Nicotinamide adenine dinucleotide (NAD) is a ubiquitous carrier of reduction equivalents. It functions as a cofactor in numerous metabolic reactions and is essential for calcium mobilization, DNA repair, and post-translational modification of proteins in eukaryotes. The final step of the NAD biosynthesis is a two-step reaction, which is catalyzed by the NAD synthetases (NADS). Initially, the enzymes mediate an adenylyl transfer from adenosine triphosphate (ATP) to the nicotinysyl moiety of nicotinic acid adenine dinucleotide (NAAD), resulting in NAD-adenylate as an intermediate. Finally, the amidation of the nicotinysyl moiety using either ammonia or glutamine as an amide source results in producing of NAD.
The crystal structure of the ammonia-dependent homodimeric NADS from S. typhimurium LT2 in complex with the reaction product NAD is reported. Pairwise structural alignment reveals similar tertiary and quaternary architecture between the NadE protein and other known NH3-dependent NADS structures. This suggests a resembling molecular mechanism of the catalyzed reaction. The 3D structure of the enzyme disclosed two NAAD/NAD binding sites at the dimer interface and empty adenosine triphosphate (ATP) binding site within each subunit. Several sulfate ions have been modeled with two anions docking similar to two phosphates of PPi in the NADS structure from B. subtilis (PDB ID 1EE1). Distortion of the region encompassing residues 209 through 221 of the S. typhimurium enzyme has functional character. In the NAAD-ATP-bound structure of the B. subtilis protein similar region exhibits a loop conformation accommodating two substrates. In the absence of ATP, but presence of NAAD as in the E.coli NAAD-complexed synthetase structure (PDB ID 1WXG), this loops is disordered. That is, adopting stable conformation by this area requires binding of both substrates fulfilling the recognition role of the loop. Association of Mg2+ in the active site is crucial for initiation of the reaction forming the intermediate, which is then cleaved by ammonia releasing all the products and leading to unfolding of the loop region.