Literature summary for 1.14.11.6 extracted from

Li, W.; Zhang, T.; Ding, J.
Molecular basis for the substrate specificity and catalytic mechanism of thymine-7-hydroxylase in fungi (2015), Nucleic Acids Res., 43, 10026-10038 .

Search for more literature for 1.14.11.6

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of C-terminally His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Codon Plus	Neurospora crassa

Crystallization (Commentary)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

Protein Variants	Comment	Organism
D216N	site-directed mutagenesis, the mutant shows reduced activity with thymine and altered substrate specificity compared to the wild-type enzyme	Neurospora crassa
E122A	site-directed mutagenesis, the mutant shows reduced activity with thymine and altered substrate specificity compared to the wild-type enzyme	Neurospora crassa
F292A	site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme	Neurospora crassa
H214A	site-directed mutagenesis, the mutant shows highly reduced activity with thymine and altered substrate specificity compared to the wild-type enzyme	Neurospora crassa
H271A	site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme	Neurospora crassa
L192A	site-directed mutagenesis, the mutant shows reduced activity with thymine and altered substrate specificity compared to the wild-type enzyme	Neurospora crassa
L223A	site-directed mutagenesis, the mutant shows reduced activity with thymine and altered substrate specificity compared to the wild-type enzyme	Neurospora crassa
additional information	construction of a C-terminally truncated enzyme mutant NcT7H, comprising residues 1-299	Neurospora crassa
N294A	site-directed mutagenesis, the mutant shows slightly increased activity compared to the wild-type enzyme	Neurospora crassa
N87A	site-directed mutagenesis, the mutant shows similar activity with thymine and altered substrate specificity compared to the wild-type enzyme	Neurospora crassa
R190A	site-directed mutagenesis, the mutant shows highly reduced activity with thymine and altered substrate specificity compared to the wild-type enzyme	Neurospora crassa
R190K	site-directed mutagenesis, the mutant shows reduced activity with thymine and altered substrate specificity compared to the wild-type enzyme	Neurospora crassa
R286A	site-directed mutagenesis, the mutant shows reduced activity with thymine and altered substrate specificity compared to the wild-type enzyme	Neurospora crassa
R286K	site-directed mutagenesis, the mutant shows reduced activity with thymine and altered substrate specificity compared to the wild-type enzyme	Neurospora crassa
S288A	site-directed mutagenesis, the mutant shows similar activity with thymine and altered substrate specificity compared to the wild-type enzyme	Neurospora crassa
V273A	site-directed mutagenesis, the mutant shows reduced activity with thymine and altered substrate specificity compared to the wild-type enzyme	Neurospora crassa
Y194F	site-directed mutagenesis, the mutant shows reduced activity with thymine and altered substrate specificity compared to the wild-type enzyme	Neurospora crassa
Y217A	site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme	Neurospora crassa
Y217F	site-directed mutagenesis, the mutant shows similar activity compared to the wild-type enzyme	Neurospora crassa

Inhibitors

Inhibitors	Comment	Organism	Structure
Ni2+	binding structure with truncated enzyme mutant	Neurospora crassa

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
additional information	-	additional information	Michaelis-Menten kinetics, isothermal titration calorimetry analysis, steady-state kinetics of enzyme mutants, overview	Neurospora crassa
0.141	-	thymine	recombinant wild-type enzyme, pH 7.5, 25°C	Neurospora crassa
0.178	-	5-Hydroxyuracil	recombinant wild-type enzyme, pH 7.5, 25°C	Neurospora crassa
0.253	-	5-Formyluracil	recombinant wild-type enzyme, pH 7.5, 25°C	Neurospora crassa

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Fe2+	dependent on, required for catalysis	Neurospora crassa
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)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
5-carboxyluracil + O2	Neurospora crassa	-	?	-	?
5-formyluracil + O2	Neurospora crassa	-	?	-	?
thymine + 2-oxoglutarate + O2	Neurospora crassa	-	5-hydroxymethyluracil + succinate + CO2	-	?

Organism

Organism	UniProt	Comment	Textmining
Neurospora crassa	Q7RYZ9	-	-

Purification (Commentary)

Purification (Comment)	Organism
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)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
5-carboxyluracil + O2	-	Neurospora crassa	?	-	?
5-formyluracil + O2	-	Neurospora crassa	?	-	?
5-hydroxyuracil + O2	-	Neurospora crassa	?	-	?
thymine + 2-oxoglutarate + O2	-	Neurospora crassa	5-hydroxymethyluracil + succinate + CO2	-	?
thymine + 2-oxoglutarate + O2	i.e. 5-methyluracil	Neurospora crassa	5-hydroxymethyluracil + succinate + CO2	-	?

Subunits

Subunits	Comment	Organism
More	enzyme structure analysis, overview	Neurospora crassa

Synonyms

Synonyms	Comment	Organism
T7H	-	Neurospora crassa
thymine-7-hydroxylase	-	Neurospora crassa

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
25	37	assay at	Neurospora crassa

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.325	-	5-Hydroxyuracil	recombinant wild-type enzyme, pH 7.5, 25°C	Neurospora crassa
0.416	-	thymine	recombinant wild-type enzyme, pH 7.5, 25°C	Neurospora crassa
0.765	-	5-Formyluracil	recombinant wild-type enzyme, pH 7.5, 25°C	Neurospora crassa

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Neurospora crassa

General Information

General Information	Comment	Organism
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]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
1.826	-	5-Hydroxyuracil	recombinant wild-type enzyme, pH 7.5, 25°C	Neurospora crassa
2.404	-	thymine	recombinant wild-type enzyme, pH 7.5, 25°C	Neurospora crassa
3.024	-	5-Formyluracil	recombinant wild-type enzyme, pH 7.5, 25°C	Neurospora crassa