DB code: S00315

RLCP classification 3.113.163000.1110 : Transfer
3.1143.70030.3060 : Transfer
CATH domain 3.40.50.620 : Rossmann fold Catalytic domain
E.C. 6.3.1.5
CSA
M-CSA
MACiE M0200

CATH domain Related DB codes (homologues)
3.40.50.620 : Rossmann fold S00314 S00549 S00316 S00317 S00318 T00085 T00249 D00300 M00177 M00178 T00106 T00114

Uniprot Enzyme Name
UniprotKB Protein name Synonyms RefSeq Pfam
P08164 NH(3)-dependent NAD(+) synthetase
EC 6.3.1.5
Spore outgrowth factor B
Sporulation protein outB
General stress protein 38
GSP38
NP_388195.1 (Protein)
NC_000964.3 (DNA/RNA sequence)
PF02540 (NAD_synthase)
[Graphical View]

KEGG enzyme name
NAD+ synthase
NAD+ synthetase
NAD+ synthase
nicotinamide adenine dinucleotide synthetase
diphosphopyridine nucleotide synthetase

UniprotKB: Accession Number Entry name Activity Subunit Subcellular location Cofactor
P08164 NADE_BACSU ATP + deamido-NAD(+) + NH(3) = AMP + diphosphate + NAD(+). Homodimer.

KEGG Pathways
Map code Pathways E.C.
MAP00760 Nicotinate and nicotinamide metabolism
MAP00910 Nitrogen metabolism

Compound table
Cofactors Substrates Products Intermediates
KEGG-id C00305 C00238 C00002 C00857 C00014 C00020 C00013 C00003
E.C.
Compound Magnesium Potassium ATP Deamido-NAD+ NH3 AMP Pyrophosphate NAD+ NAD-adenylate
Type divalent metal (Ca2+, Mg2+) univalent metal (Na+, K+) amine group,nucleotide amine group,carbohydrate,nucleotide amine group,organic ion amine group,nucleotide phosphate group/phosphate ion amide group,amine group,nucleotide
ChEBI 18420
29103
15422
18304
16134
16027
29888
15846
PubChem 888
813
5957
165491
222
6083
1023
21961011
5893
1ee1A Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Bound:_MG Unbound Bound:ATP Bound:DND Unbound Unbound Unbound Unbound Unbound
1ee1B Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Unbound Unbound Bound:DND Unbound Unbound Unbound Unbound Unbound
1fydA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Bound:_MG Unbound Unbound Unbound Unbound Bound:AMP Bound:POP Unbound Unbound
1fydB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Unbound Unbound Unbound Unbound Unbound Unbound Unbound Unbound
1ifxA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Unbound Unbound Bound:DND Unbound Unbound Unbound Unbound Unbound
1ifxB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Unbound Unbound Bound:DND Unbound Unbound Unbound Unbound Unbound
1ih8A Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Bound:2x_MG Unbound Analogue:APC Unbound Unbound Unbound Unbound Unbound Unbound
1ih8B Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Bound:_MG Unbound Unbound Unbound Unbound Unbound Unbound Unbound Unbound
1kqpA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Bound:2x_MG Unbound Unbound Unbound Unbound Unbound Bound:POP Unbound Intermediate-bound:ADJ
1kqpB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Bound:2x_MG Unbound Unbound Unbound Unbound Unbound Bound:POP Unbound Intermediate-bound:ADJ
1nsyA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Bound:_MG Unbound Unbound Unbound Unbound Bound:AMP Bound:POP Analogue:ATP Unbound
1nsyB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Bound:_MG Unbound Unbound Unbound Unbound Bound:AMP Bound:POP Analogue:ATP Unbound
2nsyA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Bound:2x_MG Unbound Unbound Unbound Unbound Unbound Bound:POP Unbound Intermediate-bound:__A-NAD
2nsyB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Bound:2x_MG Unbound Unbound Unbound Unbound Unbound Bound:POP Unbound Intermediate-bound:__A-NAD

Reference for Active-site residues
resource references E.C.
Swiss-prot;P08164 & literature [4], [6], [7]

Active-site residues
PDB Catalytic residues Cofactor-binding residues Modified residues Main-chain involved in catalysis Comment
1ee1A Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain SER 46;ASP 173 ASP 50;GLU 162(Magnesium-1 binding);THR 208(Magnesium-2);ASP 220(Potassium)
1ee1B Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain SER 46;ASP 173 ASP 50;GLU 162(Magnesium-1 binding); ; invisible 205-225
1fydA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain SER 46;ASP 173 ASP 50;GLU 162(Magnesium-1 binding);THR 208(Magnesium-2);ASP 220(Potassium)
1fydB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain SER 1046;ASP 1173 ASP 1050;GLU 1162(Magnesium-1 binding); ; invisible 1205-1225
1ifxA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain SER 46;ASP 173 ASP 50;GLU 162(Magnesium-1 binding); ; invisible 205-225
1ifxB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain SER 1046;ASP 1173 ASP 1050;GLU 1162(Magnesium-1 binding); ; invisible 1205-1225
1ih8A Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain SER 46;ASP 173 ASP 50;GLU 162(Magnesium-1 binding);THR 208(Magnesium-2);ASP 220(Potassium)
1ih8B Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain SER 1046;ASP 1173 ASP 1050;GLU 1162(Magnesium-1 binding); ; invisible 1205-1224
1kqpA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain SER 46;ASP 173 ASP 50;GLU 162(Magnesium-1 binding);THR 208(Magnesium-2);ASP 220(Potassium)
1kqpB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain SER 46;ASP 173 ASP 50;GLU 162(Magnesium-1 binding);THR 208(Magnesium-2);ASP 220(Potassium)
1nsyA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain SER 46;ASP 173 ASP 50;GLU 162(Magnesium-1 binding);THR 208(Magnesium-2);ASP 220(Potassium)
1nsyB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain SER 46;ASP 173 ASP 50;GLU 162(Magnesium-1 binding);THR 208(Magnesium-2);ASP 220(Potassium)
2nsyA Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain SER 46;ASP 173 ASP 50;GLU 162(Magnesium-1 binding);THR 208(Magnesium-2);ASP 220(Potassium)
2nsyB Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain SER 46;ASP 173 ASP 50;GLU 162(Magnesium-1 binding);THR 208(Magnesium-2);ASP 220(Potassium)

References for Catalytic Mechanism
References Sections No. of steps in catalysis
[1]
p.5130-5131
[4]
Fig.7, p.1134-1138
[7]
p.1143-1145, Fig.6

References
[1]
Resource
Comments X-RAY CRYSTALLOGRAPHY (2.0 ANGSTROMS).
Medline ID 97050817
PubMed ID 8895556
Journal EMBO J
Year 1996
Volume 15
Pages 5125-34
Authors Rizzi M, Nessi C, Mattevi A, Coda A, Bolognesi M, Galizzi A
Title Crystal structure of NH3-dependent NAD+ synthetase from Bacillus subtilis.
Related PDB 1nsy
Related UniProtKB P08164
[2]
Resource
Comments
Medline ID
PubMed ID 8916230
Journal Proteins
Year 1996
Volume 26
Pages 236-8
Authors Rizzi M, Nessi C, Bolognesi M, Coda A, Galizzi A
Title Crystallization of NAD+ synthetase from Bacillus subtilis.
Related PDB
Related UniProtKB
[3]
Resource
Comments
Medline ID
PubMed ID 9261082
Journal Structure
Year 1997
Volume 5
Pages 895-906
Authors Savage H, Montoya G, Svensson C, Schwenn JD, Sinning I
Title Crystal structure of phosphoadenylyl sulphate (PAPS) reductase: a new family of adenine nucleotide alpha hydrolases.
Related PDB
Related UniProtKB
[4]
Resource
Comments X-RAY CRYSTALLOGRAPHY (2.0 ANGSTROMS).
Medline ID 98428669
PubMed ID 9753692
Journal Structure
Year 1998
Volume 6
Pages 1129-40
Authors Rizzi M, Bolognesi M, Coda A
Title A novel deamido-NAD+-binding site revealed by the trapped NAD-adenylate intermediate in the NAD+ synthetase structure.
Related PDB 2nsy
Related UniProtKB P08164
[5]
Resource
Comments
Medline ID
PubMed ID 10544053
Journal J Struct Biol
Year 1999
Volume 127
Pages 279-82
Authors Ozment C, Barchue J, DeLucas LJ, Chattopadhyay D
Title Structural study of Escherichia coli NAD synthetase: overexpression, purification, crystallization, and preliminary crystallographic analysis.
Related PDB
Related UniProtKB
[6]
Resource
Comments X-ray crystallography
Medline ID
PubMed ID 11375500
Journal Acta Crystallogr D Biol Crystallogr
Year 2001
Volume 57
Pages 806-12
Authors Devedjiev Y, Symersky J, Singh R, Jedrzejas M, Brouillette C, Brouillette W, Muccio D, Chattopadhyay D, DeLucas L
Title Stabilization of active-site loops in NH3-dependent NAD+ synthetase from Bacillus subtilis.
Related PDB 1ee1 1fyd 1ifx 1ih8
Related UniProtKB
[7]
Resource
Comments X-ray crystallography
Medline ID
PubMed ID 12077433
Journal Acta Crystallogr D Biol Crystallogr
Year 2002
Volume 58
Pages 1138-46
Authors Symersky J, Devedjiev Y, Moore K, Brouillette C, DeLucas L
Title NH3-dependent NAD+ synthetase from Bacillus subtilis at 1 A resolution.
Related PDB 1kqp
Related UniProtKB

Comments
This enzyme catalyzes two successive transfer reactions as follows;
The first reaction: Transfer of adenylate (AMP) to carboxylate of deamido-NAD, releasing pyrophosphate.
The second reaction: Transfer of acyl group of NAD to amine of ammonia molecule, releasing AMP and H2O.
According to the literature [1] & [4], the first reaction activates the substrate, thorough the adenylation. The literature [4] proposed the catalytic mechanisms for these two transfer reactions.
The first transfer reaction, in which no protein residues are directly involved in the catalysis, proceeds as follows:
(1) Two Mg2+ ions stabilize the leaving pyrophosphate, by neutralizing the negative charges, and also activate the transferred group, alpha-phosphate of ATP, by enhancing the electrophilicity of the group through polarization.
(2) The acceptor group, the oxygen atom of the carboxylate attached to nicotin, makes a nucleophilic attack on the transferred group, the alpha-phosphate of ATP.
(3) The two Mg2+ ions and a monovalent cation (K+) stabilize the pentacovalent transition-state.
(4) Adenylated-NAD intermediate is formed, releasing the pyrophosphate.
The second reaction proceeds as follows:
(1') Asp173 acts as a general base, to deprotonate the ammonium ion.
(2') The activated ammonium makes a nucleophilic attack on the carbonyl group of the adenylated-NAD intermediate, forming a tetrahedral transition-state adduct.
(3') The protonated sidechain of Asp173 and the monovalent cation (K+) stabilize the tetrahedral transition-state.
(4') Finally, NAD and AMP are formed.
According to the literature [7], the carbonyl group of the adenylated-NAD might be protonated due to an electron-withdrawing effect by the monovalent cation, such as K+ ion.

Created Updated
2004-04-19 2009-02-26