NAD+ synthase

 

NAD+ is involved electron transport and redox reactions and in DNA ligation and protein ADP-ribosylation. In yeast and most other organisms, NAD is generated through the de novo pathway and the salvage pathway. In the de novo pathway, quinolinic acid is converted to nicotinic acid mononucleotide (NaMN). In the salvage pathway, NaMN is generated by recycling of nicotinamide. Both pathways converge on NaMN, which is then converted into deamido-NAD+. Subsequently, deamido-NAD+ is converted to NAD+ by NAD+ synthetase. This entry represents NH(3)-dependent NAD(+) synthetases from prokaryotes.

 

Reference Protein and Structure

Sequence
P08164 UniProt (6.3.1.5) IPR022926 (Sequence Homologues) (PDB Homologues)
Biological species
Bacillus subtilis subsp. subtilis str. 168 (Bacteria) Uniprot
PDB
1kqp - NH3-DEPENDENT NAD+ SYNTHETASE FROM BACILLUS SUBTILIS AT 1 A RESOLUTION (1.03 Å) PDBe PDBsum 1kqp
Catalytic CATH Domains
3.40.50.620 CATHdb (see all for 1kqp)
Cofactors
Magnesium(2+) (2), Water (2) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:6.3.1.5)

deamido-NAD(2-)
CHEBI:58437ChEBI
+
ATP(4-)
CHEBI:30616ChEBI
+
ammonium
CHEBI:28938ChEBI
→
NAD(1-)
CHEBI:57540ChEBI
+
diphosphate(3-)
CHEBI:33019ChEBI
+
hydron
CHEBI:15378ChEBI
+
adenosine 5'-monophosphate(2-)
CHEBI:456215ChEBI
Alternative enzyme names: NAD synthetase, NAD(+) synthetase, Diphosphopyridine nucleotide synthetase, Nicotinamide adenine dinucleotide synthetase,

Enzyme Mechanism

Introduction

There is no direct involvement in this mechanism by any amino acid residues in the active site. The carboxylate group of the deamino-NAD acts as a nucleophile and attacks the gamma-phosphate of the ATP in a substitution reaction, liberating pyrophosphate. The carbonyl group of the phosphorylated substrate accepts a proton from the ammonium ion, thus increasing the electrophilicity of the carbon atom. Water deprotonates the ammonia molecule, which then acts as a nucleophile and attacks the carboxyl carbon of the phosphorylated intermediate in a substitution reaction, liberating AMP. A second water molecule deprotonates the alcohol group formed in the previous step, regenerating the carbonyl group.

Catalytic Residues Roles

UniProt PDB* (1kqp)
Asp51, Glu163 Asp50A, Glu162A Form part of the magensium binding site. metal ligand
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

bimolecular nucleophilic substitution, overall reactant used, intermediate formation, overall product formed, proton transfer, intermediate terminated

References

  1. Symersky J et al. (2002), Acta Crystallogr D Biol Crystallogr, 58, 1138-1146. NH3-dependent NAD+ synthetase from Bacillus subtilis at 1 A resolution. DOI:10.2210/pdb1kqp/pdb. PMID:12077433.
  2. De Ingeniis J et al. (2012), PLoS One, 7, e39115-. Glutamine versus ammonia utilization in the NAD synthetase family. DOI:10.1371/journal.pone.0039115. PMID:22720044.
  3. Devedjiev Y et al. (2001), Acta Crystallogr D Biol Crystallogr, 57, 806-812. Stabilization of active-site loops in NH3-dependent NAD+synthetase fromBacillus subtilis. DOI:10.1107/s0907444901003523. PMID:11375500.
  4. Rizzi M et al. (1998), Structure, 6, 1129-1140. A novel deamido-NAD+-binding site revealed by the trapped NAD-adenylate intermediate in the NAD+ synthetase structure. DOI:10.1016/s0969-2126(98)00114-2. PMID:9753692.

Step 1. Carboxylate group of the deamino-NAD acts as a nucleophile and attacks the gamma-phosphate of the ATP in a substitution reaction, liberating pyrophosphate. The two Mg(II) ions stabilise the intermediates formed

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu162A metal ligand
Asp50A metal ligand

Chemical Components

ingold: bimolecular nucleophilic substitution, overall reactant used, intermediate formation, overall product formed

Step 2. The carbonyl group of the phosphorylated substrate accepts a proton from the ammonium ion, thus increasing the electrophilicity of the carbon atom.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu162A metal ligand
Asp50A metal ligand

Chemical Components

proton transfer, overall reactant used, intermediate formation

Step 3. Water deprotonates the ammonia molecule, which then acts as a nucleophile and attacks the carboxyl carbon of the phosphorylated intermediate in a substitution reaction, liberating AMP.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu162A metal ligand
Asp50A metal ligand

Chemical Components

proton transfer, ingold: bimolecular nucleophilic substitution, intermediate terminated, overall product formed

Step 4. A second water molecule deprotonates the alcohol group formed in the previous step, regenerating the carbonyl group.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu162A metal ligand
Asp50A metal ligand

Chemical Components

proton transfer, intermediate terminated, overall product formed
Download: Image, Marvin File

Step 1. Carboxylate group of the deamino-NAD acts as a nucleophile and attacks the gamma-phosphate of the ATP in a substitution reaction, liberating pyrophosphate. The two Mg(II) ions stabilise the intermediates formed

Step 2. The carbonyl group of the phosphorylated substrate accepts a proton from the ammonium ion, thus increasing the electrophilicity of the carbon atom.

Step 3. Water deprotonates the ammonia molecule, which then acts as a nucleophile and attacks the carboxyl carbon of the phosphorylated intermediate in a substitution reaction, liberating AMP.

Step 4. A second water molecule deprotonates the alcohol group formed in the previous step, regenerating the carbonyl group.

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid, Charity Hornby