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.

Background: PRMT5-MEP50 is an arginine methyltransferase with significant roles in development and cancer. Results: MEP50 binds to the histone fold domain and is essential for the efficient use of SAM by PRMT5. Conclusion: MEP50 is essential for methylation of histones H4 and H2A by PRMT5. Significance: The mechanism of histone methylation by PRMT5-MEP50 provides novel insight into methyltransferase mechanisms and therapeutic development.

Human nicotinamide phosphoribosyltransferase (NAMPT) replenishes the NAD pool and controls the activities of sirtuins (SIRT), mono- and poly-(ADP-ribose) polymerases (PARP) and NAD nucleosidase (CD38). The nature of the enzymatic transition-state (TS) is central to understanding the function of NAMPT. We determined the TS structure for pyrophosphorolysis of nicotinamide mononucleotide (NMN) by kinetic isotope effects (KIEs). With the natural substrates, NMN and pyrophosphate (PPi), the intrinsic KIEs of [1′-14C], [1-15N], [1′-3H] and [2′-3H] are 1.047, 1.029, 1.154 and 1.093, respectively. A unique quantum computational approach was used for TS analysis that included structural elements of the catalytic site. Without constraints (e.g. imposed torsion angles), the theoretical and experimental data are in good agreement. The quantum-mechanical calculations incorporated a crucial catalytic site residue (D313), two magnesium atoms and coordinated water molecules. The transition state model predicts primary 14C, α-secondary 3H, β-secondary 3H and primary 15N KIE close to the experimental values. The analysis reveals significant ribocation character at the TS. The attacking PPi nucleophile is weakly interacting (rC-O = 2.60 Å) and the N-ribosidic C1′-N bond is highly elongated at the TS (rC-N = 2.35 Å), consistent with an ANDN mechanism. Together with the crystal structure of the NMN•PPi•Mg2•enzyme complex, the reaction coordinate is defined. The enzyme holds the nucleophile and leaving group in relatively fixed positions to create a reaction coordinate with C1′-anomeric migration from nicotinamide to the PPi. The transition state is reached by a 0.85 Å migration of C1′.

Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the first reversible step in NAD biosynthesis and nicotinamide (NAM) salvage. The enzyme is designed for efficient capture of nicotinamide by coupling of ATP hydrolysis to assist in extraordinary NAM binding affinity and formation of nicotinamide mononucleotide (NMN). NAMPT provides the mechanism to replenish the NAD pool in human metabolism. In addition to its role in redox biochemistry, NAD fuels the sirtuins (SIRTs) to regulate transcription factors involved in pathways linked to inflammation, diabetes and lifespan. NAMPT-mediated lifespan expansion has caused a focus on the catalytic mechanism, regulation and inhibition of NAMPT. Structural, mechanistic and inhibitor design all contribute to a developing but yet incomplete story of NAMPT function. Although the first generation of NAMPT inhibitors has entered clinical trials, disappointing outcomes suggest more powerful and specific inhibitors will be needed. Understanding the ATP-linked mechanism of NAMPT and the catalytic site machinery may permit the design of improved NAMPT inhibitors as more efficient drugs against cancer.

Trypanosoma cruzi, the human parasite that causes Chagas disease, contains a functional pentose phosphate pathway, probably essential for protection against oxidative stress and also for R5P (ribose 5-phosphate) production for nucleotide synthesis. The haploid genome of the CL Brener clone of the parasite contains one gene coding for a Type B Rpi (ribose 5-phosphate isomerase), but genes encoding Type A Rpis, most frequent in eukaryotes, seem to be absent. The RpiB enzyme was expressed in Escherichia coli as a poly-His tagged active dimeric protein, which catalyses the reversible isomerization of R5P to Ru5P (ribulose 5-phosphate) with Km values of 4 mM (R5P) and 1.4 mM (Ru5P). 4-phospho-D-erythronohydroxamic acid, an analogue to the reaction intermediate when the Rpi acts via a mechanism involving the formation of a 1,2-cis-enediol, inhibited the enzyme competitively, with an IC50 value of 0.7 mM and a Ki of 1.2 mM. Site-directed mutagenesis allowed the demonstration of a role for His102, but not for His138, in the opening of the ribose furanosic ring. A major role in catalysis was confirmed for Cys69, since the C69A mutant was inactive in both forward and reverse directions of the reaction. The present paper contributes to the know-ledge of the mechanism of the Rpi reaction; in addition, the absence of RpiBs in the genomes of higher animals makes this enzyme a possible target for chemotherapy of Chagas disease.