Information on EC 1.14.11.6 - thymine dioxygenase

Word Map on EC 1.14.11.6
Please wait a moment until all data is loaded. This message will disappear when all data is loaded.
Specify your search results
Select one or more organisms in this record:
Show additional data
Do not include text mining results
Include (text mining) results (more...)
Include results (AMENDA + additional results, but less precise; more...)

The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.14.11.6
-
RECOMMENDED NAME
GeneOntology No.
thymine dioxygenase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
thymine + 2-oxoglutarate + O2 = 5-hydroxymethyluracil + succinate + CO2
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydroxylation
oxidation
-
-
-
-
redox reaction
-
-
-
-
reduction
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
thymine,2-oxoglutarate:oxygen oxidoreductase (7-hydroxylating)
Requires Fe2+ and ascorbate. Also acts on 5-hydroxymethyluracil to oxidize its -CH2OH group first to -CHO and then to -COOH.
CAS REGISTRY NUMBER
COMMENTARY hide
37256-67-0
-
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
the genomic DNA of kinetoplastid parasites contains a unique modified base beta-D-glucosylhydroxymethyluracil or base J. J-binding protein, JBP, 1 and 2, which regulate the levels of J in the genome, are catalyzing the first step in J biosynthesis. JBP2, like JBP1, is a thymidine hydroxylase. Unlike JBP1, JBP2 is dispensable for Leishmania, mutational analysis, overview
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
1-ethylthymine + 2-oxoglutarate + O2
? + succinate + CO2
show the reaction diagram
-
-
-
?
1-ethyluracil + 2-oxoglutarate + O2
? + succinate + CO2
show the reaction diagram
-
-
-
?
1-methylthymine + 2-oxoglutarate + O2
1-methyl-5-(hydroxymethyl)uracil + thymine + formaldehyde + succinate + CO2
show the reaction diagram
1-methyluracil + 2-oxoglutarate + O2
? + succinate + CO2
show the reaction diagram
-
-
-
?
5,6-dihydrothymine + 2-oxoglutarate + O2
5,6-dihydro-5-(hydroxymethyl)uracil + succinate + CO2
show the reaction diagram
-
-
-
?
5-(2-hydroxyethyl)uracil + 2-oxoglutarate + O2
5-(1,2-dihydroxyethyl)uracil + succinate + CO2
show the reaction diagram
5-(methylthio)uracil + 2-oxoglutarate + O2
5-(methylsulfonyl)uracil + 5-(methylsulfinyl)uracil + succinate + CO2
show the reaction diagram
-
-
-
?
5-aminouracil + 2-oxoglutarate + O2
? + succinate + CO2
show the reaction diagram
5-bromo-1-methyluracil + 2-oxoglutarate + O2
? + succinate + CO2
show the reaction diagram
-
-
-
?
5-ethynyluracil + 2-oxoglutarate + O2 + H2O
5-(carboxymethyl)uracil + succinate + CO2
show the reaction diagram
5-fluorouracil + 2-oxoglutarate + O2
? + succinate + CO2
show the reaction diagram
5-formyluracil + 2-oxoglutarate + O2
5-carboxyuracil + succinate + CO2
show the reaction diagram
5-hydroxymethyluracil + 2-oxoglutarate + O2
5-formyluracil + succinate + CO2
show the reaction diagram
5-methylcytosine + 2-oxoglutarate + O2
?
show the reaction diagram
-
-
-
-
?
5-vinyluracil + 2-oxoglutarate + O2
5-(1,2-dihydroxyethyl)uracil + succinate + CO2
show the reaction diagram
6-azathymine + 2-oxoglutarate + O2
? + succinate + CO2
show the reaction diagram
-
-
-
?
N1-methylthymine + 2-oxoglutarate + O2
N1-methyl-5-hydroxymethyluracil + succinate + CO2
show the reaction diagram
-
-
-
-
?
N1-methyluracil + 2-oxoglutarate + O2
? + succinate + CO2
show the reaction diagram
-
-
-
-
?
thymine + 2-oxoglutarate + 2 O2
uracil-5-carboxylate + succinate + CO2 + H2O
show the reaction diagram
thymine + 2-oxoglutarate + O2
5-hydroxymethyluracil + succinate + CO2
show the reaction diagram
uracil + 2-oxoglutarate + O2
? + succinate + CO2
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
5-formyluracil + 2-oxoglutarate + O2
5-carboxyuracil + succinate + CO2
show the reaction diagram
5-hydroxymethyluracil + 2-oxoglutarate + O2
5-formyluracil + succinate + CO2
show the reaction diagram
thymine + 2-oxoglutarate + 2 O2
uracil-5-carboxylate + succinate + CO2 + H2O
show the reaction diagram
Q66Q78
conversion of thymine to uracil
three step reaction with 5-hydroxymethyluracil and 5-formyluracil as intermediate products
-
?
thymine + 2-oxoglutarate + O2
5-hydroxymethyluracil + succinate + CO2
show the reaction diagram
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1-ethylthymine
-
70% inhibition at 5 mM with thymine as substrate
1-ethyluracil
-
60% inhibition at 5 mM with thymine as substrate
1-methylthymine
-
73% inhibition at 5 mM with thymine as substrate
1-Methyluracil
-
80% inhibition at 5 mM with thymine as substrate
2,4-dimethoxy-5-methylpyrimidine
-
68% inhibition at 5 mM with thymine as substrate
5,6-Dihydrothymine
-
73% inhibition at 5 mM with thymine as substrate
5-acetyluracil
-
poor competitive inhibitor
5-Aminouracil
-
100% inhibition at 5 mM with thymine as substrate
5-Bromouracil
-
95% inhibition at 5 mM with thymine as substrate
5-Carboxyuracil
-
-
5-Ethynyluracil
5-fluorouracil
-
linear non-competitive inhibitor
5-Formyluracil
5-Hydroxymethyluracil
-
competitive inhibitor against thymine, noncompetitive against oxygen
5-Hydroxyuracil
-
92% inhibition at 5 mM with thymine as substrate
5-Mercaptouracil
-
60% inhibition at 5 mM with thymine as substrate
5-methylaminouracil
-
100% inhibition at 5 mM with thymine as substrate
5-Nitrouracil
-
70% inhibition at 5 mM with thymine as substrate
6-Azathymine
-
94% inhibition at 5 mM with thymine as substrate
6-methylthymine
-
41% inhibition at 5 mM with thymine as substrate
6-Methyluracil
-
18% inhibition at 5 mM with thymine as substrate
Uracil
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(beta,gamma-methylene)adenosine 5'-triphosphate
-
at 1 mM 2.5fold stimulation of the thymine dependent reaction
Adenyl-5'-ylimidodiphosphate
-
at 1 mM 2fold stimulation of the thymine dependent reaction
ADP
-
at 1 mM 2.5fold stimulation of the thymine dependent reaction
ascorbate
catalase
-
-
-
GTP
-
at 1 mM 2fold stimulation of the thymine dependent reaction
UTP
-
at 1 mM 2fold stimulation of the thymine dependent reaction
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.6
1-ethylthymine
-
-
1
1-ethyluracil
-
-
0.45 - 1.3
1-methylthymine
0.5
1-Methyluracil
-
-
0.024
2-oxoglutarate
-
-
0.087
5,6-dihydroxythymine
-
-
0.124
5-(2-hydroxyethyl)uracil
-
-
0.322
5-fluorouracil
-
-
0.22 - 2.34
5-Formyluracil
0.118 - 1
5-Hydroxymethyluracil
0.25
6-Azathymine
-
-
0.0011
Fe2+
-
-
1.3
N1-methylthymine
-
pH 7.5, 30C, wild-type enzyme
0.11
N1-methyluracil
-
pH 7.5, 30C, wild-type enzyme
0.058 - 2.1
thymine
0.2
Uracil
-
-
additional information
additional information
-
steady-state kinetics
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.13
5-Formyluracil
Rhodotorula glutinis
-
pH 7.5, 30C, wild-type enzyme
0.42
5-Hydroxymethyluracil
Rhodotorula glutinis
-
pH 7.5, 30C, wild-type enzyme
0.43
N1-methylthymine
Rhodotorula glutinis
-
pH 7.5, 30C, wild-type enzyme
0.27
N1-methyluracil
Rhodotorula glutinis
-
pH 7.5, 30C, wild-type enzyme
0.011 - 16.7
thymine
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.83
5-acetyluracil
-
-
0.022
5-Ethynyluracil
-
-
1.39
5-Formyluracil
-
with respect to thymine
0.2
Uracil
-
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
insect stage; insect stage
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37000
-
calculated from deduced amino acid sequence
40300
-
disc gel electrophoresis under denaturing conditions
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
-
1 * 42700, gel filtration
additional information
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60
-
10% activity after 2 min
98
-
20% activity after 15 min of ascorbate or ATP-dependent stimulation of thymine dependent reaction; 50% activity after 5 min of ascorbate or ATP-dependent stimulation of thymine dependent reaction
100
-
rapid inactivation
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
stabilized by glycine and dithiothreitol
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-70C, ammonium sulfate precipitate, several weeks, no activity loss
-
0C, ammonium sulfate precipitate, 5 h, quite stable
-
20 mM potassium phosphate pH 7.5, 100 mM glycine and 0.1 mM EDTA, stable
-
4C, after dialysis against distilled water, one day or more
-
freezing at -79C, storage at -39C, 50 mM Tris-HCl buffer, pH 8.0, 1 mM glutathione, 1 mM ascorbate, 10 mM KCl, 8 days, 96% activity
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
native enzyme by ammonium sulfate fractionation, anion exchange chromatography, and gel filtration. Recombinant GST-tagged enzyme from Escherichia coli strain BL21(DE3) by glutathione affinity chromatography, the tag is cleaved off by thrombin, followed by gel filtration
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DNA and amino acid sequence determination and analysis, expression of GST-tagged enzyme in Escherichia coli strain BL21(DE3)
-
expressed in Escherichia coli BL21(DE3)Gold
-
expression in Escherichia coli, fusion protein with green fluorescent protein and with His-tag
expression of either the wild type JBP2 or the mutant JBP2 proteins in insect stage trypanosomes, which do not contain the modified base J and determined the base J content of each cell line, mutations made within the key catalytic domain ablates the ability of JBP2 to initiate de novo base J synthesis
JBP2, DNA and amino acid sequence determination and analysis, expression of wild-type and mutant enzymes, the latter contain 4 exchanges of conserved residues
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D215S
-
mutant proteins fail to restore the J-level in the JBP1 null trypanosomes to wild-type levels
D416A
site-directed mutagenesis, cells expressing the LtJBP2 mutant exhibits a serious growth defect
H213S
-
mutant proteins fail to restore the J-level in the JBP1 null trypanosomes to wild-type levels
H414A
site-directed mutagenesis, cells expressing the LtJBP2 mutant exhibits a serious growth defect
H465A
site-directed mutagenesis, cells expressing the LtJBP2 mutant exhibits a serious growth defect
R478A
site-directed mutagenesis, cells expressing the LtJBP2 mutant exhibits a serious growth defect
V482A/L548Q
site-directed and random mutagenesis, the mutant can complement the JBP2 knockout mutant strain
N187D
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
N250D
-
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
N293D
-
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
R285C
-
site-directed mutagenesis, inactive active site mutant
D191A
-
mutation abrogates the ability of JBP1 to complement JBP1 null mutants
D393A
site-directed mutagenesis, mutation inside the catalytic domain, procyclic cells expressing mutant versions of JBP2 inside the catalytic domain
H189A
-
mutation abrogates the ability of JBP1 to complement JBP1 null mutants
H189A/D191A
-
mutation abrogates the ability of JBP1 to complement JBP1 null mutants
H239A
-
mutation abrogates the ability of JBP1 to complement JBP1 null mutants
H391A
site-directed mutagenesis, mutation inside the catalytic domain, procyclic cells expressing mutant versions of JBP2, expression of the TH mutant H319A fails to stimulate base J formation
H441A
site-directed mutagenesis, mutation inside the catalytic domain, procyclic cells expressing mutant versions of JBP2 inside the catalytic domain
R255A
-
mutation abrogates the ability of JBP1 to complement JBP1 null mutants
R445A
site-directed mutagenesis, mutation inside the catalytic domain, procyclic cells expressing mutant versions of JBP2 inside the catalytic domain
V259A
-
mutation abrogates the ability of JBP1 to complement JBP1 null mutants
V459A
site-directed mutagenesis, control substitution of a residue outside the catalytic domain
additional information