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Literature summary for 1.1.3.10 extracted from

  • Wongnate, T.; Chaiyen, P.
    The substrate oxidation mechanism of pyranose 2-oxidase and other related enzymes in the glucose-methanol-choline superfamily (2013), FEBS J., 280, 3009-3027.
    View publication on PubMed
Search for more literature for 1.1.3.10

Application

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 (Commentary)

Crystallization (Comment) Organism
wild-type and mutants, structure analysis, overview Trametes ochracea

Protein Variants

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 [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 [Da] Molecular Weight Maximum [Da] Comment Organism
270000
-
-
 Trametes ochracea

Natural Substrates/ Products (Substrates)

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

Organism UniProt Comment Textmining
Trametes ochracea
-
-
-

Reaction

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
a

Substrates and Products (Substrate)

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

Subunits Comment Organism
homotetramer
-
 Trametes ochracea

Synonyms

Synonyms Comment Organism
P2O
-
 Trametes ochracea
pyranose 2-Oxidase
-
 Trametes ochracea

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
 assay at Trametes ochracea

Turnover Number [1/s]

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

pH Optimum Minimum pH Optimum Maximum Comment Organism
7
-
 assay at Trametes ochracea

Cofactor

Cofactor Comment Organism Structure
FAD the FAD cofactor in P2O is covalently linked through a histidyl linkage at His167 Trametes ochracea

General Information

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