Structural Analysis of the NH3-dependent NAD+Synthetase from Deinococcus radiodurans

Abstract

Nicotinamide adenine dinucleotide (NAD) is a ubiquitous molecule involved in both redox reactions as a cofactor and numerous regulatory processes as a substrate such as cell cycle, calcium signaling, immune response, and DNA repair. The biosynthesis of NAD is vitally important in all living organisms and has been studied extensively in variety of species for antibiotic drug development. The NAD is synthesized through two metabolic processes which are de novo and salvage pathways. Despite some variations in the early steps of two pathways, the final step of NAD synthesis from nicotinic acid adenine dinucleotide (NaAD) to NAD is highly conserved. NAD synthetase (NADS, EC. 6.3.5.1) catalyzes conversion of NaAD to NAD through two steps; first step is the adenylation of NaAD in the presence of ATP and Mg; second, the NAD-adenylate intermediate is attacked by nucleophilic ammonia leading to generate NAD and AMP (Fig. 1(a)). The NADS family is categorized into two subgroups: (i) NH3-dependent NADS present only in prokaryotes, which has only synthetase domain (S-domain), and (ii) glutamine-dependent NADS present in all eukaryotes and some prokaryotes, which has an additional glutamine amide transfer domain (GAT-domain). The crystal structures of NH3-depenent NADS from bacterial species including Bacillus subtilis (bsuNADS), Escherichia coli (ecoNADS), Bacillus anthracis (banNADS), Helicobacter pylori (hpyNADS), and Francisella tularensis (ftuNADS) have been determined. As the prokaryotic and eukaryotic NADS differ in size, enzymatic activity and substrate requirements, NADS is an attractive target for the development of a new class of antibiotics. The NADS homolog (UniProt code Q9RYV5) in Deinococcus radiodurans encodes a protein of 287 amino acid residues, with 59% sequence identity to that of E. coli. Further sequence comparisons of D. radiodurans NADS (draNADS) with bsuNADS, banNADS, and ftuNADS shows 58%, 59%, and 36% sequence identity, respectively (Fig. 2(a)). In order to obtain structural and functional information of draNADS protein, we report here the crystal structure of NH3-dependent NADS homolog from D. radiodurans at 2.60 A resolution.