Stabilization of active-site loops in NH3-dependent NAD+ synthetase from Bacillus subtilis

The NH3‐dependent NAD+ synthetase (NADS) participates in the biosynthesis of nicotinamide adenine dinucleotide (NAD+) by transforming nicotinic acid adenine dinucleotide (NaAD) to NAD+. The structural behavior of the active site, including stabilization of flexible loops 82–87 and 204–225, has been studied by determination of the crystal structures of complexes of NADS with natural substrates and a substrate analog. Both loops are stabilized independently of NaAD and solely from the ATP‐binding site. Analysis of the binding contacts suggests that the minor loop 82–87 is stabilized primarily by a hydrogen bond with the adenine base of ATP. Formation of a coordination complex with Mg2+ in the ATP‐binding site may contribute to the stabilization of the major loop 204–225. The major loop has a role in substrate recognition and stabilization, in addition to the protection of the reaction intermediate described previously. A second and novel Mg2+ position has been 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 catalytically active Mg2+.