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Literature summary for 1.14.11.6 extracted from
- Cloning and characterization of Rhodotorula glutinis thymine hydroxylase (2009), Chem. Res. Toxicol., 22, 885-893.
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| DNA and amino acid sequence determination and analysis, expression of GST-tagged enzyme in Escherichia coli strain BL21(DE3) | Rhodotorula glutinis |
Protein Variants
| Protein Variants | Comment | 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 | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | steady-state kinetics | Rhodotorula glutinis | |
| 0.067 | - |
thymine | pH 7.5, 30°C, mutant N187D | Rhodotorula glutinis |  |
| 0.11 | - |
N1-methyluracil | pH 7.5, 30°C, wild-type enzyme | Rhodotorula glutinis | |
| 0.145 | - |
thymine | pH 7.5, 30°C, wild-type enzyme | Rhodotorula glutinis | |
| 0.23 | - |
5-Hydroxymethyluracil | pH 7.5, 30°C, wild-type enzyme | Rhodotorula glutinis | |
| 1.15 | - |
thymine | pH 7.5, 30°C, mutant N250D | Rhodotorula glutinis | |
| 1.3 | - |
N1-methylthymine | pH 7.5, 30°C, wild-type enzyme | Rhodotorula glutinis | |
| 1.7 | - |
5-Formyluracil | pH 7.5, 30°C, wild-type enzyme | Rhodotorula glutinis | |
| 2.1 | - |
thymine | pH 7.5, 30°C, mutant N293D | Rhodotorula glutinis | |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| thymine + 2-oxoglutarate + O2 | Rhodotorula glutinis | - |
5-hydroxymethyluracil + succinate + CO2 | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Rhodotorula glutinis | Q66Q78 | strain ATCC 2527 | - |
Purification (Commentary)
| Purification (Comment) | 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 | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 5-formyluracil + 2-oxoglutarate + O2 | - |
Rhodotorula glutinis | 5-carboxyuracil + succinate + CO2 | - |
? | |
| 5-hydroxymethyluracil + 2-oxoglutarate + O2 | - |
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 | Rhodotorula glutinis | ? | - |
? | |
| N1-methylthymine + 2-oxoglutarate + O2 | - |
Rhodotorula glutinis | N1-methyl-5-hydroxymethyluracil + succinate + CO2 | - |
? | |
| N1-methyluracil + 2-oxoglutarate + O2 | - |
Rhodotorula glutinis | ? + succinate + CO2 | - |
? | |
| thymine + 2-oxoglutarate + O2 | - |
Rhodotorula glutinis | 5-hydroxymethyluracil + succinate + CO2 | - |
? | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| More | peptide mass spectrometry analysis, and active site structure homology modelling, overview | Rhodotorula glutinis |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| More | is a member of the alpha-ketoglutarate-dependent nonheme iron dioxygenase family that includes a series of DNA repair proteins including alkB | Rhodotorula glutinis |
| thymine hydroxylase | - |
Rhodotorula glutinis |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 30 | - |
assay at | Rhodotorula glutinis |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.011 | - |
thymine | pH 7.5, 30°C, mutant N250D | Rhodotorula glutinis | |
| 0.025 | - |
thymine | pH 7.5, 30°C, mutant N187D | Rhodotorula glutinis | |
| 0.053 | - |
thymine | pH 7.5, 30°C, mutant N293D | Rhodotorula glutinis | |
| 0.27 | - |
N1-methyluracil | pH 7.5, 30°C, wild-type enzyme | Rhodotorula glutinis | |
| 0.42 | - |
5-Hydroxymethyluracil | pH 7.5, 30°C, wild-type enzyme | Rhodotorula glutinis | |
| 0.43 | - |
N1-methylthymine | pH 7.5, 30°C, wild-type enzyme | Rhodotorula glutinis | |
| 1.12 | - |
thymine | pH 7.5, 30°C, wild-type enzyme | Rhodotorula glutinis | |
| 1.13 | - |
5-Formyluracil | pH 7.5, 30°C, wild-type enzyme | Rhodotorula glutinis | |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 7.5 | - |
- |
Rhodotorula glutinis |
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