Stabilization of active-site loops in NH3-dependent NAD+ synthetase from Bacillus subtilis.
The NH(3)-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 Mg(2+) 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 Mg(2+) 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 Mg(2+).
Devedjiev
Y
Y
Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Symersky
J
J
Singh
R
R
Jedrzejas
M
M
Brouillette
C
C
Brouillette
W
W
Muccio
D
D
Chattopadhyay
D
D
DeLucas
L
L
D019732:Amide Synthases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000641:Ammonia / Q000737:chemistry; D001412:Bacillus subtilis / Q000201:enzymology*; D001665:Binding Sites; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D008274:Magnesium / Q000737:chemistry; D011487:Protein Conformation; D013379:Substrate Specificity
D000641:Ammonia; D019732:Amide Synthases; C038479:NAD+ synthase; D008274:Magnesium