Information on EC 2.5.1.3 - thiamine phosphate synthase

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

EC NUMBER
COMMENTARY hide
2.5.1.3
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RECOMMENDED NAME
GeneOntology No.
thiamine phosphate synthase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
4-amino-2-methyl-5-(diphosphomethyl)pyrimidine + 2-(2-carboxy-4-methylthiazol-5-yl)ethyl phosphate = diphosphate + thiamine phosphate + CO2
show the reaction diagram
(2)
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-
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4-amino-2-methyl-5-(diphosphomethyl)pyrimidine + 2-[(2R,5Z)-2-carboxy-4-methylthiazol-5(2H)-ylidene]ethyl phosphate = diphosphate + thiamine phosphate + CO2
show the reaction diagram
4-amino-2-methyl-5-(diphosphomethyl)pyrimidine + 4-methyl-5-(2-phosphono-oxyethyl)thiazole = diphosphate + thiamine phosphate
show the reaction diagram
(3)
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydroxymethylpyrimidine group transfer
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-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Metabolic pathways
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-
thiamine diphosphate biosynthesis I (E. coli)
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-
thiamine diphosphate biosynthesis II (Bacillus)
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thiamine diphosphate biosynthesis III (Staphylococcus)
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thiamine diphosphate biosynthesis IV (eukaryotes)
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thiamine formation from pyrithiamine and oxythiamine (yeast)
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Thiamine metabolism
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thiamine salvage II
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thiamine salvage IV (yeast)
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vitamin B1 metabolism
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SYSTEMATIC NAME
IUBMB Comments
4-amino-2-methyl-5-diphosphomethylpyrimidine:2-[(2R,5Z)-2-carboxy-4-methylthiazol-5(2H)-ylidene]ethyl phosphate 4-amino-2-methylpyrimidine-5-methenyltransferase (decarboxylating)
The enzyme catalyses the penultimate reaction in thiamine de novo biosynthesis, condensing the pyrimidine and thiazole components. The enzyme is thought to accept the product of EC 2.8.1.10, thiazole synthase, as its substrate. However, it has been shown that in some bacteria, such as Bacillus subtilis, an additional enzyme, thiazole tautomerase (EC 5.3.99.10) converts that compound into its tautomer 2-(2-carboxy-4-methylthiazol-5-yl)ethyl phosphate, and that it is the latter that serves as the substrate for the synthase. In addition to this activity, the enzyme participates in a salvage pathway, acting on 4-methyl-5-(2-phosphono-oxyethyl)thiazole, which is produced from thiamine degradation products. In yeast this activity is found in a bifunctional enzyme (THI6) and in the plant Arabidopsis thaliana the activity is part of a trifunctional enzyme (TH1).
CAS REGISTRY NUMBER
COMMENTARY hide
9030-30-2
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
ecotype Landsberg erecta
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-
Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
bifunctional enzyme with hydroxyethylthiazole kinase and thiamine-phosphate pyrophosphorylase activity
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Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
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UniProt
Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2-methyl-4-amino-5-diphosphomethylpyrimidine + 2-(4-methyl-1,3-thiazol-5-yl)ethyl phosphate
diphosphate + ?
show the reaction diagram
2-methyl-4-amino-5-hydroxymethylpyrimidine diphosphate + (R,Z)-2-(2-carboxy-4-methylthiazol-5(2H)-ylidene)ethyl phosphate
diphosphate + thiamine phosphate + CO2
show the reaction diagram
2-methyl-4-amino-5-hydroxymethylpyrimidine diphosphate + 2-(2-carboxy-4-methylthiazol-5-yl)ethyl phosphate
diphosphate + thiamine phosphate + CO2
show the reaction diagram
2-methyl-4-amino-5-hydroxymethylpyrimidine diphosphate + 4-methyl-5-(2-phosphono-oxyethyl)-thiazole
?
show the reaction diagram
-
very low activity, almost 400-fold lower than the activity shown by the thiE-encoded thiamine-phosphate diphosphatase of Escherichia coli
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-
?
2-methyl-4-amino-5-hydroxymethylpyrimidine diphosphate + 4-methyl-5-(2-phosphono-oxyethyl)-thiazole
thiamine monophosphate + diphosphate
show the reaction diagram
2-methyl-4-amino-5-hydroxymethylpyrimidine diphosphate + 4-methyl-5-(2-phosphono-oxyethyl)-thiazole
thiamine phosphate + diphosphate
show the reaction diagram
2-methyl-4-amino-5-hydroxymethylpyrimidine diphosphate + 4-methyl-5-(2-phosphono-oxyethyl)thiazole
diphosphate + thiamine phosphate
show the reaction diagram
2-methyl-4-amino-5-hydroxymethylpyrimidine diphosphate + 4-methyl-5-beta-hydroxyethylthiazole phosphate
thiamine phosphate + diphosphate
show the reaction diagram
-
-
-
-
?
4-amino-5-(hydroxymethyl)-2-(trifluoromethyl)pyrimidine diphosphate + 4-methyl-5-(2-phosphono-oxyethyl)-thiazole
?
show the reaction diagram
-
poor substrate
-
-
?
4-amino-5-(hydroxymethyl)-2-methoxypyrimidine diphosphate + 4-methyl-5-(2-phosphono-oxyethyl)-thiazole
?
show the reaction diagram
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good substrate
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-
?
4-amino-5-hydroxymethyl-2-methylpyrimidine diphosphate + 4-methyl-5-(2-hydroxyethyl)thiazole phosphate
thiamine monophosphate + diphosphate + phosphate
show the reaction diagram
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?
additional information
?
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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
2-methyl-4-amino-5-diphosphomethylpyrimidine + 2-(4-methyl-1,3-thiazol-5-yl)ethyl phosphate
diphosphate + ?
show the reaction diagram
2-methyl-4-amino-5-hydroxymethylpyrimidine diphosphate + (R,Z)-2-(2-carboxy-4-methylthiazol-5(2H)-ylidene)ethyl phosphate
diphosphate + thiamine phosphate + CO2
show the reaction diagram
2-methyl-4-amino-5-hydroxymethylpyrimidine diphosphate + 2-(2-carboxy-4-methylthiazol-5-yl)ethyl phosphate
diphosphate + thiamine phosphate + CO2
show the reaction diagram
2-methyl-4-amino-5-hydroxymethylpyrimidine diphosphate + 4-methyl-5-(2-phosphono-oxyethyl)-thiazole
thiamine monophosphate + diphosphate
show the reaction diagram
2-methyl-4-amino-5-hydroxymethylpyrimidine diphosphate + 4-methyl-5-(2-phosphono-oxyethyl)thiazole
diphosphate + thiamine phosphate
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
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stimulates, 20% as effective as Mg2+
Co2+
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stimulates, 20% as effective as Mg2+
Zn2+
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stimulates, 20% as effective as Mg2+
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-methyl-4-amino-5-hydroxymethylpyrimidine
0.1 mM, 6.3% inhibition
2-methyl-4-amino-5-hydroxymethylpyrimidine phosphate
0.1 mM, 18.2% inhibition
3-benzylsulfanyl-phenanthro[9,10-e][1,2,4]triazine
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IC50 of 0.1 mg/ml
4-amino-5-hydroxymethyl-2-methylpyrimidine diphosphate
uncompetitive
4-[[(2-hydroxy-5-nitrophenyl)methylidene]amino]-5-methyl-2-(propan-2-yl)phenol
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IC50 of 0.034 mg/ml
5-(2-hydroxyethyl)-4-methylthiazole
0.1 mM, 12.3% inhibition
7-[[4-chloro-6-(diethylamino)-s-triazin-2-yl]amino]-3-phenyl-coumarin
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IC50 of more than 0.1 mg/ml
acetyl phosphate
diphosphate
PCMB
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0.01 mM, 98.5% inhibition, inhibition prevented by addition of 2-mercaptoethanol in 10fold molar excess
phosphocreatine
phosphoenolpyruvate
thiamin
0.1 mM, 17.3% inhibition
thiamin phosphate
0.1 mM, 59% inhibition
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0004 - 0.007
2-(4-methyl-1,3-thiazol-5-yl)ethyl phosphate
0.0007
2-methyl-4-amino-5-hydroxymethylpyrimidine diphosphate
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wild type enzyme, in buffer containing 50 mM phosphate and 10 mM MgSO4, at pH 8.0 and 37C
0.0128
4-amino-5-hydroxymethyl-2-methylpyrimidine diphosphate
37C, pH 8.0
0.0085
4-methyl-5-(2-hydroxyethyl)thiazole phosphate
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0.0012
4-methyl-5-beta-hydroxyethylthiazole phosphate
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wild type enzyme, in buffer containing 50 mM phosphate and 10 mM MgSO4, at pH 8.0 and 37C
0.00085 - 0.001
hydroxymethylpyrimidine diphosphate
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
additional information
Bacillus subtilis
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0148
4-amino-5-hydroxymethyl-2-methylpyrimidine diphosphate
37C, pH 8.0
0.0034
ATP
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.05
37C, pH 8.0, presence of ATP
0.52
37C, pH 8.0
0.7
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wild type enzyme, in buffer containing 50 mM phosphate and 10 mM MgSO4, at pH 8.0 and 37C
2.46
37C, pH 8.0, presence of Mg2+
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 9.1
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pH 7.0: about 30% of maximal activity, pH 9.1: optimum
7.5 - 8.5
the optimum pH is pH 8.5, and the activity at pH 7.5 is about 60% of that at pH 8.5
7.7 - 10
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about 40% of activity maximum at pH 7.7 and pH 10.0
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
80 - 120
activity increases with an increase in temperature from 80C to 120C, and the highest activity is observed around 110C. The activity at 100C is about 75% of the highest activity
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8.5
calculated
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Candida glabrata (strain ATCC 2001 / CBS 138 / JCM 3761 / NBRC 0622 / NRRL Y-65)
Candida glabrata (strain ATCC 2001 / CBS 138 / JCM 3761 / NBRC 0622 / NRRL Y-65)
Candida glabrata (strain ATCC 2001 / CBS 138 / JCM 3761 / NBRC 0622 / NRRL Y-65)
Candida glabrata (strain ATCC 2001 / CBS 138 / JCM 3761 / NBRC 0622 / NRRL Y-65)
Candida glabrata (strain ATCC 2001 / CBS 138 / JCM 3761 / NBRC 0622 / NRRL Y-65)
Candida glabrata (strain ATCC 2001 / CBS 138 / JCM 3761 / NBRC 0622 / NRRL Y-65)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Pyrococcus furiosus (strain ATCC 43587 / DSM 3638 / JCM 8422 / Vc1)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
17000
-
gel filtration
29000
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x * 29000, SDS-PAGE
55000
2 * 57800, calculated, 2 * 55000, SDS-PAGE
57800
2 * 57800, calculated, 2 * 55000, SDS-PAGE
58058
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x * 58058, calculation from nucleotide sequence
95000
gel filtration
470000
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hydroxyethylthiazole kinase is a bifunctional enzyme with thiamine-phosphate pyrophosphorylase activity, gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
2 * 57800, calculated, 2 * 55000, SDS-PAGE
homohexamer
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-
homooctamer
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8 * 60000 Da
octamer
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8 * 60000, SDS-PAGE, hydroxyethylthiazole kinase is a bifunctional enzyme with thiamine-phosphate pyrophosphorylase activity
additional information
the activities of bifunctional phosphomethylpyrimidine kinase/thiamin-phosphate diphosphorylase are located in two distinct, N-terminal kinase and C-terminal synthase, domains
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging-drop vapor diffusion, crystal structure at 1.25 A resolution
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unliganded and in complex with substrates and products, hanging drop vapor diffusion method, using 0.1 M HEPES (pH 7.5), 0.2-0.25 M MgCl2, and 25-32% PEG400, at 4C
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
21
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room temperature, completely inactivated overnight
45
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5 min, without addition of both substrates, completely inactivated
100
5 min, 20% loss of activity. 15 min, 50% loss of activity. 2 h, 90% loss of activity
additional information
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-15C, 6 months, less than 10% loss of activity
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-20C, stable for several months
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-80C, about 64.4% loss of activity after 1 month
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
bifunctional enzyme with hydroxyethylthiazole kinase and thiamine-phosphate pyrophosphorylase activity
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enzyme is purified and characterised: The two activities of the purified protein (EC 2.5.1.3 and EC 2.7.1.50, respectively) are inseparable, though their pH optima differ
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from E. coli overexpression strain
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Ni-NTA column chromatography
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strep - tactin column chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
bifunctional enzyme with hydroxyethylthiazole kinase and thiamine-phosphate pyrophosphorylase activity
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bifunctional enzyme with hydroxyethylthiazole kinase and thiamine-phosphate pyrophosphorylase activity, expression in Escherichia coli
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expressed in DELTAthiE-Escherichia coli cells
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expressed in Escherichia coli
expressed in Escherichia coli BL21(DE3) cells
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expression in Escherichia coli
overexpression of YjbQ complements the thiamin auxotrophy of the Escherichia coli MC1061DELTAthiE strain, in spite of their thiamine phosphate synthase activity being several orders of magnitude lower than that of the thiE-coded enzyme
overexpression of YjbQ complements the thiamin auxotrophy of the Escherichia coli MC1061DELTAthiE strain, in spite of their thiamine phosphate synthase activity being several orders of magnitude lower than that of the thiE-coded enzyme; overexpression of YjbQ complements the thiamin auxotrophy of the Escherichia coli MC1061DELTAthiE strain, in spite of their thiamine phosphate synthase activity being several orders of magnitude lower than that of the thiE-coded enzyme
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
R320M
activity is 1% compared to wild-type enzyme
H341L
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no activity
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R320M
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activity is 1% compared to wild-type enzyme
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M134K
phosphomethylpyrimidine kinase activity similar to wild-type, thiamin-phosphate diphosphorylase activity almost completely abolished
M134K/T472D
83-97% residual phosphomethylpyrimidine kinase activity, 19-32% residual thiamin-phosphate diphosphorylase activity
Q373L
phosphomethylpyrimidine kinase activity similar to wild-type, 64-75% residual thiamin-phosphate diphosphorylase activity
Q98L
phosphomethylpyrimidine kinase activity similar to wild-type, thiamin-phosphate diphosphorylase activity almost completely abolished
Q98L/Q373L
phosphomethylpyrimidine kinase activity similar to wild-type, 7-13% residual thiamin-phosphate diphosphorylase activity
S444A
almost complete loss of phosphomethylpyrimidine kinase activity, 80-91% residual thiamin-phosphate diphosphorylase activity
T472D
phosphomethylpyrimidine kinase activity similar to wild-type, 46-54% residual thiamin-phosphate diphosphorylase activity
additional information
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in enzyme deletion mutant, no growth difference is observed when compared to the wild-type strain
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