Structures of Escherichia coli NAD synthetase with substrates and products reveal mechanistic rearrangements.
Nicotinamide adenine dinucleotide synthetases (NADS) catalyze the amidation of nicotinic acid adenine dinucleotide (NAAD) to yield the enzyme cofactor nicotinamide adenine dinucleotide (NAD). Here we describe the crystal structures of the ammonia-dependent homodimeric NADS from Escherichia coli alone and in complex with natural substrates and with the reaction product NAD. The structures disclosed two NAAD/NAD binding sites at the dimer interface and an adenosine triphosphate (ATP) binding site within each subunit. Comparison with the Bacillus subtilis NADS showed pronounced chemical differences in the NAAD/NAD binding sites and less prominent differences in the ATP binding pockets. In addition, the E. coli NADS structures revealed unexpected dynamical rearrangements in the NAAD/NAD binding pocket upon NAAD-to-NAD conversion, which define a catalysis state and a substrate/product exchange state. The two states are adopted by concerted movement of the nicotinysyl moieties of NAAD and NAD, Phe-170, and residues 224-228, which may be triggered by differential coordination of a magnesium ion to NAAD and NAD. Phylogenetic structure comparisons suggest that the present results are relevant for designing species-specific antibiotics.
Jauch
Ralf
R
Max-Planck-Institut für Biophysikalische Chemie, Abteilung Molekulare Entwicklungsbiologie, Am Fassberg 11, D-37077 Göttingen, Germany.
Humm
Andreas
A
Huber
Robert
R
Wahl
Markus C
MC
D000255:Adenosine Triphosphate / Q000737:chemistry; D019732:Amide Synthases / Q000737:chemistry*; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000378:metabolism; D001665:Binding Sites; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D006801:Humans; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009243:NAD / Q000031:analogs & derivatives* / Q000737:chemistry; D010649:Phenylalanine / Q000737:chemistry; D010802:Phylogeny; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
D009243:NAD; D010649:Phenylalanine; C018348:nicotinic acid adenine dinucleotide; D000255:Adenosine Triphosphate; D019732:Amide Synthases; C038479:NAD+ synthase