Recycling Nicotinamide. The Transition-State Structure of Human Nicotinamide Phosphoribosyltransferase

Abstract

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′.

Physiological and pathophysiological roles of NAMPT and NAD metabolism

“…Several research groups have characterized the structural and enzymological features of mammalian NAMPT. [24][25][26][27][28][29][30][31] Structural and mutagenesis studies have shown that mutations in NAMPT that impair dimerization attenuate enzymatic activity; 26 furthermore, Asp219 is important in defining the substrate specificity of NAMPT, 29 which does not include nicotinic acid. 14,32 Autophosphorylation of NAMPT (by use of ATP) at His247 24,26 creates a reaction intermediate that is hydrolysed during each catalytic cycle, which increases the affinity of NAMPT for NAM and its enzymatic activity up to 1,000-fold.…”

Kinetic Isotope Effects and Transition State Structure for Hypoxanthine-Guanine-Xanthine Phosphoribosyltransferase from Plasmodium falciparum

“…20–22 In every case, these transition states were studied from the direction of nucleoside monophosphate pyrophosphorolytic cleavage from the base and phosphorylated ribosyl groups. For orotate phosphoribosyltransferases, insignificant isotope effects were observed with pyrophosphate as the substrate, but use of phosphonoacetic acid, a slow-reacting analogue of pyrophosphate, permitted measurement of the KIE values.…”

Comparative Functional Genomic Analysis of Two Vibrio Phages Reveals Complex Metabolic Interactions with the Host Cell

“…Both eukaryotic and prokaryotic sirtuins are known for post-translational modifications using NAD + as a co-substrate. Specifically, deacetylation of acetyl-lysine by the sirtuins produces nicotinamide as a byproduct, which can also be recycled for NAD + biosynthesis as mentioned above (Burgos et al, 2013). Deacetylation of acetyl-lysine has been strongly correlated with the activation of acetyl-coA synthetase ( ACS ) in prokaryotes, which is characterized as a Sir2-dependent enzyme (Starai, 2002).…”

“…By being obligate parasites, bacteriophages are known to carry genes toward their own benefit (Gazzaniga et al, 2009) for host metabolic manipulation. Sirtuins are able to deacetylase acetyl-lysine in proteins (Burgos et al, 2013) and their presence in large bacteriophage genomes is still enigmatic. It is natural for phages to carry molecular tools for metabolic reprogramming of their host, since they need them in order to replicate their genome, before capsid packaging.…”