Identification of structural determinants of nicotinamide phosphoribosyl transferase (NAMPT) activity and substrate selectivity

DOI: 10.1016/j.jsb.2023.108004

Authors: Dorothée Houry (University of Bergen) , Arne Raasakka (University of Bergen) , Eugenio Ferrario (University of Bergen) , Marc Niere (University of Bergen) , Ersilia Bifulco (University of Bergen) , Petri Kursula (University of Bergen; University of Oulu) , Mathias Ziegler (University of Bergen; Hans Knöll Institute)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Structural Biology , VOL 66

State: Published (Approved)
Published: July 2023

Abstract: NAD homeostasis in mammals requires the salvage of nicotinamide (Nam), which is cleaved from NAD± by sirtuins, PARPs, and other NAD±-dependent signaling enzymes. Nam phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step in vitamin B3 salvage, whereby Nam reacts with phosphoribosyl pyrophosphate (PRPP) to form nicotinamide mononucleotide. NAMPT has a high affinity towards Nam, which is further enhanced by autophosphorylation of His247. The mechanism of this enhancement has remained unknown. Here, we present high-resolution crystal structures and biochemical data that provide reasoning for the increased affinity of the phosphorylated NAMPT for its substrate. Structural and kinetic analyses suggest a mechanism that includes Mg2+ coordination by phospho-His247, such that PRPP is stabilized in a position highly favorable for catalysis. Under these conditions, nicotinic acid (NA) can serve as a substrate. Moreover, we demonstrate that a stretch of 10 amino acids, present only in NAMPTs from deuterostomes, facilitates conformational plasticity and stabilizes the chemically unstable phosphorylation of His247. Thereby the apparent substrate affinity is considerably enhanced compared to prokaryotic NAMPTs. Collectively, our study provides a structural basis for the important function of NAMPT to recycle Nam into NAD biosynthesis with high affinity.

Journal Keywords: NAMPT; phosphoribosyltransferas; enicotinamide; nicotinic acid; SAXS; X-ray crystallography; NAD biosynthesis; sirtuins; PARP

Diamond Keywords: Enzymes

Subject Areas: Biology and Bio-materials


Instruments: I03-Macromolecular Crystallography

Other Facilities: P13 at EMBL/DESY

Added On: 26/07/2023 11:58

Discipline Tags:

Structural biology Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)