Nicotinamide riboside kinase structures reveal new pathways to NAD+

Abstract

The eukaryotic nicotinamide riboside kinase (Nrk) pathway, which is induced in response to nerve damage and promotes replicative life span in yeast, converts nicotinamide riboside to nicotinamide adenine dinucleotide (NAD+) by phosphorylation and adenylylation. Crystal structures of human Nrk1 bound to nucleoside and nucleotide substrates and products revealed an enzyme structurally similar to Rossmann fold metabolite kinases and allowed the identification of active site residues, which were shown to be essential for human Nrk1 and Nrk2 activity in vivo. Although the structures account for the 500-fold discrimination between nicotinamide riboside and pyrimidine nucleosides, no enzyme feature was identified to recognize the distinctive carboxamide group of nicotinamide riboside. Indeed, nicotinic acid riboside is a specific substrate of human Nrk enzymes and is utilized in yeast in a novel biosynthetic pathway that depends on Nrk and NAD+ synthetase. Additionally, nicotinic acid riboside is utilized in vivo by Urh1, Pnp1, and Preiss-Handler salvage. Thus, crystal structures of Nrk1 led to the identification of new pathways to NAD+.

Figure 1. Nrk1 Is a Rossmann Fold Metabolite Kinase

(A) Ribbon diagram of Nrk1 bound to ADP with rainbow coloring from the N terminus (violet) to the C terminus (red). Helices A through E, strands 1 through 5, and the lid domain are indicated. (B) Structural superposition of Nrk1 (blue) with human Uck2 (red).

Figure 2. Nucleoside and Nucleotide Recognition by Nrk1

Stereo diagrams of Nrk1 are provided in a consistent orientation using the main chain backbone colors from Figure 1A. (A) ADP•Mg² product complex with the corresponding difference electron density map, contoured at 3.0 Å. (B) NR substrate complex. (C) Bisubstrate analog complex of AppNHp•Mg² plus NR. (D) NMN product complex.

Figure 3. Active Site Requirement of Nrk1 and Nrk2

The conserved carboxylates of human Nrk1, Asp36 and Glu98, and the corresponding carboxylates of Nrk2 are required for biological activity in yeast.

Figure 4. NR/Uridine Discrimination by Nrk1

In stereo, the 2- and 4-oxy functions (black) of a hypothetical uridine substrate are shown superimposed onto NR coordinates in the Nrk1 NR co-crystal structure. At a distance of 2.55 Å, the 4 oxygen would be in van der Waals conflict with the carbonyl oxygen of Gln135, which is in a cis peptide linkage with Pro136.

Figure 5. NR and Tiazofurin Recognition by Nrk1

(A) Coordinates of tiazofurin, in yellow, are superimposed with those of NR in isomorphous crystal structures. The sulfur atom of tiazofurin was localized by the 3 Å anomalous difference Fourier map, contoured in purple at 2.7 Å. (B) A surface representation of the Nam binding-site of NR reveals no specific recognition of the carboxamide group, which is exposed to solvent.

Figure 6. NaR Utilization In Vivo

In (A), the vitamin requirement of de novo mutant bna1 and glutamine-dependent NAD⁺ synthetase mutant qns1 is illustrated. The fact that bna1, bna1 nrk1, bna1 npt1, and qns1 strains are satisfied by addition of NR is shown in (B). In (C), the vitamin activity of NaR is demonstrated for the de novo mutant bna1, even when either the Nrk pathway or the Preiss-Handler pathway is mutationally inactivated by nrk1 or npt1 mutation, respectively. Establishing the uniqueness of NaR as a vitamin, NaR fails to support the growth of qns1. In (D), the intracellular NAD⁺ concentration is calculated for wild-type, npt1, nrk1, urh1 pnp1, and nrk1 urh1 pnp1 mutants in vitamin-free (gray bars) and in vitamin-free media supplemented with 10 μM NaR (black bars). The unique lack of utilization by the nrk1 urh1 pnp1 strain shows that NaR makes use of Nrk1, Urh1, and Pnp1 for conversion to NAD⁺.

Figure 7. NAD⁺ Metabolism in Yeast: Two New Pathways to NaMN

Previously reported NAD⁺ metabolic pathways are shown in black[15]. In blue and green, respectively, are Nrk1-dependent and Nrk1-independent routes from NaR to NaMN.