DB code: S00220

RLCP classification 5.1304.3086000.333 : Elimination
4.1244.165000.313 : Addition
CATH domain 3.20.20.70 : TIM Barrel Catalytic domain
E.C. 2.5.1.3
CSA 1g4p 2tps
M-CSA 1g4p 2tps
MACiE

CATH domain Related DB codes (homologues)
3.20.20.70 : TIM Barrel S00215 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

Uniprot Enzyme Name
UniprotKB Protein name Synonyms RefSeq Pfam
P39594 Thiamine-phosphate pyrophosphorylase
TMP pyrophosphorylase
TMP-PPase
EC 2.5.1.3
Thiamine-phosphate synthase
NP_391708.1 (Protein)
NC_000964.3 (DNA/RNA sequence)
PF02581 (TMP-TENI)
[Graphical View]

KEGG enzyme name
thiamine-phosphate diphosphorylase
thiamine phosphate pyrophosphorylase
thiamine monophosphate pyrophosphorylase
TMP-PPase

UniprotKB: Accession Number Entry name Activity Subunit Subcellular location Cofactor
P39594 THIE_BACSU 2-methyl-4-amino-5-hydroxymethylpyrimidine diphosphate + 4-methyl-5-(2-phosphono-oxyethyl)thiazole = diphosphate + thiamine phosphate. Monomer. Binds 1 magnesium ion per subunit.

KEGG Pathways
Map code Pathways E.C.
MAP00730 Thiamine metabolism

Compound table
Cofactors Substrates Products Intermediates
KEGG-id C00305 C04752 C04327 C00013 C01081
E.C.
Compound Magnesium 2-Methyl-4-amino-5-hydroxymethylpyrimidine diphosphate 4-Methyl-5-(2-phosphono-oxyethyl)-thiazole Diphosphate Thiamin monophosphate
Type divalent metal (Ca2+, Mg2+) amine group,aromatic ring (with nitrogen atoms),phosphate group/phosphate ion aromatic ring (with nitrogen atoms),phosphate group/phosphate ion phosphate group/phosphate ion amine group,aromatic ring (with nitrogen atoms),phosphate group/phosphate ion
ChEBI 18420
 18420
 		16629
 16629
 		17857
 17857
 		29888
 29888
 		9533
 9533
PubChem 888
 888
 		217
 217
 		1137
 1137
 		1023
 21961011
 1023
 21961011
 		1131
 1131
1g4eA Unbound Unbound Unbound Unbound Unbound Unbound
1g4eB Unbound Unbound Unbound Unbound Unbound Unbound
1g4pA Bound:_MG Analogue:FQP Unbound Unbound Unbound Unbound
1g4pB Bound:_MG Analogue:FQP Unbound Unbound Unbound Unbound
1g4sA Bound:_MG Unbound Unbound Bound:POP Bound:TPS Unbound
1g4sB Bound:_MG Unbound Unbound Bound:POP Bound:TPS Unbound
1g4tA Bound:_MG Unbound Unbound Bound:POP Analogue:FTP Unbound
1g4tB Bound:_MG Unbound Unbound Bound:POP Analogue:FTP Unbound
1g67A Bound:_MG Unbound Bound:TZP Bound:POP Unbound Intermediate-bound:ICP
1g67B Bound:_MG Unbound Bound:TZP Bound:POP Unbound Intermediate-bound:ICP
1g69A Bound:_MG Unbound Bound:TZP Bound:POP Unbound Intermediate-bound:ICP
1g69B Bound:_MG Unbound Bound:TZP Bound:POP Unbound Intermediate-bound:ICP
1g6cA Bound:_MG Unbound Bound:TZP Bound:POP Unbound Intermediate-analogue:IFP
1g6cB Bound:_MG Unbound Bound:TZP Bound:POP Unbound Intermediate-analogue:IFP
2tpsA Bound:_MG Unbound Unbound Bound:POP Bound:TPS Unbound
2tpsB Bound:_MG Unbound Unbound Bound:POP Bound:TPS Unbound

Reference for Active-site residues
resource references E.C.
PDB;2tps & Swiss-prot;P39594 & literature [3], [5]

Active-site residues
PDB Catalytic residues Cofactor-binding residues Modified residues Main-chain involved in catalysis Comment
1g4eA GLN 57;ARG 59;LYS 61;ASN 92; ; ASP 93; (Magnesium binding) mutant S130A, invisible 111-112, 157-161
1g4eB GLN 1057;ARG 1059;LYS 1061;ASN 1092; ; ASP 1093; (Magnesium binding) mutant S1130A, invisible 1112, 1156-1161
1g4pA GLN 57;ARG 59;LYS 61;ASN 92; ; ASP 93;ASP 112(Magnesium binding) mutant S130A, invisible 156-161
1g4pB GLN 1057;ARG 1059;LYS 1061;ASN 1092; ; ASP 1093;ASP 1112(Magnesium binding) mutant S1130A, invisible 1156-1162
1g4sA GLN 57;ARG 59;LYS 61;ASN 92; ;LYS 159 ASP 93;ASP 112(Magnesium binding) mutant S130A
1g4sB GLN 1057;ARG 1059;LYS 1061;ASN 1092; ;LYS 1159 ASP 1093;ASP 1112(Magnesium binding) mutant S1130A
1g4tA GLN 57;ARG 59;LYS 61;ASN 92;SER 130;LYS 159 ASP 93;ASP 112(Magnesium binding)
1g4tB GLN 1057;ARG 1059;LYS 1061;ASN 1092;SER 1130;LYS 1159 ASP 1093;ASP 1112(Magnesium binding)
1g67A GLN 57;ARG 59;LYS 61;ASN 92; ;LYS 159 ASP 93;ASP 112(Magnesium binding) mutant S130A
1g67B GLN 1057;ARG 1059;LYS 1061;ASN 1092; ;LYS 1159 ASP 1093;ASP 1112(Magnesium binding) mutant S1130A
1g69A GLN 57;ARG 59;LYS 61;ASN 92; ;LYS 159 ASP 93;ASP 112(Magnesium binding) mutant S130A
1g69B GLN 1057;ARG 1059;LYS 1061;ASN 1092; ;LYS 1159 ASP 1093;ASP 1112(Magnesium binding) mutant S1130A
1g6cA GLN 57;ARG 59;LYS 61;ASN 92; ;LYS 159 ASP 93;ASP 112(Magnesium binding) mutant S130A
1g6cB GLN 1057;ARG 1059;LYS 1061;ASN 1092; ;LYS 1159 ASP 1093;ASP 1112(Magnesium binding) mutant S1130A
2tpsA GLN 57;ARG 59;LYS 61;ASN 92;SER 130;LYS 159 ASP 93;ASP 112(Magnesium binding)
2tpsB GLN 57;ARG 59;LYS 61;ASN 92;SER 130;LYS 159 ASP 93;ASP 112(Magnesium binding)

References for Catalytic Mechanism
References Sections No. 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]
Resource
Comments CHARACTERIZATION.
Medline ID 97284509
PubMed ID 9139923
Journal J Bacteriol
Year 1997
Volume 179
Pages 3030-5
Authors Zhang Y, Taylor SV, Chiu HJ, Begley TP
Title Characterization of the Bacillus subtilis thiC operon involved in thiamine biosynthesis
Related PDB
Related UniProtKB P39594
[2]
Resource
Comments
Medline ID
PubMed ID 10382260
Journal Arch Microbiol
Year 1999
Volume 171
Pages 293-300
Authors Begley TP, Downs DM, Ealick SE, McLafferty FW, Van Loon AP, Taylor S, Campobasso N, Chiu HJ, Kinsland C, Reddick JJ, Xi J
Title Thiamin biosynthesis in prokaryotes.
Related PDB
Related UniProtKB
[3]
Resource
Comments X-RAY CRYSTALLOGRAPHY (1.25 ANGSTROMS).
Medline ID 99280703
PubMed ID 10350464
Journal Biochemistry
Year 1999
Volume 38
Pages 6460-70
Authors Chiu HJ, Reddick JJ, Begley TP, Ealick SE
Title Crystal structure of thiamin phosphate synthase from Bacillus subtilis at 1.25 A resolution
Related PDB 2tps
Related UniProtKB P39594
[4]
Resource
Comments
Medline ID
PubMed ID 11054297
Journal J Mol Biol
Year 2000
Volume 303
Pages 627-41
Authors Copley RR, Bork P
Title Homology among (betaalpha)(8) barrels: implications for the evolution of metabolic pathways.
Related PDB
Related UniProtKB
[5]
Resource
Comments X-ray crystallography
Medline ID
PubMed ID 11513589
Journal Biochemistry
Year 2001
Volume 40
Pages 10103-14
Authors Peapus DH, Chiu HJ, Campobasso N, Reddick JJ, Begley TP, Ealick SE
Title Structural characterization of the enzyme-substrate, enzyme-intermediate, and enzyme-product complexes of thiamin phosphate synthase.
Related PDB 1g4e 1g4p 1g4s 1g4t 1g67 1g69 1g6c
Related UniProtKB
[6]
Resource
Comments
Medline ID
PubMed ID 11513588
Journal Biochemistry
Year 2001
Volume 40
Pages 10095-102
Authors Reddick JJ, Nicewonger R, Begley TP
Title Mechanistic studies on thiamin phosphate synthase: evidence for a dissociative mechanism.
Related PDB
Related UniProtKB
[7]
Resource
Comments
Medline ID
PubMed ID 14675553
Journal Curr Opin Struct Biol
Year 2003
Volume 13
Pages 739-47
Authors Settembre E, Begley TP, Ealick SE
Title Structural biology of enzymes of the thiamin biosynthesis pathway.
Related PDB
Related UniProtKB

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.

Created Updated
2005-03-22 2009-02-26