Crystal structure of NH₃-dependent NAD⁺ synthetase from Bacillus subtilis

NAD⁺ synthetase catalyzes the last step in the biosynthesis of nicotinamide adenine dinucleotide. The three-dimensional structure of NH₃-dependent NAD⁺ synthetase from Bacillus subtilis, in its free form and in complex with ATP, has been solved by X-ray crystallography (at 2.6 and 2.0 Å resolution, respectively) using a combination of multiple isomorphous replacement and density modification techniques. The enzyme consists of a tight homodimer with α/β subunit topology. The catalytic site is located at the parallel β-sheet topological switch point, where one AMP molecule, one pyrophosphate and one Mg²⁺ ion are observed. Residue Ser46, part of the neighboring 'P-loop', is hydrogen bonded to the pyrophosphate group, and may play a role in promoting the adenylation of deamido-NAD⁺ during the first step of the catalyzed reaction. The deamido-NAD⁺ binding site, located at the subunit interface, is occupied by one ATP molecule, pointing towards the catalytic center. A conserved structural fingerprint of the catalytic site, comprising Ser46, is very reminiscent of a related protein region observed in glutamine-dependent GMP synthetase, supporting the hypothesis that NAD⁺ synthetase belongs to the newly discovered family of 'N-type' ATP pyrophosphatases.