Nicotinamide/nicotinic acid mononucleotide adenylyltransferase, new insights into an ancient enzyme

Abstract

Nicotinamide/nicotinic acid mononucleotide adenylyltransferase (NMNAT) has long been known as the master enzyme in NAD biosynthesis in living organisms. A burst of investigations on NMNAT, going beyond enzymology, have paralleled increasing discoveries of key roles played by NAD homeostasis in a number or patho-physiological conditions. The availability of in-depth kinetics and structural enzymology analyses carried out on NMNATs from different organisms offer a powerful tool for uncovering fascinating evolutionary relationships. On the other hand, additional functions featuring NMNAT have emerged from investigations aimed at unraveling the molecular mechanisms responsible for complex biological phenomena such as neurodegeneration. NMNAT appears to be a multifunctional protein that sits both at the core of central metabolism and at a crossroads of multiple cellular processes. The resultant wealth of biochemical data has built a robust framework upon which design of NMNAT activators, inhibitors or enzyme variants of potential medical interest can be based.

Fig. 1

Structural architecture of archaea NMNAT, considered the prototype of the family. M. jannaschii NMNAT in complex with ATP (left panel) and M. thermoautotrophicum NMNAT in complex with NAD (right panel) are shown in a ribbon representation. The ATP and NAD are shown as ball-and-sticks and the magnesium ion as a green sphere. The loop playing an important role in the oligomeric assembly and the small C-terminal domain, are colored in blue. All figures were generated by PyMol (http://www.pymol.org)

Fig. 2

Ribbon representation of NMNAT from different sources with the small C-terminal domain colored in blue. The structural element, either a long loop or a loop and a small β strand that connects the α/β domain to the C-terminal domain is depicted in orange. Such a structural element is subjected to conformational changes that accompany catalysis and also provides a key contribution to the oligomeric assembly stabilization in all the NMNATs featured with a quaternary structure, either dimers, tetramers or hexamers

Fig. 3

View of the ATP binding site in M. jannaschii NMNAT (PDB code 1F9A). The key residues for ATP binding are shown in a ball-and-stick representation and the catalytically essential magnesium ion as a magenta sphere. The strictly conserved residues of the (H/T)XXH and SXXXXR sequence fingerprints, are shown with a green background. Dotted lines indicate specific interactions described in the text

Fig. 4

View of the NaAD and NAD binding site in different NMNATs. Protein residues and solvent molecules playing a key role in dinucleotide binding are shown as ball-and-stick and sphere, respectively. The strictly conserved tryptophan π-stacking with the pyridine ring is depicted with a green background. a M. thermautotrophicum NMNAT-NAD complex (PDB code 1M8 K). b S. aureus NaMNAT-NaAD complex (PDB code 2H2A). c hNMNAT1-NAD complex (PDB code 1KQN). d hNMNAT3-NaAD complex (PDB code 1NUP)

Fig. 5

Oligomeric assembly and electrostatic potential in hexameric NMNATs viewed along the three-fold axis. Positive and negative electrostatic potential are represented in blue and red, respectively. a M. jannaschii NMNAT. b Human NMNAT1 (PDB code 1KKU). For one of the subunits, the region not visible in the crystal structure due to high conformational flexibility is indicated by a dotted line. The sequence of the nuclear localization sequence (NLS) is indicated with the residue subjected to phosphorylation (Ser136) in red

Fig. 6

Human NMNAT1 and chaperone UspA share significant structure similarity. a A structure superposition of human NMNAT1 and NMNAT3 shows that they share 96% structure identity. b Crystal structure of UspA. c A structural superposition of NMNAT1 and UspA shows 13% sequence identity and a RMSD of 3.0 Å over the entire length of the UspA protein. The three best overlapping alpha-helices are marked with H1 (UspA 15–24) (hNMNAT1 24–33) RMSD of 0.44 Å based on 10 Ca, H2 (UspA 64–74) (hNMNAT1 65–75) RMSD of 0.40 Å based on 11 Ca, and H3 (UspA 90–98) (hNMNAT1 95–103) RMSD of 0.39 Å based on 9 Ca (arrows in c). The structure superposition was done using DALILITE (http://www.ebi.ac.uk) and the figure generated with the program PyMol (http://www.pymol.org)