Stabilization of active-site loops in NH3-dependent NAD(+) synthetase fromBacillus subtilis

The NH3-dependent NAD(+) synthetase (NADS) participates in the biosynthesis of nicotinamide adenine dinucleotide (NAD(+)) by transforming nicotinic ac id adenine dinucleotide (NaAD) to NAD(+). The structural behavior of the ac tive site, including stabilization of flexible loops 82-87 and 204-225, has been studied by determination of the crystal structures of complexes of NA DS with natural substrates and a substrate analog. Both loops are stabilize d independently of NaAD and solely from the ATP-binding site. Analysis of t he binding contacts suggests that the minor loop 82-87 is stabilized primar ily by a hydrogen bond with the adenine base of ATP. Formation of a coordin ation complex with Mg2+ in the ATP-binding site may contribute to the stabi lization of the major loop 204-225. The major loop has a role in substrate recognition and stabilization, in addition to the protection of the reactio n intermediate described previously. A second and novel Mg2+ position has b een observed closer to the NaAD-binding site in the structure crystallized at pH 7.5, where the enzyme is active. This could therefore be the catalyti cally active Mg2+.