Application | Comment | Organism |
---|---|---|
biotechnology | enzyme P2O is a useful biocatalyst in several biotechnological applications, including biotransformation of carbohydrates such as D-glucose and D-galactose to generate 2-oxo-sugars that can be further reduced at the C1 position to yield D-fructose and D-tagatose, respectively | Trametes ochracea |
energy production | enzyme P2O has the potential to be useful for biofuel cell applications | Trametes ochracea |
synthesis | enzyme P2O is a useful biocatalyst in several biotechnological applications, including biotransformation of carbohydrates such as D-glucose and D-galactose to generate 2-oxo-sugars that can be further reduced at the C1 position to yield D-fructose and D-tagatose, respectively | Trametes ochracea |
Crystallization (Comment) | Organism |
---|---|
wild-type and mutants, structure analysis, overview | Trametes ochracea |
Protein Variants | Comment | Organism |
---|---|---|
H167A | site-directed mutagenesis, reductive activity of the mutant is highly reduced compared to the wild-type enzyme, the mutation ablates the covalent histidyl-FAD linkage. The H167A mutant enzyme oxidizes D-glucose regiospecifically at the C2 position, similarly to the wild-type enzyme, but noncovalent flavin in H167A mainly decreases the flavin reduction rate constant by 22fold. In the crystal complex of the H167A mutant and 2FG (C3 oxidation), the substrate-recognition loop swings further away from the active site to assume the open conformation, whereas in the C2 oxidation complex of the H167A mutant and 3FG, the gating segment (residues 452-456) swings towards the active site to provide a binding pocket for the substrate | Trametes ochracea |
H167A/H548A | site-directed mutagenesis, reductively inactive mutant, contains noncovalently linked FAD | Trametes ochracea |
H167A/H548D | site-directed mutagenesis, reductively inactive mutant, contains noncovalently linked FAD | Trametes ochracea |
H167A/H548N | site-directed mutagenesis, reductively inactive mutant, contains noncovalently linked FAD | Trametes ochracea |
H167A/H548R | site-directed mutagenesis, reductive activity of the mutant is reduced compared to the wild-type enzyme, contains noncovalently linked FAD, highest reductive activity at pH 10.5 | Trametes ochracea |
H167A/H548S | site-directed mutagenesis, reductively inactive mutant, contains noncovalently linked FAD | Trametes ochracea |
H548A | site-directed mutagenesis, reductive activity of the mutant is reduced compared to the wild-type enzyme, contains noncovalently and covalently linked FAD | Trametes ochracea |
H548D | site-directed mutagenesis, reductive activity of the mutant is reduced compared to the wild-type enzyme, contains noncovalently and covalently linked FAD | Trametes ochracea |
H548N | site-directed mutagenesis, reductive activity of the mutant is reduced compared to the wild-type enzyme, contains noncovalently and covalently linked FAD | Trametes ochracea |
H548R | site-directed mutagenesis, reductive activity of the mutant is slightly reduced compared to the wild-type enzyme, contains some noncovalently and mostly covalently linked FAD | Trametes ochracea |
H548S | site-directed mutagenesis, reductive activity of the mutant is reduced compared to the wild-type enzyme, contains noncovalently and covalently linked FAD | Trametes ochracea |
additional information | mutagenesis of Phe454 and Tyr456 results in inactive enzymes, indicating that this region is functionally important for P2O | Trametes ochracea |
N593H | site-directed mutagenesis, the enzyme contains a covalently linked FAD, similar to the wild-type enzyme | Trametes ochracea |
T169A | site-directed mutagenesis, the hydrogen bond between Thr169 and the N5 atom of FAD is absent in the mutant.The kinetic mechanism of the T169A mutant with D-glucose or D-galactose indicates that a 2-keto-sugar product remains bound at the active site during the oxidative half-reaction | Trametes ochracea |
T169G | site-directed mutagenesis | Trametes ochracea |
T169N | site-directed mutagenesis | Trametes ochracea |
T169S | site-directed mutagenesis | Trametes ochracea |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | the steady-state kinetics of P2O can be classified as a ping pong bi-bi type, because the 2-keto-sugar product is released prior to the oxygen reaction, transient kinetics and isotope effects, overview | Trametes ochracea | |
3.7 | - |
D-galactose | pH 7.0, 25°C, reductive half-reaction, wild-type enzyme | Trametes ochracea | |
4.2 | - |
D-galactose | pH 7.0, 25°C, reductive half-reaction, mutant T169G | Trametes ochracea | |
5.9 | - |
D-galactose | pH 7.0, 25°C, reductive half-reaction, mutant T169S | Trametes ochracea | |
6.6 | - |
D-galactose | pH 7.0, 25°C, reductive half-reaction, mutant T169N | Trametes ochracea | |
45 | - |
D-glucose | pH 7.0, 25°C, reductive half-reaction, wild-type enzyme and mutant T169S | Trametes ochracea | |
47 | - |
D-glucose | pH 7.0, 25°C, reductive half-reaction, mutant T169N | Trametes ochracea |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
270000 | - |
- |
Trametes ochracea |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
D-galactose + O2 | Trametes ochracea | mutants T169S, T169N, and T169G | 2-dehydro-D-galactose + H2O2 | - |
? | |
D-glucose + O2 | Trametes ochracea | - |
2-dehydro-D-glucose + H2O2 | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Trametes ochracea | - |
- |
- |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
D-glucose + O2 = 2-dehydro-D-glucose + H2O2 | ping pong bi bi reaction mechanism, sugar oxidation and flavin reduction activation and mechanism, regiospecificity and selectivity of sugar oxidation, overview. The 2-oxo-sugar product is released prior to the oxygen reaction, overview. The enzyme shows a hydride transfer mechanism in which there is stepwise formation of D-glucose alkoxide prior to the hydride transfer, and a C4a-hydroperoxyflavin as an intermediate during the oxidative half-reaction, the C4a-hydroperoxyflavin merely eliminates H2O2 to generate oxidized FAD. The breakage of the flavin N (5)-H bond controls the overall process of H2O2 elimination from C4a-hydroperoxyflavin | Trametes ochracea |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
D-galactose + O2 | mutants T169S, T169N, and T169G | Trametes ochracea | 2-dehydro-D-galactose + H2O2 | - |
? | |
D-glucose + O2 | - |
Trametes ochracea | 2-dehydro-D-glucose + H2O2 | - |
? |
Subunits | Comment | Organism |
---|---|---|
homotetramer | - |
Trametes ochracea |
Synonyms | Comment | Organism |
---|---|---|
P2O | - |
Trametes ochracea |
pyranose 2-Oxidase | - |
Trametes ochracea |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Trametes ochracea |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.006 | - |
D-glucose | pH 6.0, 25°C, mutant H167A/H548R | Trametes ochracea | |
0.018 | - |
D-glucose | pH 7.0, 25°C, mutant H167A/H548R | Trametes ochracea | |
0.057 | - |
D-glucose | pH 8.0, 25°C, mutant H167A/H548R | Trametes ochracea | |
0.3 | - |
D-galactose | pH 7.0, 25°C, reductive half-reaction, wild-type enzyme | Trametes ochracea | |
0.5 | - |
D-galactose | pH 7.0, 25°C, reductive half-reaction, mutant T169S | Trametes ochracea | |
0.63 | - |
D-glucose | pH 9.5, 25°C, mutant H167A/H548R | Trametes ochracea | |
0.7 | - |
D-glucose | pH 7.0, 25°C, reductive half-reaction, mutant T169G | Trametes ochracea | |
0.9 | - |
D-galactose | pH 7.0, 25°C, reductive half-reaction, mutant T169N | Trametes ochracea | |
1.6 | - |
D-glucose | pH 10.25, 25°C, mutant H167A/H548R | Trametes ochracea | |
2.13 | - |
D-glucose | pH 10.5, 25°C, mutant H167A/H548R | Trametes ochracea | |
2.7 | - |
D-galactose | pH 7.0, 25°C, reductive half-reaction, mutant T169G | Trametes ochracea | |
9.7 | - |
D-glucose | pH 7.0, 25°C, reductive half-reaction, mutant T169N | Trametes ochracea | |
13.8 | - |
D-glucose | pH 7.0, 25°C, reductive half-reaction, mutant T169S | Trametes ochracea | |
15.3 | - |
D-glucose | pH 7.0, 25°C, reductive half-reaction, wild-type enzyme | Trametes ochracea |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7 | - |
assay at | Trametes ochracea |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FAD | the FAD cofactor in P2O is covalently linked through a histidyl linkage at His167 | Trametes ochracea |
General Information | Comment | Organism |
---|---|---|
evolution | the enzyme belongs to the glucose-methanol-choline (GMC) oxidoreductase superfamily, comparison of P2O and other enzymes in the GMC family, overview. Although all of the GMC enzymes share similar structural folding and use the hydride transfer mechanism for flavin reduction, they appear to have subtle differences in the fine-tuned details of how they catalyze substrate oxidation | Trametes ochracea |