BRENDA - Enzyme Database show
show all sequences of 1.14.11.6

Cloning and characterization of Rhodotorula glutinis thymine hydroxylase

Neidigh, J.W.; Darwanto, A.; Williams, A.A.; Wall, N.R.; Sowers, L.C.; Chem. Res. Toxicol. 22, 885-893 (2009)

Data extracted from this reference:

Cloned(Commentary)
Commentary
Organism
DNA and amino acid sequence determination and analysis, expression of GST-tagged enzyme in Escherichia coli strain BL21(DE3)
Rhodotorula glutinis
Engineering
Amino acid exchange
Commentary
Organism
N187D
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
Rhodotorula glutinis
N250D
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
Rhodotorula glutinis
N293D
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
Rhodotorula glutinis
R285C
site-directed mutagenesis, inactive active site mutant
Rhodotorula glutinis
KM Value [mM]
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
additional information
-
additional information
steady-state kinetics
Rhodotorula glutinis
0.067
-
thymine
pH 7.5, 30C, mutant N187D
Rhodotorula glutinis
0.11
-
N1-methyluracil
pH 7.5, 30C, wild-type enzyme
Rhodotorula glutinis
0.145
-
thymine
pH 7.5, 30C, wild-type enzyme
Rhodotorula glutinis
0.23
-
5-Hydroxymethyluracil
pH 7.5, 30C, wild-type enzyme
Rhodotorula glutinis
1.15
-
thymine
pH 7.5, 30C, mutant N250D
Rhodotorula glutinis
1.3
-
N1-methylthymine
pH 7.5, 30C, wild-type enzyme
Rhodotorula glutinis
1.7
-
5-Formyluracil
pH 7.5, 30C, wild-type enzyme
Rhodotorula glutinis
2.1
-
thymine
pH 7.5, 30C, mutant N293D
Rhodotorula glutinis
Natural Substrates/ Products (Substrates)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
thymine + 2-oxoglutarate + O2
Rhodotorula glutinis
-
5-hydroxymethyluracil + succinate + CO2
-
-
?
Organism
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
Rhodotorula glutinis
Q66Q78
strain ATCC 2527
-
Purification (Commentary)
Commentary
Organism
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
Rhodotorula glutinis
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
5-formyluracil + 2-oxoglutarate + O2
-
697315
Rhodotorula glutinis
5-carboxyuracil + succinate + CO2
-
-
-
?
5-hydroxymethyluracil + 2-oxoglutarate + O2
-
697315
Rhodotorula glutinis
5-formyluracil + succinate + CO2
-
-
-
?
additional information
no activity with N1-methyl-5-hydroxymethyluracil, 5-methylcytosine, N3-methyluracil, 6-methyluracil, uracil, 5-carboxyuracil, and thymidine, substrate specificity, overview. Analysis of enzyme reaction products by GC/MS
697315
Rhodotorula glutinis
?
-
-
-
-
N1-methylthymine + 2-oxoglutarate + O2
-
697315
Rhodotorula glutinis
N1-methyl-5-hydroxymethyluracil + succinate + CO2
-
-
-
?
N1-methyluracil + 2-oxoglutarate + O2
-
697315
Rhodotorula glutinis
? + succinate + CO2
-
-
-
?
thymine + 2-oxoglutarate + O2
-
697315
Rhodotorula glutinis
5-hydroxymethyluracil + succinate + CO2
-
-
-
?
Subunits
Subunits
Commentary
Organism
More
peptide mass spectrometry analysis, and active site structure homology modelling, overview
Rhodotorula glutinis
Temperature Optimum [C]
Temperature Optimum [C]
Temperature Optimum Maximum [C]
Commentary
Organism
30
-
assay at
Rhodotorula glutinis
Turnover Number [1/s]
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
0.011
-
thymine
pH 7.5, 30C, mutant N250D
Rhodotorula glutinis
0.025
-
thymine
pH 7.5, 30C, mutant N187D
Rhodotorula glutinis
0.053
-
thymine
pH 7.5, 30C, mutant N293D
Rhodotorula glutinis
0.27
-
N1-methyluracil
pH 7.5, 30C, wild-type enzyme
Rhodotorula glutinis
0.42
-
5-Hydroxymethyluracil
pH 7.5, 30C, wild-type enzyme
Rhodotorula glutinis
0.43
-
N1-methylthymine
pH 7.5, 30C, wild-type enzyme
Rhodotorula glutinis
1.12
-
thymine
pH 7.5, 30C, wild-type enzyme
Rhodotorula glutinis
1.13
-
5-Formyluracil
pH 7.5, 30C, wild-type enzyme
Rhodotorula glutinis
pH Optimum
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
7.5
-
-
Rhodotorula glutinis
Cloned(Commentary) (protein specific)
Commentary
Organism
DNA and amino acid sequence determination and analysis, expression of GST-tagged enzyme in Escherichia coli strain BL21(DE3)
Rhodotorula glutinis
Engineering (protein specific)
Amino acid exchange
Commentary
Organism
N187D
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
Rhodotorula glutinis
N250D
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
Rhodotorula glutinis
N293D
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
Rhodotorula glutinis
R285C
site-directed mutagenesis, inactive active site mutant
Rhodotorula glutinis
KM Value [mM] (protein specific)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
additional information
-
additional information
steady-state kinetics
Rhodotorula glutinis
0.067
-
thymine
pH 7.5, 30C, mutant N187D
Rhodotorula glutinis
0.11
-
N1-methyluracil
pH 7.5, 30C, wild-type enzyme
Rhodotorula glutinis
0.145
-
thymine
pH 7.5, 30C, wild-type enzyme
Rhodotorula glutinis
0.23
-
5-Hydroxymethyluracil
pH 7.5, 30C, wild-type enzyme
Rhodotorula glutinis
1.15
-
thymine
pH 7.5, 30C, mutant N250D
Rhodotorula glutinis
1.3
-
N1-methylthymine
pH 7.5, 30C, wild-type enzyme
Rhodotorula glutinis
1.7
-
5-Formyluracil
pH 7.5, 30C, wild-type enzyme
Rhodotorula glutinis
2.1
-
thymine
pH 7.5, 30C, mutant N293D
Rhodotorula glutinis
Natural Substrates/ Products (Substrates) (protein specific)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
thymine + 2-oxoglutarate + O2
Rhodotorula glutinis
-
5-hydroxymethyluracil + succinate + CO2
-
-
?
Purification (Commentary) (protein specific)
Commentary
Organism
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
Rhodotorula glutinis
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
5-formyluracil + 2-oxoglutarate + O2
-
697315
Rhodotorula glutinis
5-carboxyuracil + succinate + CO2
-
-
-
?
5-hydroxymethyluracil + 2-oxoglutarate + O2
-
697315
Rhodotorula glutinis
5-formyluracil + succinate + CO2
-
-
-
?
additional information
no activity with N1-methyl-5-hydroxymethyluracil, 5-methylcytosine, N3-methyluracil, 6-methyluracil, uracil, 5-carboxyuracil, and thymidine, substrate specificity, overview. Analysis of enzyme reaction products by GC/MS
697315
Rhodotorula glutinis
?
-
-
-
-
N1-methylthymine + 2-oxoglutarate + O2
-
697315
Rhodotorula glutinis
N1-methyl-5-hydroxymethyluracil + succinate + CO2
-
-
-
?
N1-methyluracil + 2-oxoglutarate + O2
-
697315
Rhodotorula glutinis
? + succinate + CO2
-
-
-
?
thymine + 2-oxoglutarate + O2
-
697315
Rhodotorula glutinis
5-hydroxymethyluracil + succinate + CO2
-
-
-
?
Subunits (protein specific)
Subunits
Commentary
Organism
More
peptide mass spectrometry analysis, and active site structure homology modelling, overview
Rhodotorula glutinis
Temperature Optimum [C] (protein specific)
Temperature Optimum [C]
Temperature Optimum Maximum [C]
Commentary
Organism
30
-
assay at
Rhodotorula glutinis
Turnover Number [1/s] (protein specific)
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
0.011
-
thymine
pH 7.5, 30C, mutant N250D
Rhodotorula glutinis
0.025
-
thymine
pH 7.5, 30C, mutant N187D
Rhodotorula glutinis
0.053
-
thymine
pH 7.5, 30C, mutant N293D
Rhodotorula glutinis
0.27
-
N1-methyluracil
pH 7.5, 30C, wild-type enzyme
Rhodotorula glutinis
0.42
-
5-Hydroxymethyluracil
pH 7.5, 30C, wild-type enzyme
Rhodotorula glutinis
0.43
-
N1-methylthymine
pH 7.5, 30C, wild-type enzyme
Rhodotorula glutinis
1.12
-
thymine
pH 7.5, 30C, wild-type enzyme
Rhodotorula glutinis
1.13
-
5-Formyluracil
pH 7.5, 30C, wild-type enzyme
Rhodotorula glutinis
pH Optimum (protein specific)
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
7.5
-
-
Rhodotorula glutinis
Other publictions for EC 1.14.11.6
No.
1st author
Pub Med
title
organims
journal
volume
pages
year
Activating Compound
Application
Cloned(Commentary)
Crystallization (Commentary)
Engineering
General Stability
Inhibitors
KM Value [mM]
Localization
Metals/Ions
Molecular Weight [Da]
Natural Substrates/ Products (Substrates)
Organic Solvent Stability
Organism
Oxidation Stability
Posttranslational Modification
Purification (Commentary)
Reaction
Renatured (Commentary)
Source Tissue
Specific Activity [micromol/min/mg]
Storage Stability
Substrates and Products (Substrate)
Subunits
Temperature Optimum [C]
Temperature Range [C]
Temperature Stability [C]
Turnover Number [1/s]
pH Optimum
pH Range
pH Stability
Cofactor
Ki Value [mM]
pI Value
IC50 Value
Activating Compound (protein specific)
Application (protein specific)
Cloned(Commentary) (protein specific)
Cofactor (protein specific)
Crystallization (Commentary) (protein specific)
Engineering (protein specific)
General Stability (protein specific)
IC50 Value (protein specific)
Inhibitors (protein specific)
Ki Value [mM] (protein specific)
KM Value [mM] (protein specific)
Localization (protein specific)
Metals/Ions (protein specific)
Molecular Weight [Da] (protein specific)
Natural Substrates/ Products (Substrates) (protein specific)
Organic Solvent Stability (protein specific)
Oxidation Stability (protein specific)
Posttranslational Modification (protein specific)
Purification (Commentary) (protein specific)
Renatured (Commentary) (protein specific)
Source Tissue (protein specific)
Specific Activity [micromol/min/mg] (protein specific)
Storage Stability (protein specific)
Substrates and Products (Substrate) (protein specific)
Subunits (protein specific)
Temperature Optimum [C] (protein specific)
Temperature Range [C] (protein specific)
Temperature Stability [C] (protein specific)
Turnover Number [1/s] (protein specific)
pH Optimum (protein specific)
pH Range (protein specific)
pH Stability (protein specific)
pI Value (protein specific)
Expression
General Information
General Information (protein specific)
Expression (protein specific)
KCat/KM [mM/s]
KCat/KM [mM/s] (protein specific)
745735
Bullard
Base J glucosyltransferase do ...
Leishmania major
Mol. Biochem. Parasitol.
204
77-80
2015
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4
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1
2
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745921
Li
Molecular basis for the subst ...
Neurospora crassa
Nucleic Acids Res.
43
10026-10038
2015
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1
1
19
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1
4
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1
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19
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1
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5
1
1
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3
1
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-
1
1
-
3
3
697315
Neidigh
Cloning and characterization o ...
Rhodotorula glutinis
Chem. Res. Toxicol.
22
885-893
2009
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1
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4
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9
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1
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2
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1
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6
1
1
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8
1
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1
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4
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9
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1
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1
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6
1
1
-
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8
1
-
-
-
-
-
-
-
-
-
700084
Vainio
Evidence that J-binding protei ...
Leishmania tarentolae
Mol. Biochem. Parasitol.
164
157-161
2009
-
-
1
-
6
-
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1
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2
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2
1
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1
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6
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1
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-
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-
-
2
1
-
-
-
-
-
-
-
-
-
1
1
-
-
-
700501
Cliffe
JBP1 and JBP2 are two distinct ...
Trypanosoma brucei
Nucleic Acids Res.
37
1452-1462
2009
-
-
1
-
5
-
-
-
1
1
-
2
-
2
-
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-
1
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2
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1
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5
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2
2
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2
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2
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-
2
-
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-
-
-
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-
-
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-
-
-
-
689246
Yu
The protein that binds to DNA ...
Leishmania tarentolae, Trypanosoma brucei
Nucleic Acids Res.
35
2107-2115
2007
-
-
2
-
8
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2
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2
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2
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2
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2
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8
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2
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2
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2
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658220
Smiley
Genes of the thymidine salvage ...
Rhodotorula glutinis
Biochim. Biophys. Acta
1723
256-264
2005
-
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1
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1
1
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5
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2
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1
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1
1
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2
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439133
Lai
-
Characterization of a novel, s ...
Rhodotorula glutinis
J. Am. Chem. Soc.
117
5023-5030
1995
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1
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1
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3
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1
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1
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6
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439134
Thornburg
Mechanism-based inactivation o ...
Rhodotorula glutinis
Biochemistry
32
14034-14042
1993
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1
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1
-
3
-
1
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1
1
6
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1
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1
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1
1
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1
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3
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1
1
6
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1
-
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-
-
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-
439135
Thornburg
A non-heme iron protein with h ...
Rhodotorula glutinis
Biochemistry
32
14023-14033
1993
-
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2
10
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1
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3
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2
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1
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11
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2
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2
2
10
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1
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3
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1
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11
-
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439136
Thornburg
Mechanism-based inhibition of ...
Rhodotorula glutinis
J. Am. Chem. Soc.
111
7632-7633
1989
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3
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-
439137
Warn-Cramer
Markedly different ascorbate d ...
Rhodotorula glutinis
J. Biol. Chem.
258
10551-10557
1983
2
-
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1
2
3
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2
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1
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1
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1
2
3
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1
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1
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3
1
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439138
Holme
Studies on the partial reactio ...
Neurospora crassa, Neurospora crassa STA 4
Biochim. Biophys. Acta
704
278-283
1982
1
-
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1
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1
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6
-
7
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1
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12
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1
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1
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1
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6
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1
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12
-
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-
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439139
Hsu
Uracils uncoupling of the deca ...
Neurospora crassa, Neurospora crassa uc-1
J. Biol. Chem.
256
6098-6101
1981
2
-
-
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1
2
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1
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2
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3
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1
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6
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1
1
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1
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1
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6
-
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439140
Wondrack
Substitution of nucleoside tri ...
Rhodotorula glutinis
J. Biol. Chem.
254
26-29
1979
7
-
-
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-
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-
1
-
1
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2
-
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Wondrack
Thymine 7-hydroxylase and pyri ...
Rhodotorula glutinis
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1978
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439142
Bankel
Thymine 7-hydroxylase from Neu ...
Neurospora crassa, Neurospora crassa STA 4
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439143
Holme
A kinetic study of thymine 7-h ...
Neurospora crassa, Neurospora crassa STA 4
Biochemistry
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1975
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Liu
Catalysis of three sequential ...
Neurospora crassa 1A, Neurospora crassa
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1973
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Bankel
Oxygenases involved in thymine ...
Neurospora crassa, Neurospora crassa STA 4
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1972
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439146
McCroskey
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Neurospora crassa, Neurospora crassa 1A
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1971
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Abbott
Conversion of thymine to 5-hyd ...
Neurospora crassa, Neurospora crassa 1A
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1964
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