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Literature summary extracted from
- Molecular basis for the substrate specificity and catalytic mechanism of thymine-7-hydroxylase in fungi (2015), Nucleic Acids Res., 43, 10026-10038 .
Cloned(Commentary)
| EC Number | Cloned (Comment) | Organism |
|---|---|---|
| 1.14.11.6 | recombinant expression of C-terminally His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Codon Plus | Neurospora crassa |
Crystallization (Commentary)
| EC Number | Crystallization (Comment) | Organism |
|---|---|---|
| 1.14.11.6 | purified recombinant full-length or truncated enzyme (unmodified or SeMet-labeled) in apo form, or complexed with 2-oxoglutarate, thymine, 5-hydroxymethyluracil, or 5-formyluracil, drop vapour diffusion method, 16°C, the reservoir solution consists of 0.2 M (NH4)2SO4, 0.1 M MES, pH 6.5, and 30% w/v PEGMME 5000 for the apoenzyme, for the 2-oxoglutarate complexed enzyme of 0.1 M bis-Tris, pH 5.5, and 25% w/v PEG 3,350, and 2-oxoglutarate in a 1:4 ratio protein/2-OG, or for enzyme in complex with other ligands in a 1:4:8 ratio, X-ray diffraction structure determination and analysis at 2.05-2.43 A resolution | Neurospora crassa |
Protein Variants
| EC Number | Protein Variants | Comment | Organism |
|---|---|---|---|
| 1.14.11.6 | D216N | site-directed mutagenesis, the mutant shows reduced activity with thymine and altered substrate specificity compared to the wild-type enzyme | Neurospora crassa |
| 1.14.11.6 | E122A | site-directed mutagenesis, the mutant shows reduced activity with thymine and altered substrate specificity compared to the wild-type enzyme | Neurospora crassa |
| 1.14.11.6 | F292A | site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme | Neurospora crassa |
| 1.14.11.6 | H214A | site-directed mutagenesis, the mutant shows highly reduced activity with thymine and altered substrate specificity compared to the wild-type enzyme | Neurospora crassa |
| 1.14.11.6 | H271A | site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme | Neurospora crassa |
| 1.14.11.6 | L192A | site-directed mutagenesis, the mutant shows reduced activity with thymine and altered substrate specificity compared to the wild-type enzyme | Neurospora crassa |
| 1.14.11.6 | L223A | site-directed mutagenesis, the mutant shows reduced activity with thymine and altered substrate specificity compared to the wild-type enzyme | Neurospora crassa |
| 1.14.11.6 | additional information | construction of a C-terminally truncated enzyme mutant NcT7H, comprising residues 1-299 | Neurospora crassa |
| 1.14.11.6 | N294A | site-directed mutagenesis, the mutant shows slightly increased activity compared to the wild-type enzyme | Neurospora crassa |
| 1.14.11.6 | N87A | site-directed mutagenesis, the mutant shows similar activity with thymine and altered substrate specificity compared to the wild-type enzyme | Neurospora crassa |
| 1.14.11.6 | R190A | site-directed mutagenesis, the mutant shows highly reduced activity with thymine and altered substrate specificity compared to the wild-type enzyme | Neurospora crassa |
| 1.14.11.6 | R190K | site-directed mutagenesis, the mutant shows reduced activity with thymine and altered substrate specificity compared to the wild-type enzyme | Neurospora crassa |
| 1.14.11.6 | R286A | site-directed mutagenesis, the mutant shows reduced activity with thymine and altered substrate specificity compared to the wild-type enzyme | Neurospora crassa |
| 1.14.11.6 | R286K | site-directed mutagenesis, the mutant shows reduced activity with thymine and altered substrate specificity compared to the wild-type enzyme | Neurospora crassa |
| 1.14.11.6 | S288A | site-directed mutagenesis, the mutant shows similar activity with thymine and altered substrate specificity compared to the wild-type enzyme | Neurospora crassa |
| 1.14.11.6 | V273A | site-directed mutagenesis, the mutant shows reduced activity with thymine and altered substrate specificity compared to the wild-type enzyme | Neurospora crassa |
| 1.14.11.6 | Y194F | site-directed mutagenesis, the mutant shows reduced activity with thymine and altered substrate specificity compared to the wild-type enzyme | Neurospora crassa |
| 1.14.11.6 | Y217A | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Neurospora crassa |
| 1.14.11.6 | Y217F | site-directed mutagenesis, the mutant shows similar activity compared to the wild-type enzyme | Neurospora crassa |
Inhibitors
| EC Number | Inhibitors | Comment | Organism | Structure |
|---|---|---|---|---|
| 1.14.11.6 | Ni2+ | binding structure with truncated enzyme mutant | Neurospora crassa |  |
KM Value [mM]
| EC Number | KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|---|
| 1.14.11.6 | additional information | - |
additional information | Michaelis-Menten kinetics, isothermal titration calorimetry analysis, steady-state kinetics of enzyme mutants, overview | Neurospora crassa | |
| 1.14.11.6 | 0.141 | - |
thymine | recombinant wild-type enzyme, pH 7.5, 25°C | Neurospora crassa | |
| 1.14.11.6 | 0.178 | - |
5-Hydroxyuracil | recombinant wild-type enzyme, pH 7.5, 25°C | Neurospora crassa | |
| 1.14.11.6 | 0.253 | - |
5-Formyluracil | recombinant wild-type enzyme, pH 7.5, 25°C | Neurospora crassa | |
Metals/Ions
| EC Number | Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|---|
| 1.14.11.6 | Fe2+ | dependent on, required for catalysis | Neurospora crassa | |
| 1.14.11.6 | additional information | Ni2+ and Ca2+ cannot substitute for Fe2+, binding structure with truncated enzyme mutant. Residues His214, Asp216 and His271 play essential roles in the metal ion binding | Neurospora crassa |
Natural Substrates/ Products (Substrates)
| EC Number | Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|---|
| 1.14.11.6 | 5-carboxyluracil + O2 | Neurospora crassa | - |
? | - |
? | |
| 1.14.11.6 | 5-formyluracil + O2 | Neurospora crassa | - |
? | - |
? | |
| 1.14.11.6 | thymine + 2-oxoglutarate + O2 | Neurospora crassa | - |
5-hydroxymethyluracil + succinate + CO2 | - |
? | |
Organism
| EC Number | Organism | UniProt | Comment | Textmining |
|---|---|---|---|---|
| 1.14.11.6 | Neurospora crassa | Q7RYZ9 | - |
- |
Purification (Commentary)
| EC Number | Purification (Comment) | Organism |
|---|---|---|
| 1.14.11.6 | recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) Condon Plus by nickel affinity chromatography and gel filtration | Neurospora crassa |
Substrates and Products (Substrate)
| EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|---|
| 1.14.11.6 | 5-carboxyluracil + O2 | - |
Neurospora crassa | ? | - |
? | |
| 1.14.11.6 | 5-formyluracil + O2 | - |
Neurospora crassa | ? | - |
? | |
| 1.14.11.6 | 5-hydroxyuracil + O2 | - |
Neurospora crassa | ? | - |
? | |
| 1.14.11.6 | thymine + 2-oxoglutarate + O2 | - |
Neurospora crassa | 5-hydroxymethyluracil + succinate + CO2 | - |
? | |
| 1.14.11.6 | thymine + 2-oxoglutarate + O2 | i.e. 5-methyluracil | Neurospora crassa | 5-hydroxymethyluracil + succinate + CO2 | - |
? | |
Subunits
| EC Number | Subunits | Comment | Organism |
|---|---|---|---|
| 1.14.11.6 | More | enzyme structure analysis, overview | Neurospora crassa |
Synonyms
| EC Number | Synonyms | Comment | Organism |
|---|---|---|---|
| 1.14.11.6 | T7H | - |
Neurospora crassa |
| 1.14.11.6 | thymine-7-hydroxylase | - |
Neurospora crassa |
Temperature Optimum [°C]
| EC Number | Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|---|
| 1.14.11.6 | 25 | 37 | assay at | Neurospora crassa |
Turnover Number [1/s]
| EC Number | Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|---|
| 1.14.11.6 | 0.325 | - |
5-Hydroxyuracil | recombinant wild-type enzyme, pH 7.5, 25°C | Neurospora crassa | |
| 1.14.11.6 | 0.416 | - |
thymine | recombinant wild-type enzyme, pH 7.5, 25°C | Neurospora crassa | |
| 1.14.11.6 | 0.765 | - |
5-Formyluracil | recombinant wild-type enzyme, pH 7.5, 25°C | Neurospora crassa | |
pH Optimum
| EC Number | pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|---|
| 1.14.11.6 | 7.5 | - |
assay at | Neurospora crassa |
General Information
| EC Number | General Information | Comment | Organism |
|---|---|---|---|
| 1.14.11.6 | additional information | molecular basis for substrate specificity and catalytic mechanism of the enzyme and molecular mechanism of substrate recognition, overview. Residues Phe292, Tyr217 and Arg190 play critical roles in substrate binding and catalysis, and the interactions of the C5 modification group of substrates with the cosubstrate and enzyme contribute to the slightly varied binding affinity and activity towards different substrates. After the catalysis, the products are released and new cosubstrate and substrate are reloaded to conduct the next oxidation reaction. Active site structure, and 2-oxoglutarate binding structure, binding affinity of the enzyme for different substrates, overview. Residue Arg286 plays an important role in the binding of 2-oxoglutarate, Arg190 plays a vital role in the binding of both 2-oxoglutarate and the substrate, and Phe292 and Tyr217 play critical roles in the substrate binding | Neurospora crassa |
kcat/KM [mM/s]
| EC Number | kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|---|
| 1.14.11.6 | 1.826 | - |
5-Hydroxyuracil | recombinant wild-type enzyme, pH 7.5, 25°C | Neurospora crassa | |
| 1.14.11.6 | 2.404 | - |
thymine | recombinant wild-type enzyme, pH 7.5, 25°C | Neurospora crassa | |
| 1.14.11.6 | 3.024 | - |
5-Formyluracil | recombinant wild-type enzyme, pH 7.5, 25°C | Neurospora crassa | |
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