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Enzyme Nomenclature
Information on EC 3.5.99.2 - aminopyrimidine aminohydrolase
for references in articles please use BRENDA:EC3.5.99.2
Word Map on EC 3.5.99.2
- 3.5.99.2
- transketolase
-
polioencephalomalacia
- thiaminolyticus
-
cerebrocortical
-
tenas
-
pasture
-
alosa
- venerupis
- 4-amino-5-hydroxymethyl-2-methylpyrimidine
- decussata
-
2.5.1.2
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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria
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EC NUMBER 
COMMENTARY 
3.5.99.2
-
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RECOMMENDED NAME
GeneOntology No.
aminopyrimidine aminohydrolase
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
4-amino-5-aminomethyl-2-methylpyrimidine + H2O = 4-amino-5-hydroxymethyl-2-methylpyrimidine + NH3
(1)
-
-
-
thiamine + H2O = 4-amino-5-hydroxymethyl-2-methylpyrimidine + 5-(2-hydroxyethyl)-4-methylthiazole
(2)
-
-
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
hydrolysis of C-N bond
hydrolysis of C-N bond
-
-
-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
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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.
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CAS REGISTRY NUMBER
COMMENTARY
9024-80-0
-
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
trifunctional enzyme EC 3.5.99.2/EC 2.7.1.49/EC 2.7.4.7
SwissProt
trifunctional enzyme: EC 3.5.99.2/EC 2.7.1.49/EC 2.7.4.7
SwissProt
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
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
metabolism
TenA plays a pivotal role in the thiamin biosynthetic route
metabolism
-
thiamin-degradation products are hydrolyzed by thiaminase II, yielding 4-amino-5-hydroxymethyl-2-methylpyrimidine. This compound is an intermediate in thiamin biosynthesis. The enzyme is involved in the biosynthesis of thiamin diphosphate, the active form of the cofactor
metabolism
-
thiaminase type II, TenA, catalyzes the deamination of aminopyrimidines, including the cleavage of thiamine to 4-amino-5-hydroxymethyl-2-methylpyrimidine and 5-(2-hydroxyethyl)-4-methylthiazole in the metabolism of thiamine (vitamin B1), in Staphylococcus aureus
additional information
-
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
additional information
-
putative active-site residues are Asp44, Cys137, Tyr167 and Glu208
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
4-amino-5-aminomethyl-2-methyl-pyrimidine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + NH3
4-amino-5-aminomethyl-2-methylpyrimidine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + 5-(2-hydroxyethyl)-4-methylthiazole
the wild type enzyme is poorly active on 4-amino-5-aminoethyl-2-methylpyrimidine
-
-
?
4-amino-5-aminomethyl-2-methylpyrimidine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + NH3
pyrimidinemethanol + aniline
pyrimidinemethylaniline + H2O
-
-
-
-
r
thiamine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + 5-(2-hydroxyethyl)-4-methylthiazole
4-amino-5-aminomethyl-2-methyl-pyrimidine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + NH3
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
-
-
?
4-amino-5-aminomethyl-2-methyl-pyrimidine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + NH3
hydrolyzed more than two times faster than thiamine
-
-
?
4-amino-5-aminomethyl-2-methylpyrimidine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + NH3
-
-
-
-
?
4-amino-5-aminomethyl-2-methylpyrimidine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + NH3
-
-
-
?
4-amino-5-aminomethyl-2-methylpyrimidine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + NH3
-
-
-
-
?
thiamine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + 5-(2-hydroxyethyl)-4-methylthiazole
-
-
-
-
r
thiamine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + 5-(2-hydroxyethyl)-4-methylthiazole
-
-
-
-
?
thiamine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + 5-(2-hydroxyethyl)-4-methylthiazole
-
-
-
ir
thiamine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + 5-(2-hydroxyethyl)-4-methylthiazole
-
-
-
-
?
thiamine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + 5-(2-hydroxyethyl)-4-methylthiazole
-
-
-
?
thiamine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + 5-(2-hydroxyethyl)-4-methylthiazole
-
-
-
-
?
thiamine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + 5-(2-hydroxyethyl)-4-methylthiazole
-
-
-
-
?
thiamine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + 5-(2-hydroxyethyl)-4-methylthiazole
moulds
-
-
-
-
?
thiamine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + 5-(2-hydroxyethyl)-4-methylthiazole
-
-
-
-
?
thiamine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + 5-(2-hydroxyethyl)-4-methylthiazole
-
-
-
?
thiamine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + 5-(2-hydroxyethyl)-4-methylthiazole
-
-
-
-
?
thiamine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + 5-(2-hydroxyethyl)-4-methylthiazole
Trichosporon aneurinolyticum
-
-
-
-
?
additional information
?
-
the enzyme also catalyses the reaction of EC 2.7.4.7
-
-
-
additional information
?
-
-
no activity with N-formyl-4-amino-5-aminomethyl-2-methylpyrimidine, thiamin monophosphate, thiamin diphosphate, thiamin disulfide, desthiothiamin, and thiochrome
-
-
-
additional information
?
-
no activity with N-formyl-4-amino-5-aminomethyl-2-methylpyrimidine, thiamin monophosphate, thiamin diphosphate, thiamin disulfide, desthiothiamin, and thiochrome
-
-
-
additional information
?
-
Thi20p is involved in thiamine synthesis from pyrithiamine and oxythiamin
-
-
-
additional information
?
-
-
THI20 is a trifunctional enzyme that is composed of a ThiD-like N-terminal domain that catalyzes the phosphorylation of 4-amino-5-hydroxymethyl-2-methylpyrimidine to 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate and of 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate to 4-amino-5-hydroxymethyl-2-methylpyrimidine diphosphate, and a TenA-like C-terminal domain with thiaminase activity
-
-
-
additional information
?
-
-
thiamin-degradation products are hydrolyzed by thiaminase II yielding 4-amino-5-hydroxymethyl-2-methylpyrimidine. HMP. The C-terminal domain is homologous to Bacillus subtilis TenA, which catalyzes the production of HMP from thiamin degradation products in the salvage pathway
-
-
-
additional information
?
-
-
TenA does not accept TMP and TPP as substrates
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-
-
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
4-amino-5-aminomethyl-2-methyl-pyrimidine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + NH3
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
-
-
?
4-amino-5-aminomethyl-2-methylpyrimidine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + NH3
thiamine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + 5-(2-hydroxyethyl)-4-methylthiazole
4-amino-5-aminomethyl-2-methylpyrimidine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + NH3
-
-
-
-
?
4-amino-5-aminomethyl-2-methylpyrimidine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + NH3
P25052
-
-
-
?
4-amino-5-aminomethyl-2-methylpyrimidine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + NH3
-
-
-
-
?
thiamine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + 5-(2-hydroxyethyl)-4-methylthiazole
-
-
-
-
r
thiamine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + 5-(2-hydroxyethyl)-4-methylthiazole
-
-
-
-
?
thiamine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + 5-(2-hydroxyethyl)-4-methylthiazole
-
-
-
-
?
thiamine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + 5-(2-hydroxyethyl)-4-methylthiazole
P25052
-
-
-
?
thiamine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + 5-(2-hydroxyethyl)-4-methylthiazole
-
-
-
-
?
thiamine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + 5-(2-hydroxyethyl)-4-methylthiazole
-
-
-
-
?
thiamine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + 5-(2-hydroxyethyl)-4-methylthiazole
moulds
-
-
-
-
?
thiamine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + 5-(2-hydroxyethyl)-4-methylthiazole
-
-
-
-
?
thiamine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + 5-(2-hydroxyethyl)-4-methylthiazole
Q08224
-
-
-
?
thiamine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + 5-(2-hydroxyethyl)-4-methylthiazole
-
-
-
-
?
thiamine + H2O
4-amino-5-hydroxymethyl-2-methylpyrimidine + 5-(2-hydroxyethyl)-4-methylthiazole
Trichosporon aneurinolyticum
-
-
-
-
?
additional information
?
-
-
no activity with N-formyl-4-amino-5-aminomethyl-2-methylpyrimidine, thiamin monophosphate, thiamin diphosphate, thiamin disulfide, desthiothiamin, and thiochrome
-
-
-
additional information
?
-
P25052
no activity with N-formyl-4-amino-5-aminomethyl-2-methylpyrimidine, thiamin monophosphate, thiamin diphosphate, thiamin disulfide, desthiothiamin, and thiochrome
-
-
-
additional information
?
-
-
THI20 is a trifunctional enzyme that is composed of a ThiD-like N-terminal domain that catalyzes the phosphorylation of 4-amino-5-hydroxymethyl-2-methylpyrimidine to 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate and of 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate to 4-amino-5-hydroxymethyl-2-methylpyrimidine diphosphate, and a TenA-like C-terminal domain with thiaminase activity
-
-
-
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
-
inhibited by many aromatic and heterocyclic amines
-
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
EDTA
-
in a concentration range of 0.02 mM to 1000 mM
L-cysteine
-
in a concentration range of 0.02 mM to 1000 mM
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.058
4-amino-5-aminomethyl-2-methylpyrimidine
wild type enzyme, in 30 mM Tris, 50 mM NaCl (pH 8.0), at 20°C
0.068
4-amino-5-aminomethyl-2-methylpyrimidine
mutant enzyme F47Y, in 30 mM Tris, 50 mM NaCl (pH 8.0), at 20°C
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.001
4-amino-5-aminomethyl-2-methylpyrimidine
mutant enzyme F47Y, in 30 mM Tris, 50 mM NaCl (pH 8.0), at 20°C
0.003
4-amino-5-aminomethyl-2-methylpyrimidine
wild type enzyme, in 30 mM Tris, 50 mM NaCl (pH 8.0), at 20°C
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
0.091
-
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
-
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
-
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
-
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
-
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
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.5
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
37
60
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LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
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PDB
SCOP
CATH
ORGANISM
UNIPROT
Staphylococcus epidermidis (strain ATCC 12228)
Streptococcus pneumoniae serotype 4 (strain ATCC BAA-334 / TIGR4)
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
100000
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SUBUNITS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
homotetramer
additional information
-
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
dimer
-
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, domain organization, overview
homotetramer
4 * 25000, SDS-PAGE; 4 * 25643, calculated from amino acid sequence
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Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
dialyzing the purified enzyme solution against 52% saturated ammonium sulfate solution, two weeks
-
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
-
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
LITERATURE
60
80
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Ni-Sepharose column chromatography and Superdex 200 gel filtration
recombinant C-terminally His6-tagged THI20 from Escherichia coli strain BL21 (DE3) by nickel affinity chromatography and gel filtration
-
recombinant C-terminally Strep-tagged TenA from Escherichia coli strain BL21(DE3) by affinity chromatography
-
Strep-Tactin Sepharose column chromatography and Superdex 200 gel filtration
-
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression of C-terminally His6-tagged THI20 in Escherichia coli strain BL21 (DE3)
-
expression of C-terminally Strep-tagged TenA in Escherichia coli strain BL21(DE3)
-
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY
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
-
the activity of the monomeric mutant is not significantly altered in comparison to the wild type enzyme
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LINKS TO OTHER DATABASES (specific for EC-Number 3.5.99.2)
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