EzCatDB: S00220
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DB codeS00220
RLCP classification5.1304.3086000.333 : Elimination
4.1244.165000.313 : Addition
CATH domainDomain 13.20.20.70 : TIM BarrelCatalytic domain
E.C.2.5.1.3
CSA1g4p,2tps

CATH domainRelated DB codes (homologues)
3.20.20.70 : TIM BarrelS00215,S00217,S00218,S00219,S00532,S00198,S00745,S00537,S00538,S00539,S00826,S00841,S00235,S00239,S00240,S00243,S00244,S00199,S00200,S00201,S00221,S00222,S00847,S00224,S00225,S00226,D00014,D00029,M00141,T00015,T00239,D00664,D00665,D00804,D00863,T00089

Enzyme Name
UniProtKBKEGG

P39594
Protein nameThiamine-phosphate pyrophosphorylasethiamine-phosphate diphosphorylase
thiamine phosphate pyrophosphorylase
thiamine monophosphate pyrophosphorylase
TMP-PPase
SynonymsTMP pyrophosphorylase
TMP-PPase
EC 2.5.1.3
Thiamine-phosphate synthase
RefSeqNP_391708.1 (Protein)
NC_000964.3 (DNA/RNA sequence)
PfamPF02581 (TMP-TENI)
[Graphical view]

KEGG pathways
MAP codePathways
MAP00730Thiamine metabolism

UniProtKB:Accession NumberP39594
Entry nameTHIE_BACSU
Activity2-methyl-4-amino-5-hydroxymethylpyrimidine diphosphate + 4-methyl-5-(2-phosphono-oxyethyl)thiazole = diphosphate + thiamine phosphate.
SubunitMonomer.
Subcellular location
CofactorBinds 1 magnesium ion per subunit.

Compound table: links to PDB-related databases & PoSSuM

CofactorsSubstratesProductsintermediates
KEGG-idC00305C04752C04327C00013C01081
CompoundMagnesium2-Methyl-4-amino-5-hydroxymethylpyrimidine diphosphate4-Methyl-5-(2-phosphono-oxyethyl)-thiazoleDiphosphateThiamin monophosphate
Typedivalent metal (Ca2+, Mg2+)amine group,aromatic ring (with nitrogen atoms),phosphate group/phosphate ionaromatic ring (with nitrogen atoms),phosphate group/phosphate ionphosphate group/phosphate ionamine group,aromatic ring (with nitrogen atoms),phosphate group/phosphate ion
ChEBI18420
16629
17857
29888
9533

PubChem888
217
1137
1023
21961011
1131

              
1g4eAUnboundUnboundUnboundUnboundUnboundUnbound
1g4eBUnboundUnboundUnboundUnboundUnboundUnbound
1g4pABound:_MGAnalogue:FQPUnboundUnboundUnboundUnbound
1g4pBBound:_MGAnalogue:FQPUnboundUnboundUnboundUnbound
1g4sABound:_MGUnboundUnboundBound:POPBound:TPSUnbound
1g4sBBound:_MGUnboundUnboundBound:POPBound:TPSUnbound
1g4tABound:_MGUnboundUnboundBound:POPAnalogue:FTPUnbound
1g4tBBound:_MGUnboundUnboundBound:POPAnalogue:FTPUnbound
1g67ABound:_MGUnboundBound:TZPBound:POPUnboundIntermediate-bound:ICP
1g67BBound:_MGUnboundBound:TZPBound:POPUnboundIntermediate-bound:ICP
1g69ABound:_MGUnboundBound:TZPBound:POPUnboundIntermediate-bound:ICP
1g69BBound:_MGUnboundBound:TZPBound:POPUnboundIntermediate-bound:ICP
1g6cABound:_MGUnboundBound:TZPBound:POPUnboundIntermediate-analogue:IFP
1g6cBBound:_MGUnboundBound:TZPBound:POPUnboundIntermediate-analogue:IFP
2tpsABound:_MGUnboundUnboundBound:POPBound:TPSUnbound
2tpsBBound:_MGUnboundUnboundBound:POPBound:TPSUnbound

Active-site residues
resource
PDB;2tps & Swiss-prot;P39594 & literature [3], [5]
pdbCatalytic residuesCofactor-binding residuescomment
           
1g4eAGLN   57;ARG   59;LYS   61;ASN   92;        ;        
ASP   93;        (Magnesium binding)
mutant S130A, invisible 111-112, 157-161
1g4eBGLN 1057;ARG 1059;LYS 1061;ASN 1092;        ;        
ASP 1093;        (Magnesium binding)
mutant S1130A, invisible 1112, 1156-1161
1g4pAGLN   57;ARG   59;LYS   61;ASN   92;        ;        
ASP   93;ASP  112(Magnesium binding)
mutant S130A, invisible 156-161
1g4pBGLN 1057;ARG 1059;LYS 1061;ASN 1092;        ;        
ASP 1093;ASP 1112(Magnesium binding)
mutant S1130A, invisible 1156-1162
1g4sAGLN   57;ARG   59;LYS   61;ASN   92;        ;LYS  159
ASP   93;ASP  112(Magnesium binding)
mutant S130A
1g4sBGLN 1057;ARG 1059;LYS 1061;ASN 1092;        ;LYS 1159
ASP 1093;ASP 1112(Magnesium binding)
mutant S1130A
1g4tAGLN   57;ARG   59;LYS   61;ASN   92;SER  130;LYS  159
ASP   93;ASP  112(Magnesium binding)
 
1g4tBGLN 1057;ARG 1059;LYS 1061;ASN 1092;SER 1130;LYS 1159
ASP 1093;ASP 1112(Magnesium binding)
 
1g67AGLN   57;ARG   59;LYS   61;ASN   92;        ;LYS  159
ASP   93;ASP  112(Magnesium binding)
mutant S130A
1g67BGLN 1057;ARG 1059;LYS 1061;ASN 1092;        ;LYS 1159
ASP 1093;ASP 1112(Magnesium binding)
mutant S1130A
1g69AGLN   57;ARG   59;LYS   61;ASN   92;        ;LYS  159
ASP   93;ASP  112(Magnesium binding)
mutant S130A
1g69BGLN 1057;ARG 1059;LYS 1061;ASN 1092;        ;LYS 1159
ASP 1093;ASP 1112(Magnesium binding)
mutant S1130A
1g6cAGLN   57;ARG   59;LYS   61;ASN   92;        ;LYS  159
ASP   93;ASP  112(Magnesium binding)
mutant S130A
1g6cBGLN 1057;ARG 1059;LYS 1061;ASN 1092;        ;LYS 1159
ASP 1093;ASP 1112(Magnesium binding)
mutant S1130A
2tpsAGLN   57;ARG   59;LYS   61;ASN   92;SER  130;LYS  159
ASP   93;ASP  112(Magnesium binding)
 
2tpsBGLN   57;ARG   59;LYS   61;ASN   92;SER  130;LYS  159
ASP   93;ASP  112(Magnesium binding)
 

References for Catalytic Mechanism
ReferencesSectionsNo. of steps in catalysis
[2]p.298
[3]Fig.8, p.6468
[5]Scheme 2, p.10111
[6]FIGURE 2, p.10096
[6]FIGURE 14, p.10101

references
[1]
CommentsCHARACTERIZATION.
Medline ID97284509
PubMed ID9139923
JournalJ Bacteriol
Year1997
Volume179
Pages3030-5
AuthorsZhang Y, Taylor SV, Chiu HJ, Begley TP
TitleCharacterization of the Bacillus subtilis thiC operon involved in thiamine biosynthesis
Related UniProtKBP39594
[2]
PubMed ID10382260
JournalArch Microbiol
Year1999
Volume171
Pages293-300
AuthorsBegley TP, Downs DM, Ealick SE, McLafferty FW, Van Loon AP, Taylor S, Campobasso N, Chiu HJ, Kinsland C, Reddick JJ, Xi J
TitleThiamin biosynthesis in prokaryotes.
[3]
CommentsX-RAY CRYSTALLOGRAPHY (1.25 ANGSTROMS).
Medline ID99280703
PubMed ID10350464
JournalBiochemistry
Year1999
Volume38
Pages6460-70
AuthorsChiu HJ, Reddick JJ, Begley TP, Ealick SE
TitleCrystal structure of thiamin phosphate synthase from Bacillus subtilis at 1.25 A resolution
Related PDB2tps
Related UniProtKBP39594
[4]
PubMed ID11054297
JournalJ Mol Biol
Year2000
Volume303
Pages627-41
AuthorsCopley RR, Bork P
TitleHomology among (betaalpha)(8) barrels: implications for the evolution of metabolic pathways.
[5]
CommentsX-ray crystallography
PubMed ID11513589
JournalBiochemistry
Year2001
Volume40
Pages10103-14
AuthorsPeapus DH, Chiu HJ, Campobasso N, Reddick JJ, Begley TP, Ealick SE
TitleStructural characterization of the enzyme-substrate, enzyme-intermediate, and enzyme-product complexes of thiamin phosphate synthase.
Related PDB1g4e,1g4p,1g4s,1g4t,1g67,1g69,1g6c
[6]
PubMed ID11513588
JournalBiochemistry
Year2001
Volume40
Pages10095-102
AuthorsReddick JJ, Nicewonger R, Begley TP
TitleMechanistic studies on thiamin phosphate synthase: evidence for a dissociative mechanism.
[7]
PubMed ID14675553
JournalCurr Opin Struct Biol
Year2003
Volume13
Pages739-47
AuthorsSettembre E, Begley TP, Ealick SE
TitleStructural biology of enzymes of the thiamin biosynthesis pathway.

comments
The literature [2] & [3] suggested that this enzyme catalyzes SN1-like transfer reaction, forming a carbenium intermediate. In contrast, the other literature [5] & [6] suggested that it catalyzes two successive reactions, (A) Elimination of pyrophosphate group, forming a double-bond at C7'-C5' of the first substrate (HMP-PP), and (B) Addition of thiazole group to the double-bond.
According to the literature [5], despite the important role of Ser130 in catalysis, the mutant S130A does not prevent the first reaction, forming an intermediate. Thus, the residue must be involved more importantly in the second reaction.
The reaction probably proceeds as follows (see [5] & [6]);
(A) Eliminative double-bond formation: Elimination of phosphate group
(A1) The eliminated group, pyrophosphate, is stabilized by magnesium ion, which is bound to Asp93 and Asp112, along with Arg59, Lys61, Ser130, and Lys159. The pyrimidine ring (of leaving group) is stabilized by Gln57.
(A2) Bond cleavage between C7'-O5 (of alpha-phosphate) occurs first, forming a carbocation intermediate. (E1-like reaction)
(A3) The beta-phosphate oxygen acts as a general base to deprotonate the N4' amine (deprotonation site) of the pyrimidine ring, leading to the formation of double bonds at C7'-C5' bond and C4'-N4' bond, forming an iminemethide intermediate.
(B) Addition of thiazole group to the intermediate.
(B1) During this reaction, Ser130 must modulate the interaction between the C7' of the pyrimidine ring and the eliminated pyrophosphate. The N4' imine of the pyrimidine ring (of unsaturated group) is stabilized by Gln57.
(B2) The added group, the nitrogen atom of the thiazole group of the second substrate, makes a nucleophilic attack on the C7' atom of the iminemethide intermediate.
(B3) The beta-phosphate oxygen acts as a general acid to protonate the N4' imine, in a concerted mode, to complete the reaction.

createdupdated
2005-03-222009-02-26
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 2008)
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)
Funded by Grant-in-Aid for Publication of Scientific Research Results/Japan Society for the Promotion of Science (JSPS) (April 2012 - March 2013)
Supported by the commission for the Development of Artificial Gene Synthesis Technology for Creating Innovative Biomaterial from the Ministry of Economy, Trade and Industry (METI) (October 2012 - )
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