A phosphoenzyme mimic, overlapping catalytic sites and reaction coordinate motion for human NAMPT

Abstract

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.

Role of Human Hypoxanthine Guanine Phosphoribosyltransferase in Activation of the Antiviral Agent T-705 (Favipiravir)

“…This is also evident from our gene knockdown experiments, which further revealed that two other phosphoribosyltransferases, APRT and NAMPT, are irrelevant for the antiviral activity of these 2-pyrazinecarboxamide compounds. NAMPT was included because of the structural analogy between T-705 and nicotinamide (Burgos et al, 2009). These gene knockdown experiments confirmed the role for ADK in activating ribavirin (Willis et al, 1978).…”

Metal Fluorides as Analogues for Studies on Phosphoryl Transfer Enzymes

“…4B) have a covalent His247·BeF3 − , though, in contrast to all other trifluoroberyllate structures, magnesium is coordinated to one fluorine without any direct linkage to His247. [8] …”

Nicotinamide Phosphoribosyltransferase in Malignancy: A Review

“…27,28 iNampt is phosphorylated at histidine 247, resulting in a 160,000-fold increased enzymatic affinity for NAM. 29 iNampt and eNampt undergo other posttranslational modifications, including acetylation and ubiquitization. The significance of these modifications is presently poorly understood.…”