Information on EC 3.5.99.2 - aminopyrimidine aminohydrolase

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

EC NUMBER
COMMENTARY hide
3.5.99.2
-
RECOMMENDED NAME
GeneOntology No.
aminopyrimidine aminohydrolase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
4-amino-5-aminomethyl-2-methylpyrimidine + H2O = 4-amino-5-hydroxymethyl-2-methylpyrimidine + NH3
show the reaction diagram
(1)
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-
-
thiamine + H2O = 4-amino-5-hydroxymethyl-2-methylpyrimidine + 5-(2-hydroxyethyl)-4-methylthiazole
show the reaction diagram
(2)
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of C-N bond
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
base-degraded thiamine salvage
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Metabolic pathways
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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
4-amino-5-aminomethyl-2-methylpyrimidine aminohydrolase
Previously known as thiaminase II, this enzyme is involved in the regeneration of the thiamine pyrimidine from degraded products, rather than in thiamine degradation, and participates in thiamine salvage pathways.
CAS REGISTRY NUMBER
COMMENTARY hide
9024-80-0
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
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Manually annotated by BRENDA team
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UniProt
Manually annotated by BRENDA team
moulds
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-
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Manually annotated by BRENDA team
Trichosporon aneurinolyticum
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-
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
thi20 deletion mutant cells can not grow in the presence of the synthetic thiamine antagonists, pyrithiamine and oxythiamin, indicating that the THI20 gene is necessary for the utilization of pyrithiamine and oxythiamine as precursors for thiamin
metabolism
physiological function
thiaminase II activity of Thi20p is involved in the thiamine salvage pathway
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
2-northiamine + H2O
? + hydroxyethylthiazole
show the reaction diagram
-
-
-
-
?
2-thiothiamine + H2O
?
show the reaction diagram
-
-
-
-
?
4-amino-5-aminomethyl-2-methyl-pyrimidine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + NH3
show the reaction diagram
4-amino-5-aminomethyl-2-methylpyrimidine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + 5-(2-hydroxyethyl)-4-methylthiazole
show the reaction diagram
the wild type enzyme is poorly active on 4-amino-5-aminoethyl-2-methylpyrimidine
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-
?
4-amino-5-aminomethyl-2-methylpyrimidine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + NH3
show the reaction diagram
dimethialium + H2O
?
show the reaction diagram
-
-
-
-
?
oxothiamin + H2O
?
show the reaction diagram
oxythiamin + H2O
?
show the reaction diagram
oxythiamine + H2O
?
show the reaction diagram
-
-
-
-
?
pyrimidinemethanol + aniline
pyrimidinemethylaniline + H2O
show the reaction diagram
-
-
-
-
r
pyrithiamine + H2O
?
show the reaction diagram
-
-
-
-
?
thiamine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + 5-(2-hydroxyethyl)-4-methylthiazole
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
4-amino-5-aminomethyl-2-methyl-pyrimidine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + NH3
show the reaction diagram
Q08224
physiological substrate, hydrolyzed more than two times faster than thiamine. Thiaminase II activity of Thi20p is involved in the thiamine salvage pathway by catalyzing the hydrolysis of 4-amino-5-hydroxymethyl-2-methylpyrimidine precursors in Saccharomyces cerevisiae
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-
?
4-amino-5-aminomethyl-2-methylpyrimidine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + NH3
show the reaction diagram
oxothiamin + H2O
?
show the reaction diagram
oxythiamin + H2O
?
show the reaction diagram
thiamine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + 5-(2-hydroxyethyl)-4-methylthiazole
show the reaction diagram
additional information
?
-
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
As2O3
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0.01 mM 33% inhibition
Ca2+
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0.01 mM 45% inhibition
Cd2+
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0.01 mM 88% inhibition
Fe3+
-
0.01 mM 45% inhibition
ICH2COOH
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0.01 mM 42% inhibition
Li+
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0.01 mM 30% inhibition
Mg2+
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0.01 mM 30% inhibition
Mn2+
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0.01 mM 30% inhibition
Ni2+
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0.01 mM 73% inhibition
p-chloromercuribenzoate
additional information
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inhibited by many aromatic and heterocyclic amines
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
EDTA
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in a concentration range of 0.02 mM to 1000 mM
L-cysteine
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in a concentration range of 0.02 mM to 1000 mM
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.058 - 0.068
4-amino-5-aminomethyl-2-methylpyrimidine
0.003 - 2.25
thiamine
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.001 - 0.003
4-amino-5-aminomethyl-2-methylpyrimidine
0.0017 - 4.83
thiamine
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00292 - 0.00304
thiamine
642
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0046
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in 100 mM Tris-HCl, pH 7.5, at 37°C
0.091
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with oxothiamin as substrate, at 30°C in 45 mM Tris/HCl buffer, pH 7.5, containing 0.9 mM dithiothreitol and 0.45 M glycinebetaine
0.162
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with thiamine as substrate, at 30°C in 45 mM Tris/HCl buffer, pH 7.5, containing 0.9 mM dithiothreitol and 0.45 M glycinebetaine
0.205
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with oxythiamin as substrate, at 30°C in 45 mM Tris/HCl buffer, pH 7.5, containing 0.9 mM dithiothreitol and 0.45 M glycinebetaine
0.292
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with 4-amino-5-aminomethyl-2-methylpyrimidine as substrate, at 30°C in 45 mM Tris/HCl buffer, pH 7.5, containing 0.9 mM dithiothreitol and 0.45 M glycinebetaine
additional information
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mean thiaminase activity in the alewives studied increases from 6.9 to 16.0 nmol/min/g from the time of their collection in Cayuga lake to the start of laboratory experiments 1.5-2.5 years later, the latter value is more than twice that of previously reported levels of thiaminase activity from alewives collected in the wild, data suggest that the variability in thiaminase activity is not related to stress from salt reduction or food limitation; whole body activity is measured, no significant diferrences in thiaminase activity are found between the control fishes and the alewives which are exposed to food limitation and reduced salt content in the water
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30 - 40
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40 - 45
Trichosporon aneurinolyticum
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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)
Staphylococcus aureus (strain MRSA252)
Staphylococcus epidermidis (strain ATCC 12228)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
24000
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4 * 24000, SDS-PAGE
25000
4 * 25000, SDS-PAGE
25643
4 * 25643, calculated from amino acid sequence
26840
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x * 26840, sequence calculation
30000
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x * 30000, SDS-PAGE
82900
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static light scattering
100000
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homotetramer
monomer
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1 * 100000, SDS-PAGE
trimer
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in vitro
additional information
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THI20 is a trifunctional enzyme containing an N-terminal HMP kinase/HMP-P kinase (ThiD-like) domain and a C-terminal thiaminase II (TenA-like) domain. The ThiD-like dimer observed in THI20 resembles other ThiD structures, the TenA-like domain, which is tetrameric in all previously reported structures, forms a dimer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
dialyzing the purified enzyme solution against 52% saturated ammonium sulfate solution, two weeks
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hanging drop vapour diffusion method
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vapor diffusion method, using 0.1 m Tris (pH 8.5), 1.1 M lithium sulfate
purified recombinant C-terminally His6-tagged THI20, hanging drop vapour diffusion method, 10 mg/ml protein in 10 mM Tris buffer, pH 7.6, with 30 mM NaCl, is mixed with 10-14% PEG 8000, 0.2 M calcium acetate, and 0.1 M imidazole, pH 7.5, microseeding, 2-3 days, X-ray diffraction structure determination and analysis at 2.68 A resolution, molecular replacement, modelling
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hanging drop vapor diffusion method
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purified recombinant C-terminally Strep-tagged TenA, hanging drop vapour diffusion method, 0.002 ml of 10 mg/ml protein in 100 mM Tris-HCl buffer pH 8.0, is mixed with 0.002 l of reservoir solution containing 0.2 M sodium acetate, 0.1 M Tris-HCl pH 8.5 and 28% w/v PEG 3350, 20°C, 7 days, X-ray diffraction structure determination and analysis at 2.60 A resolution, molecular replacement, modelling
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Ni-NTA column chromatography and gel filtration
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Ni-Sepharose column chromatography and Superdex 200 gel filtration
Ni2+ -affinity column chromatography
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recombinant C-terminally His6-tagged THI20 from Escherichia coli strain BL21 (DE3) by nickel affinity chromatography and gel filtration
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recombinant C-terminally Strep-tagged TenA from Escherichia coli strain BL21(DE3) by affinity chromatography
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Strep-Tactin Sepharose column chromatography and Superdex 200 gel filtration
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3) cells
expressed in Escherichia coli BL21-CodonPlus (DE3)-RIPL cells
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expressed in Escherichia coli BLR (DE3) cells
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expressed in Escherichia coli strain BL21(D3)
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expression in Escherichia coli
expression of C-terminally His6-tagged THI20 in Escherichia coli strain BL21 (DE3)
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expression of C-terminally Strep-tagged TenA in Escherichia coli strain BL21(DE3)
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overexpression in Escherichia coli
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
F47Y
poorly active mutant. At pH 6.0, its activity is absent. Moreover, the enzyme does not present any activity on thiamin degradation
D111A/K115A
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the activity of the monomeric mutant is not significantly altered in comparison to the wild type enzyme
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