer the ADP‐ribose group to acetyl groups and proteins, respectively. These have roles in transcriptional control and DNA repair in eukaryotes.
The final step in the biosynthesis of nicotinamideadenine dinucleotide, a major coenzyme in cellular redox reactions and involved in intracellular signaling, is catalyzed by the enzyme nicotinamide mononucleotide adenylyltransferase (NMNAT). The X-ray structure of human NMNAT in complex with nicotinamide mononucleotide was solved by the singlewavelength anomalous dispersion method at a resolution of 2.9 A î . Human NMNAT is a symmetric hexamer whose subunit is formed by a large six-stranded parallel L L-sheet with helices on both sides. Human NMNAT displays a different oligomerization compared to the archaeal enzyme. The protein^nicotinamide mononucleotide interaction pattern provides insight into ligand binding in the human enzyme. ß
Since the studies of Gutman and Gutman (2, 3), measurements of the serum acid phosphatase have become a recognized and widely used ancillary procedure for the diagnosis of cancer of the prostate and a practical means for the evaluation of the functional status of the tumor in many patients with metastasizing cancer of the prostate. While an abnormally increased value of serum acid phosphatase is considered to be a specific index for the presence of carcinoma of the prostate, a normal serum acid phosphatase value in itself cannot be interpreted as signifying absence of this disease. According to Bodansky and Bodansky (4) Attempts have been made to render the determination of serum acid phosphatase more specific for cancer of the prostate. Thus, a search has been made for substrates which would be split prefer-
Nicotinamide mononucleotide adenylyltransferase catalyses the ®nal step in the synthesis of nicotinamide-adenine dinucleotide (NAD + ) by transferring the adenylyl moiety of ATP to nicotinamide mononucleotide (NMN) with the release of pyrophosphate. The human enzyme was crystallized in the presence of NAD + . Crystals grew in the orthorhombic space group C222 1 , with unit-cell parameters a = 140.3, b = 235.5, c = 89.3 A Ê , and diffract to a maximum resolution of 3.0 A Ê . Packing considerations suggest a trimer or higher multimer to be present in the asymmetric unit of the crystal. Two archaeal homologues have been described to form hexamers.
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