Information on EC 2.7.1.49 - hydroxymethylpyrimidine kinase

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria

EC NUMBER
COMMENTARY hide
2.7.1.49
-
RECOMMENDED NAME
GeneOntology No.
hydroxymethylpyrimidine kinase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + 4-amino-5-hydroxymethyl-2-methylpyrimidine = ADP + 4-amino-5-phosphonooxymethyl-2-methylpyrimidine
show the reaction diagram
; CTP, UTP and GTP can act as donors
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
phospho group transfer
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
hydroxymethylpyrimidine salvage
-
-
Metabolic pathways
-
-
thiamine formation from pyrithiamine and oxythiamine (yeast)
-
-
Thiamine metabolism
-
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thiamine salvage IV (yeast)
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vitamin B1 metabolism
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SYSTEMATIC NAME
IUBMB Comments
ATP:4-amino-5-hydroxymethyl-2-methylpyrimidine 5-phosphotransferase
CTP, UTP and GTP can act as donors.
CAS REGISTRY NUMBER
COMMENTARY hide
9026-55-5
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
gene annotated ThiD, instead of YjbV
-
-
Manually annotated by BRENDA team
rape
SwissProt
Manually annotated by BRENDA team
BL21(DE3) (pLysS)
-
-
Manually annotated by BRENDA team
strain 3D7
-
-
Manually annotated by BRENDA team
bv. viciae strain 3841
-
-
Manually annotated by BRENDA team
strain YPH500, 2 isozymes Thi20p and Thi21p encoded by 2 redundant genes THI20 and THI21
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-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + 2-methyl-4-amino-5-hydroxymethylpyrimidine
ADP + 2-methyl-4-amino-5-phosphomethylpyrimidine
show the reaction diagram
ATP + 2-methyl-4-amino-5-phosphomethylpyrimidine
ADP + 2-methyl-4-amino-5-diphosphomethylpyrimidine
show the reaction diagram
-
the enzyme shows lower affinity to 2-methyl-4-amino-5-hydroxymethylpyrimidine monophosphate than to 2-methyl-4-amino-5-hydroxymethylpyrimidine
-
-
ir
ATP + 4-amino-5-hydroxy-2-methylpyrimidine
ADP + 4-amino-2-methyl-5-phosphopyrimidine
show the reaction diagram
-
-
-
?
ATP + 4-amino-5-hydroxymethyl-2-methylpyrimidine
ADP + 4-amino-5-phosphomethyl-2-methylpyrimidine
show the reaction diagram
ATP + 4-amino-5-hydroxymethyl-2-methylpyrimidine
ADP + 4-amino-5-phosphonooxymethyl-2-methylpyrimidine
show the reaction diagram
-
-
-
?
ATP + 4-amino-5-phosphomethyl-2-methylpyrimidine
ADP + 4-amino-5-diphosphomethyl-2-methylpyrimidine
show the reaction diagram
ATP + bacimethrin
ADP + ?
show the reaction diagram
-
-
-
-
?
ATP + pyridoxal
ADP + pyridoxal 5'-phosphate
show the reaction diagram
ATP + pyridoxamine
ADP + pyridoxamine 5'-phosphate
show the reaction diagram
-
-
-
-
?
ATP + pyridoxine
ADP + pyridoxine 5'-phosphate
show the reaction diagram
CTP + 5-hydroxy-2-methylpyrimidine
CDP + 2-methyl-5-phosphopyrimidine
show the reaction diagram
-
-
-
?
GTP + 5-hydroxy-2-methylpyrimidine
GDP + 2-methyl-5-phosphopyrimidine
show the reaction diagram
-
-
-
?
UTP + 5-hydroxy-2-methylpyrimidine
UDP + 5-hydroxy-2-methylpyrimidine monophosphate
show the reaction diagram
-
-
-
?
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
ATP + 2-methyl-4-amino-5-hydroxymethylpyrimidine
ADP + 2-methyl-4-amino-5-phosphomethylpyrimidine
show the reaction diagram
ATP + 4-amino-5-hydroxymethyl-2-methylpyrimidine
ADP + 4-amino-5-phosphomethyl-2-methylpyrimidine
show the reaction diagram
ATP + 4-amino-5-hydroxymethyl-2-methylpyrimidine
ADP + 4-amino-5-phosphonooxymethyl-2-methylpyrimidine
show the reaction diagram
Q08224
-
-
-
?
ATP + 4-amino-5-phosphomethyl-2-methylpyrimidine
ADP + 4-amino-5-diphosphomethyl-2-methylpyrimidine
show the reaction diagram
additional information
?
-
-
the enzyme has both a biosynthetic and a salvage pathway function
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-
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
no metal requirement
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(6R)-6-[(2E)-4-oxo-6-phenylhex-2-en-1-yl]-5,6-dihydro-2H-pyran-2-one
lactone isolated from Cryptocarya rugulosa, covalent inhibition
(6R)-6-[(2E)-6-(4-ethynylphenyl)-4-oxohex-2-en-1-yl]-5,6-dihydro-2H-pyran-2-one
derivative of a lactone isolated from Cryptocarya rugulosa, covalent inhibition
(6S)-6-[(2E)-6-(4-ethynylphenyl)-4-oxohex-2-en-1-yl]tetrahydro-2H-pyran-2-one
derivative of a lactone isolated from Cryptocarya rugulosa, covalent inhibition
2-methyl-4-amino-5-hydroxymethylpyrimidine
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competitive inhibition
pyridoxine
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inhibits competitively the phosphorylation of 2-methyl-4-amino-5-hydroxymethylpyrimidine
rugulactone
selectively modifies the enzyme not at the active site cysteine, but on a remote cysteine residue
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additional information
-
stable to treatment with p-hydroxymercuribenzoate
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.012 - 0.11
2-methyl-4-amino-5-hydroxymethylpyrimidine
1.85 - 1.99
4-Amino-5-hydroxymethyl-2-methylpyrimidine
0.117 - 0.1431
ATP
0.111 - 0.26
pyridoxal
2.4
pyridoxamine
-
pH 6.2, 37C
0.0285 - 2.07
pyridoxine
additional information
additional information
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-
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.07 - 0.43
2-methyl-4-amino-5-hydroxymethylpyrimidine
0.044 - 0.045
4-Amino-5-hydroxymethyl-2-methylpyrimidine
0.43
ATP
Escherichia coli
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pH 8.0, 25C
0.165 - 0.328
pyridoxal
0.015 - 0.36
pyridoxine
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.09
2-methyl-4-amino-5-hydroxymethylpyrimidine
-
pH 6.2, 37C
0.0027
pyridoxine
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pH 6.2, 37C
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.025
(6R)-6-[(2E)-4-oxo-6-phenylhex-2-en-1-yl]-5,6-dihydro-2H-pyran-2-one
Staphylococcus aureus
P66915
pH not specified in the publication, temperature not specified in the publication
0.014
(6R)-6-[(2E)-6-(4-ethynylphenyl)-4-oxohex-2-en-1-yl]-5,6-dihydro-2H-pyran-2-one
Staphylococcus aureus
P66915
pH not specified in the publication, temperature not specified in the publication
0.032
(6S)-6-[(2E)-6-(4-ethynylphenyl)-4-oxohex-2-en-1-yl]tetrahydro-2H-pyran-2-one
Staphylococcus aureus
P66915
pH not specified in the publication, temperature not specified in the publication
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.108
-
using 2-methyl-4-amino-5-hydroxymethylpyrimidine as a substrate, at 37C
0.169
-
using bacimethrin as a substrate, at 37C
additional information
-
thiamine synthesis from hydroxymethylpyrimidine activities in wild-type and mutant strains
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.6
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substrate pyridoxine
6
-
substrate 2-methyl-4-amino-5-hydroxymethylpyrimidine
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 6
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about half-maximal activity at pH 5.0 and pH 6.0, pyridoxine as substrate
5.5 - 6.5
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about half-maximal activity at pH 5.5 and pH 6.5, 2-methyl-4-amino-5-hydroxymethylpyrimidine as substrate
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8.51
-
calculated from amino acid sequence
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720)
Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
27500
SDS-PAGE
31000
gel filtration
33000
-
4 * 33000, SDS-PAGE
33534
-
4 * 33534, predicted from amino acid sequence
35000
-
calculated from amino acid sequence
43000 - 44000
-
gel filtration
47500
-
2 * 47500, gel filtration
57800
-
calculated from amino acid sequence
65000
-
x * 65000, recombinant His-tagged isozymes Thi20p or Thi21p, SDS-PAGE
95000
-
gel filtration
additional information
-
gel filtration indicates the enzyme is tetrameric in the native state
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
tetramer
-
4 * 33000, SDS-PAGE; 4 * 33534, predicted from amino acid sequence
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
to 2.7 A resolution. Enzyme is composed of a ThiD-like N-terminal domain that catalyzes the phosphorylation of 4-amino-5-hydroxymethyl-2-methylpyrimidine and a TenA-like C-terminal domain with thiaminase activity. The structure reveals an overall dimeric organization in which N-terminal domains and C-terminal domains form ThiD-like and TenA-like local dimers. A relatively flexible linker region composed of a loop and a short helix joins the two domains. This linker region and a flexible N-terminal extension occupy the interface between the ThiD-like and TenA-like dimers. The N-terminal extension is composed of a single beta-strand that packs against and extends the length of the internal beta-sheet of the ThiD-like domain and a short alpha-helix
native protein and in complex with its substrates to 1.4-1.85 A resolution. The protein shows a typical ribokinase fold with a central large beta-sheet consisting of nine strands, flanked by three and five structurally conserved alpha-helices
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
55
-
stable to heating for 10 min
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
protected from inactivation by cysteine
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-70C, stored in 10% glycerol, loses half of its activity after 1 month
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant enzyme
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
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recombinant His-tagged isozymes Thi20p or Thi21p from Escherichia coli
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Superdex 200 HR 10/50 gel filtration
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
; Escherichia coli BL21(DE3) (pLysS) transformed with pTRD63 carrying ORF2, single gene thiD/J encodes 2 distinct enzyme activities, HMP kinase and HMP-P kinase, functional complemetation of Escherichia coli NI500, overexpression of ThiD
cDNA clone pBTH1 encoding bifunctional hydroxymethylpyrimidine kinase/thiamin-phosphate pyrophosphorylase established in Escherichia coli DH5alpha, expressed in Escherichia coli NI500 and Salmonella typhimurium DM456 by functional complementation
coexpression of and functional complementation by His-tagged soluble Thi20p or Thi21p isozymes with a thiamin synthetase in an Escherichia coli strain SN372L-3-9-10 deficient in thiamin biosynthesis
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expressed in Escherichia coli
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expressed in Escherichia coli strain BLR (DE3)
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expression in Escherichia coli
gene annotated ThiD, instead of YjbV, subcloning in Escherichia coli strain DH5alpha, overexpression of His-tagged enzyme in Escherichia coli strain BL21(DE3)
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overexpression in Escherichia coli
pET overexpression system
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single-copy gene in BAC clone T23L3
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C110A
residue C110 is mandatory for pyridoxal, but not for pyridoxine, or 4-amino-5-hydroxymethyl-2-methylpyrimidine turnover
C214A
mutant does not turn over pyridoxal, pyridoxine, or 4-amino-5-hydroxymethyl-2-methylpyrimidine. Residue C214 acts as the catalytic base in the phosphorylation reaction
C110A
-
residue C110 is mandatory for pyridoxal, but not for pyridoxine, or 4-amino-5-hydroxymethyl-2-methylpyrimidine turnover
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C214A
-
mutant does not turn over pyridoxal, pyridoxine, or 4-amino-5-hydroxymethyl-2-methylpyrimidine. Residue C214 acts as the catalytic base in the phosphorylation reaction
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M134K
-
detrimental to HMP kinase activity
M134K/T472D
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double mutant with 25% activity compared to the wild type enzyme
Q373L
-
30% reduced HMP kinase activity
Q98L
-
detrimental to HMP kinase activity
Q98L/Q373L
-
double mutant with 10% activity compared to the wild type enzyme
S444A
-
without any effect on activity
T472D
-
50% reduced HMP kinase activity
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis
-
useful reagent for the preparation of intermediates on the thiamin biosynthetic pathway
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