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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 angstroms resolution,
respectively) using a combination of multiple isomorphous replacement and
density modification techniques. The enzyme consists of a tight homodimer with
alpha/beta subunit topology. The catalytic site is located at the parallel
beta-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.
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