EzCatDB: S00315

DB codeS00315
RLCP classification3.113.163000.1110
3.1143.70030.3060
CATH domainDomain 13.40.50.620Catalytic domain
E.C.6.3.1.5
MACiEM0200

CATH domainRelated DB codes (homologues)
3.40.50.620S00314,S00549,S00316,S00317,S00318,T00085,T00249,D00300,M00177,M00178,T00106,T00114

Enzyme Name
Swiss-protKEGG

P08164
Protein nameNH(3)-dependent NAD(+) synthetaseNAD+ synthase
NAD+ synthetase
NAD+ synthase
nicotinamide adenine dinucleotide synthetase
diphosphopyridine nucleotide synthetase
SynonymsEC 6.3.1.5
Spore outgrowth factor B
Sporulation protein outB
General stress protein 38
GSP38

KEGG pathways
MAP codePathways
MAP00760Nicotinate and nicotinamide metabolism
MAP00910Nitrogen metabolism

Swiss-prot:Accession NumberP08164
Entry nameNADE_BACSU
ActivityATP + deamido-NAD(+) + NH(3) = AMP + diphosphate + NAD(+).
SubunitHomodimer.
Subcellular location
Cofactor


CofactorsSubstratesProductsintermediates
KEGG-idC00305C00238C00002C00857C00014C00020C00013C00003
CompoundMagnesiumPotassiumATPDeamido-NAD+NH3AMPPyrophosphateNAD+NAD-adenylate
Typedivalent metal (Ca2+, Mg2+)univalent metal (Na+, K+)amine group,nucleotideamine group,carbohydrate,nucleotideamine group,organic ionamine group,nucleotidephosphate group/phosphate ionamide group,amine group,nucleotide
1ee1ABound:_MGUnboundBound:ATPBound:DNDUnboundUnboundUnboundUnboundUnbound
1ee1BUnboundUnboundUnboundBound:DNDUnboundUnboundUnboundUnboundUnbound
1fydABound:_MGUnboundUnboundUnboundUnboundBound:AMPBound:POPUnboundUnbound
1fydBUnboundUnboundUnboundUnboundUnboundUnboundUnboundUnboundUnbound
1ifxAUnboundUnboundUnboundBound:DNDUnboundUnboundUnboundUnboundUnbound
1ifxBUnboundUnboundUnboundBound:DNDUnboundUnboundUnboundUnboundUnbound
1ih8ABound:2x_MGUnboundAnalogue:APCUnboundUnboundUnboundUnboundUnboundUnbound
1ih8BBound:_MGUnboundUnboundUnboundUnboundUnboundUnboundUnboundUnbound
1kqpABound:2x_MGUnboundUnboundUnboundUnboundUnboundBound:POPUnboundIntermediate-bound:ADJ
1kqpBBound:2x_MGUnboundUnboundUnboundUnboundUnboundBound:POPUnboundIntermediate-bound:ADJ
1nsyABound:_MGUnboundUnboundUnboundUnboundBound:AMPBound:POPAnalogue:ATPUnbound
1nsyBBound:_MGUnboundUnboundUnboundUnboundBound:AMPBound:POPAnalogue:ATPUnbound
2nsyABound:2x_MGUnboundUnboundUnboundUnboundUnboundBound:POPUnboundIntermediate-bound:__A-NAD
2nsyBBound:2x_MGUnboundUnboundUnboundUnboundUnboundBound:POPUnboundIntermediate-bound:__A-NAD

Active-site residues
resource
Swiss-prot;P08164 & literature [4], [6], [7]
pdbCatalytic residuesCofactor-binding residuescomment
1ee1ASER   46;ASP  173
ASP   50;GLU  162(Magnesium-1 binding);THR  208(Magnesium-2);ASP  220(Potassium)

1ee1BSER   46;ASP  173
ASP   50;GLU  162(Magnesium-1 binding);                     ;                   
invisible 205-225
1fydASER   46;ASP  173
ASP   50;GLU  162(Magnesium-1 binding);THR  208(Magnesium-2);ASP  220(Potassium)

1fydBSER 1046;ASP 1173
ASP 1050;GLU 1162(Magnesium-1 binding);                     ;                   
invisible 1205-1225
1ifxASER   46;ASP  173
ASP   50;GLU  162(Magnesium-1 binding);                     ;                   
invisible 205-225
1ifxBSER 1046;ASP 1173
ASP 1050;GLU 1162(Magnesium-1 binding);                     ;                   
invisible 1205-1225
1ih8ASER   46;ASP  173
ASP   50;GLU  162(Magnesium-1 binding);THR  208(Magnesium-2);ASP  220(Potassium)

1ih8BSER 1046;ASP 1173
ASP 1050;GLU 1162(Magnesium-1 binding);                     ;                   
invisible 1205-1224
1kqpASER   46;ASP  173
ASP   50;GLU  162(Magnesium-1 binding);THR  208(Magnesium-2);ASP  220(Potassium)

1kqpBSER   46;ASP  173
ASP   50;GLU  162(Magnesium-1 binding);THR  208(Magnesium-2);ASP  220(Potassium)

1nsyASER   46;ASP  173
ASP   50;GLU  162(Magnesium-1 binding);THR  208(Magnesium-2);ASP  220(Potassium)

1nsyBSER   46;ASP  173
ASP   50;GLU  162(Magnesium-1 binding);THR  208(Magnesium-2);ASP  220(Potassium)

2nsyASER   46;ASP  173
ASP   50;GLU  162(Magnesium-1 binding);THR  208(Magnesium-2);ASP  220(Potassium)

2nsyBSER   46;ASP  173
ASP   50;GLU  162(Magnesium-1 binding);THR  208(Magnesium-2);ASP  220(Potassium)


References for Catalytic Mechanism
ReferencesSectionsNo. of steps in catalysis
[1]p.5130-5131
[4]Fig.7, p.1134-1138
[7]p.1143-1145, Fig.6

references
[1]
CommentsX-RAY CRYSTALLOGRAPHY (2.0 ANGSTROMS).
Medline ID97050817
PubMed ID8895556
JournalEMBO J
Year1996
Volume15
Pages5125-34
AuthorsRizzi M, Nessi C, Mattevi A, Coda A, Bolognesi M, Galizzi A
TitleCrystal structure of NH3-dependent NAD+ synthetase from Bacillus subtilis.
Related PDB1nsy
Related Swiss-protP08164
[2]
PubMed ID8916230
JournalProteins
Year1996
Volume26
Pages236-8
AuthorsRizzi M, Nessi C, Bolognesi M, Coda A, Galizzi A
TitleCrystallization of NAD+ synthetase from Bacillus subtilis.
[3]
PubMed ID9261082
JournalStructure
Year1997
Volume5
Pages895-906
AuthorsSavage H, Montoya G, Svensson C, Schwenn JD, Sinning I
TitleCrystal structure of phosphoadenylyl sulphate (PAPS) reductase: a new family of adenine nucleotide alpha hydrolases.
[4]
CommentsX-RAY CRYSTALLOGRAPHY (2.0 ANGSTROMS).
Medline ID98428669
PubMed ID9753692
JournalStructure
Year1998
Volume6
Pages1129-40
AuthorsRizzi M, Bolognesi M, Coda A
TitleA novel deamido-NAD+-binding site revealed by the trapped NAD-adenylate intermediate in the NAD+ synthetase structure.
Related PDB2nsy
Related Swiss-protP08164
[5]
PubMed ID10544053
JournalJ Struct Biol
Year1999
Volume127
Pages279-82
AuthorsOzment C, Barchue J, DeLucas LJ, Chattopadhyay D
TitleStructural study of Escherichia coli NAD synthetase: overexpression, purification, crystallization, and preliminary crystallographic analysis.
[6]
CommentsX-ray crystallography
PubMed ID11375500
JournalActa Crystallogr D Biol Crystallogr
Year2001
Volume57
Pages806-12
AuthorsDevedjiev Y, Symersky J, Singh R, Jedrzejas M, Brouillette C, Brouillette W, Muccio D, Chattopadhyay D, DeLucas L
TitleStabilization of active-site loops in NH3-dependent NAD+ synthetase from Bacillus subtilis.
Related PDB1ee1,1fyd,1ifx,1ih8
[7]
CommentsX-ray crystallography
PubMed ID12077433
JournalActa Crystallogr D Biol Crystallogr
Year2002
Volume58
Pages1138-46
AuthorsSymersky J, Devedjiev Y, Moore K, Brouillette C, DeLucas L
TitleNH3-dependent NAD+ synthetase from Bacillus subtilis at 1 A resolution.
Related PDB1kqp

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.

createdupdated
2004-04-192009-02-26
Copyright: Nozomi Nagano, JST & CBRC-AIST
Funded by PRESTO/Japan Science and Technology Corporation (JST) (December 2001 - November 2004)
Funded by Grant-in-Aid for Publication of Scientific Research Results/Japan Society for the Promotion of Science (JSPS) (April 2005 - March 2006)
Funded by Grant-in-Aid for Scientific Research (B)/Japan Society for the Promotion of Science (JSPS) (April 2005 - March 2008)
Funded by BIRD/Japan Science and Technology Corporation (JST) (September 2005 - September 2010)
Funded by BIRD/Japan Science and Technology Corporation (JST) (October 2007 - September 2010)
Funded by Grant-in-Aid for Publication of Scientific Research Results/Japan Society for the Promotion of Science (JSPS) (April 2011 - March 2012)

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