Human nicotinamide phosphoribosyltransferase (NAMPT, EC 2.4.2.12) catalyses the reversible synthesis of nicotinamide mononucleotide (NMN) and inorganic pyrophosphate (PPi) from nicotinamide (NAM) and α-D-5-phosphoribosyl-1-pyrophosphate (PRPP). NAMPT, by capturing the energy provided by its facultative ATPase activity, allows the production of NMN at products/ substrates ratios thermodynamically forbidden in the absence of ATP. By coupling ATP hydrolysis to NMN synthesis, the catalytic efficiency of the system is improved 1100-fold and substrate affinity dramatically increased (K m NAM from 855 nM to 5 nM) and the K eq shifted −2.1 kcal/mol toward NMN formation. ADP/ATP isotopic exchange experiments support the formation of a high-energy phosphorylated intermediate (phospho-H247) as the mechanism for altered catalysis efficiency during ATP hydrolysis. NAMPT captures only a small portion of the energy generated by ATP hydrolysis to shift the dynamic chemical equilibrium. Although the weak energetic coupling of ATP hydrolysis appears to be a non optimized enzymatic function, closer analysis of this remarkable protein reveals an enzyme designed to capture NAM with high efficiency at the expense of ATP hydrolysis. NMN is a rate-limiting precursor for recycling to the essential regulatory cofactor, nicotinamide adenine dinucleotide (NAD + ). NMN synthesis by NAMPT is powerfully inhibited by both NAD + (K i = 0.14 µM) and NADH (K i = 0.22 µM), an apparent regulatory feedback mechanism.The properties of NAD + in electron transfer are now established. As a cofactor in many metabolic pathways, NAD + holds a key position in energy metabolism and its regulation. Over the last decade, new involvements for NAD + were discovered. It is used by poly and mono (ADP-ribose) polymerases as a substrate for protein covalent modifications (1-5).Sirtuins (SIRT) use NAD + in protein deacetylation reactions with implications for metabolic control, cancer and longevity. While redox reactions do not influence the net level of NAD + + NADH concentration, enzymes that catalyze ADP-ribosylation and deacetylation cleave the N-ribosyl bond to nicotinamide and can lead to a rapid depletion of this substrate. NAD + levels influence all of its associated pathways and the NAD + pool needs constant replenishment from NAM recycling since dietary sources of the vitamin may be limiting.In prokaryotes and lower eukaryotes, NAD + replenishment is achieved primarily by de novo synthesis. Quinolinic acid, derived from tryptophan, is converted into nicotinic acid mononucleotide (NAMN) by the quinolinic acid phosphoribosyltransferase (QAPT). NAMN, obtained from exogenous nicotinic acid (NA) by reaction with nicotinic acid phosphoribosyltransferase (NAPT), is converted into NAD + , via the Preiss-Handler pathway † This work was supported by research grant GM41916 from the NIH. *To whom correspondence should be addressed at the Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel: (718) 430-281...

Pharmacological strategies that boost intracellular NAD + are highly coveted for their therapeutic potential. One approach is activation of nicotinamide phosphoribosyltransferase (NAMPT) to increase production of nicotinamide mononucleotide (NMN), the predominant NAD + precursor in mammalian cells. A high-throughput screen for NAMPT activators and hit-to-lead campaign yielded SBI-797812, a compound that is structurally similar to active-site directed NAMPT inhibitors and blocks binding of these inhibitors to NAMPT. SBI-797812 shifts the NAMPT reaction equilibrium towards NMN formation, increases NAMPT affinity for ATP, stabilizes phosphorylated NAMPT at His247, promotes consumption of the pyrophosphate by-product, and blunts feedback inhibition by NAD + . These effects of SBI-797812 turn NAMPT into a “super catalyst” that more efficiently generates NMN. Treatment of cultured cells with SBI-797812 increases intracellular NMN and NAD + . Dosing of mice with SBI-797812 elevates liver NAD + . Small molecule NAMPT activators such as SBI-797812 are a pioneering approach to raise intracellular NAD + and realize its associated salutary effects.

Nucleoplasmin (Npm) is an abundant histone chaperone in vertebrate oocytes and embryos. During embryogenesis, regulation of Npm histone binding is critical for its function in storing and releasing maternal histones to establish and maintain the zygotic epigenome. Here we demonstrate that Xenopus laevis Npm post-translational modifications (PTMs) specific to the oocyte and egg promote either histone deposition or sequestration, respectively. Mass spectrometry and Npm phosphomimetic mutations used in chromatin assembly assays identified hyperphosphorylation on the N-terminal tail as a critical regulator for sequestration. C-terminal tail phosphorylation and PRMT5-catalyzed arginine methylation enhance nucleosome assembly by promoting histone interaction with the second acidic tract of Npm. Electron microscopy reconstructions of Npm and TTLL4 activity towards the C-terminal tail demonstrate that oocyte- and egg-specific PTMs cause Npm conformational changes. Our results reveal that PTMs regulate Npm chaperoning activity by modulating Npm conformation and Npm-histone interaction leading to histone sequestration in the egg.

Nicotinamide phosphoribosyltransferase (NAMPT) is highly evolved to capture nicotinamide (NAM) and replenish the nicotinamide adenine dinucleotide (NAD ؉ ) pool during ADP-ribosylation and transferase reactions. ATP-phosphorylation of an active-site histidine causes catalytic activation, increasing NAM affinity by 160,000. Crystal structures of NAMPT with catalytic site ligands identify the phosphorylation site, establish its role in catalysis, demonstrate unique overlapping ATP and phosphoribosyltransferase sites, and establish reaction coordinate motion. NAMPT structures with beryllium fluoride indicate a covalent H247-BeF3 ؊ as the phosphohistidine mimic. Activation of NAMPT by H247-phosphorylation causes stabilization of the enzyme-phosphoribosylpyrophosphate complex, permitting efficient capture of NAM. Reactant and product structures establish reaction coordinate motion for NAMPT to be migration of the ribosyl anomeric carbon from the pyrophosphate leaving group to the nicotinamide-N1 while the 5-phosphoryl group, the pyrophosphate moiety, and the nicotinamide ring remain fixed in the catalytic site.ϩ is an essential cofactor in metabolic redox chemistry. It also functions in DNA repair reactions, poly-and mono-ADPribose polymerases, formation of cyclic ADP-ribose, and the Sirtuins (SIRT) (1-5). These reactions can deplete NAD ϩ by cleaving the N-ribosyl bond to generate free nicotinamide (NAM). Nicotinamide phosphoribosyltransferase (NAMPT; also known as pre-␤ cell colony enhancing factor, PBEF, and visfatin, an adipokine) is designed to efficiently recycle NAM (K m ϭ 5 nM) by reaction with ␣-D-5-phosphoribosyl-1-pyrophosphate (PRPP, Fig. 1) to sustain the pool of NAD ϩ (6).In mammals, NAMPT is the rate-limiting enzyme for NAD ϩ salvage from NAM and its overexpression increased cell lifespan (7) via activation of SIRT1 (8). Recently, NAMPT was also identified as the enzyme regulating mitochondrial NAD ϩ levels (9) and extending cell lifespan via the functions of SIR3 and SIR4. Because of its role in NAD ϩ maintenance, NAMPT is a target in cancer research (10). Its inhibition by FK866 causes depletion of NAD ϩ and a decrease in SIRT1 activity (8) resulting in cell senescence.