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.1 The biosynthesis of NAD + is vitally important in all living organisms and has been studied extensively in variety of species for antibiotic drug development.2 The NAD + is synthesized through two metabolic processes which are de novo and salvage pathways.3 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 2+; second, the NAD-adenylate intermediate is attacked by nucleophilic ammonia leading to generate NAD + and AMP ( Fig. 1(a)). 4 The NADS family is categorized into two subgroups: (i) NH 3 -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 NH 3 -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. [5][6][7][8][9] 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 NH 3 -dependent NADS homolog from D. radiodurans at 2.60 Å resolution. Materials and MethodsProtein Expression and Purification. The nadE gene encoding NADS was amplified by polymerase chain reaction using genomic DNA of D. radiodurans as a template. It was inserted into the NdeI/BamHI-digested expression vector pET-28b(+) (Novagen), resulting in a twenty-residue hexahistidine-containing tag at its N-terminus. The recombinant draNADS was expressed in E. coli BL21 (DE3) star pLysS cells (Invitrogen). Overexpression of the recombinant protein was induced with 1.0 mM isopropyl β-D-thiogalactopyranoside (IPTG) and the cells were continuously cultured at 303 K for 4 h. After harvest of the cells by centrifugation at 4200 g for 10 minutes at 277 K, the pellet was resuspended in a lysis buffer [20 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride] and homogenized by an ultras...