Structure of Human NMN Adenylyltransferase

Abstract

Nicotinamide mononucleotide adenylyltransferase (NMNAT), a member of the nucleotidyltransferase ␣/␤-phosphodiesterases superfamily, catalyzes a universal step (NMN ؉ ATP ‫؍‬ NAD ؉ PP i ) in NAD biosynthesis. Localized within the nucleus, the activity of the human enzyme is greatly altered in tumor cells, rendering it a promising target for cancer chemotherapy. By using a combination of single isomorphous replacement and density modification techniques, the human NMNAT structure was solved by x-ray crystallography to a 2.5-Å resolution, revealing a hexamer that is composed of ␣/␤-topology subunits. The active site topology of the enzyme, analyzed through homology modeling and structural comparison with other NMNATs, yielded convincing evidence for a substrate-induced conformational change. We also observed remarkable structural conservation in the ATP-recognition motifs GXXXPX-(T/H)XXH and SXTXXR, which we take to be the universal signature for NMNATs. Structural comparison of human and prokaryotic NMNATs may also lead to the rational design of highly selective antimicrobial drugs.Beyond its pivotal role as a redox cofactor in energy transduction and cellular metabolism, NAD is also intimately involved in signaling pathways. The NAD(P) derivatives nicotinic acid adenine dinucleotide phosphate and cyclic ADP-ribose are likewise among the most potent calcium-mobilizing agents (1, 2). NAD is the substrate for poly(ADP-ribosyl)ation, a vitally important post-translation modification occurring within the nuclei of higher eukaryotes by poly(ADP-ribose) polymerase (3). NAD is also the substrate for mono-ADP-ribosyltransferase, an enzyme that transfers a single ADP-ribose unit from NAD onto target proteins in both mammalian and prokaryotic cells (4). Most recently, high levels of NAD were shown to extend yeast cell life-span, a phenomenon linked to the action of NAD-dependent catalysis of protein deacetylation (5, 6).Like ATP and GTP, NAD performs multiple tasks in both energy and signaling transduction, indicating why NAD homeostasis must be tightly regulated in all living organisms. Any impairment in NAD synthesis seriously impairs cellular metabolism, eventually resulting in cell death (7). The biosynthesis of this key redox coenzyme is accomplished either through a de novo pathway or through NAD-recycling salvage routes with notable differences between prokaryotes and eukaryotes (8). All the known biochemical pathways converge to the reaction catalyzed by NMN adenylyltransferase (EC 2.7.7.1), abbreviated NMNAT 1 (8). This transferase catalyzes the nucleophilic attack by the 5Ј phosphate NMN or nicotinic acid mononucleotide on the ␣-phosphoryl of ATP, releasing PP i and NAD or nicotinic acid adenine dinucleotide (9). Although the eukaryotic enzyme forms NAD and nicotinic acid adenine dinucleotide at similar rates, its prokaryotic counterpart prefers the deamidated substrate (nicotinic acid mononucleotide) (8). Because the reaction is fully reversible, NAD can also be reconverted to ATP (10).Human NMNAT, the only NAD/n...

Structural Characterization of a Human Cytosolic NMN/NaMN Adenylyltransferase and Implication in Human NAD Biosynthesis

“…Upon binding NMN or NAD/ NaAD, residues 50 -56 and 144 -149 around the NMN binding site made various degrees of adjustment (usually less than 1 Å) and in general closed in on the bound substrate so that the contacts with the ligand are optimized. Similar ligand bindinginduced movements around the NMN binding site were also observed in hsPNAT-1 (19,20).…”

“…Significant progress has been made during the last few years in the enzymological and structural studies of PNATs in various organisms across all three kingdoms of life (15)(16)(17)(18)(19)(20)(21)(22). In human, two PNAT isoforms (gi 11245478 and gi 12620200) have been cloned and purified, and their kinetic properties were analyzed (23)(24)(25)(26).…”

“…Human PNAT-1 has also been reported to interact specifically with poly(ADP-ribose) polymerase (24,27) and may be subjected to further regulation by phosphorylation (24). Crystal structures of hsPNAT-1 have been solved in its apo form (20) and in complex with NMN (21), NAD, NaAD, or TAD (19). These structures revealed a hexameric organization of the protein and provided a structural basis for its dual specificity in recognizing both NMN and NaMN substrates (19 1 The abbreviations used are: NAD, nicotinamide adenine dinucleotide; NaAD, nicotinic acid adenine dinucleotide; NMN, nicotinamide mononucleotide; NaMN, nicotinic acid mononucleotide; NMNAT, NMN adenylyltransferase; NaMNT, NaMN adenylyltransferase; PNAT, pyridine nucleotide adenylyltransferase; AMP-CPP, adenosine 5Ј-(␣,␤-methylene)triphosphate; PPAT, phosphopantetheine adenylyltransferase; r.m.s.d., root mean square deviation; GFP, green fluorescent protein; EGFP, enhanced GFP; NLS, nuclear localization signal; c.c., correlation coefficient.…”

Mutational, Structural, and Kinetic Studies of the ATP-binding Site of Methanobacterium thermoautotrophicum Nicotinamide Mononucleotide Adenylyltransferase

“…The structures of M. thermoautotrophicum and M. jannaschii NMNATase were determined in complex with the product NAD ϩ or the substrate ATP, respectively (9,15). More recently, crystal structures were reported for the apo-form of human NMNATase (16) and its complexes with NMN ϩ (17), NAD ϩ (18), NaAD ϩ (18), and the oncolytic agent tiazofurin (18). All these analyses revealed a hexameric assembly and a highly similar overall fold.…”

“…Here, the two histidine residues of the NMNATase 13 HWGH 16 motif directly interact with the ␣-and ␤-phosphates of ATP in the ground state, His-13 with the ␤-phosphate and His-16 with the ␣-phosphate. Also in contrast to what was observed by Fersht (30) in the E-Tyr-AMP product complex in which His-48 is still bound to the ribose 5Ј-oxygen, in the NAD complex of M. thermoautotrophicum NMNATase (9), His-19 is interacting with an oxygen atom of the AMP-phosphate, which corresponds to the ␣-phosphate of the substrate ATP.…”