CRYSTAL-STRUCTURE OF NH3-DEPENDENT NAD(-SUBTILIS() SYNTHETASE FROM BACILLUS)

NAD(+) synthetase catalyzes the last step in the biosynthesis of nicot inamide adenine dinucleotide. The three-dimensional structure of NH3-d ependent NAD(+) synthetase from Bacillus subtilis, in its free form an d in complex with ATP, has been solved by X-ray crystallography (at 2. 6 and 2.0 Angstrom resolution, respectively) using a combination of mu ltiple isomorphous replacement and density modification techniques, Th e enzyme consists of a tight homodimer with alpha/beta subunit topolog y The catalytic site is located at the parallel beta-sheet topological switch point, where one alpha/beta molecule, one pyrophosphate and on e Mg2+ ion are observed, Residue Ser46, part of the neighboring 'P-loo p', 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 towa rds the catalytic center, A conserved structural fingerprint of the ca talytic site, comprising Ser46 is very reminiscent of a related protei n region observed in glutamine-dependent GMP synthetase, supporting th e hypothesis that NAD(+) synthetase belongs to the newly discovered fa mily of 'N-type' ATP pyrophosphatases.