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Literature summary 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

Application

EC Number Application Comment Organism
1.1.3.10 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
1.1.3.10 energy production enzyme P2O has the potential to be useful for biofuel cell applications Trametes ochracea
1.1.3.10 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)

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

Protein Variants

EC Number Protein Variants Comment Organism
1.1.3.10 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
1.1.3.10 H167A/H548A site-directed mutagenesis, reductively inactive mutant, contains noncovalently linked FAD Trametes ochracea
1.1.3.10 H167A/H548D site-directed mutagenesis, reductively inactive mutant, contains noncovalently linked FAD Trametes ochracea
1.1.3.10 H167A/H548N site-directed mutagenesis, reductively inactive mutant, contains noncovalently linked FAD Trametes ochracea
1.1.3.10 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
1.1.3.10 H167A/H548S site-directed mutagenesis, reductively inactive mutant, contains noncovalently linked FAD Trametes ochracea
1.1.3.10 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
1.1.3.10 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
1.1.3.10 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
1.1.3.10 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
1.1.3.10 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
1.1.3.10 additional information mutagenesis of Phe454 and Tyr456 results in inactive enzymes, indicating that this region is functionally important for P2O Trametes ochracea
1.1.3.10 N593H site-directed mutagenesis, the enzyme contains a covalently linked FAD, similar to the wild-type enzyme Trametes ochracea
1.1.3.10 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
1.1.3.10 T169G site-directed mutagenesis Trametes ochracea
1.1.3.10 T169N site-directed mutagenesis Trametes ochracea
1.1.3.10 T169S site-directed mutagenesis Trametes ochracea
1.1.3.12 H460A/H462A inactive Pseudomonas sp. MA-1

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.1.3.10 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
1.1.3.10 3.7
-
 D-galactose pH 7.0, 25°C, reductive half-reaction, wild-type enzyme Trametes ochracea
1.1.3.10 4.2
-
 D-galactose pH 7.0, 25°C, reductive half-reaction, mutant T169G Trametes ochracea
1.1.3.10 5.9
-
 D-galactose pH 7.0, 25°C, reductive half-reaction, mutant T169S Trametes ochracea
1.1.3.10 6.6
-
 D-galactose pH 7.0, 25°C, reductive half-reaction, mutant T169N Trametes ochracea
1.1.3.10 45
-
 D-glucose pH 7.0, 25°C, reductive half-reaction, wild-type enzyme and mutant T169S Trametes ochracea
1.1.3.10 47
-
 D-glucose pH 7.0, 25°C, reductive half-reaction, mutant T169N Trametes ochracea

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
1.1.3.10 270000
-
-
 Trametes ochracea
1.1.3.12 54000
-
 1 * 54000, SDS-PAGE Microbacterium luteolum
1.1.3.12 55000
-
 1 * 55000, SDS-PAGE Mesorhizobium loti

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.1.3.10 D-galactose + O2 Trametes ochracea mutants T169S, T169N, and T169G 2-dehydro-D-galactose + H2O2
-
 ?
1.1.3.10 D-glucose + O2 Trametes ochracea
-
 2-dehydro-D-glucose + H2O2
-
 ?
1.1.3.12 pyridoxine + O2 Mesorhizobium loti
-
 pyridoxal + H2O2
-
 ?
1.1.3.12 pyridoxine + O2 Microbacterium luteolum
-
 pyridoxal + H2O2
-
 ?
1.1.3.12 pyridoxine + O2 Pseudomonas sp. MA-1
-
 pyridoxal + H2O2
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.1.3.10 Trametes ochracea
-
-
-
1.1.3.12 Mesorhizobium loti
-
-
-
1.1.3.12 Microbacterium luteolum
-
-
-
1.1.3.12 Pseudomonas sp. MA-1
-
-
-

Reaction

EC Number Reaction Comment Organism Reaction ID
1.1.3.10 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)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.1.3.10 D-galactose + O2 mutants T169S, T169N, and T169G Trametes ochracea 2-dehydro-D-galactose + H2O2
-
 ?
1.1.3.10 D-glucose + O2
-
 Trametes ochracea 2-dehydro-D-glucose + H2O2
-
 ?
1.1.3.12 additional information 2,6-dihydroxypridine, 3-pyridinemethanol, and 4-pyridinemethanol give about 0.15% of the activity as compared with pyridoxine. The enzyme cannot use pyridoxal 5'-phosphate as a substrate Microbacterium luteolum ?
-
 ?
1.1.3.12 additional information 2,6-dihydroxypridine, 3-pyridinemethanol, and 4-pyridinemethanol give about 0.15% of the activity as compared with pyridoxine. The enzyme cannot use pyridoxal 5'-phosphate as a substrate Pseudomonas sp. MA-1 ?
-
 ?
1.1.3.12 additional information 2,6-dihydroxypridine, 3-pyridinemethanol, and 4-pyridinemethanol give only about 0.15% of the activity as compared with pyridoxine. The enzyme cannot use pyridoxal 5'-phosphate as a substrate Mesorhizobium loti ?
-
 ?
1.1.3.12 pyridoxine + O2
-
 Mesorhizobium loti pyridoxal + H2O2
-
 ?
1.1.3.12 pyridoxine + O2
-
 Microbacterium luteolum pyridoxal + H2O2
-
 ?
1.1.3.12 pyridoxine + O2
-
 Pseudomonas sp. MA-1 pyridoxal + H2O2
-
 ?

Subunits

EC Number Subunits Comment Organism
1.1.3.10 homotetramer
-
 Trametes ochracea
1.1.3.12 monomer 1 * 54000, SDS-PAGE Microbacterium luteolum
1.1.3.12 monomer 1 * 55000, SDS-PAGE Mesorhizobium loti

Synonyms

EC Number Synonyms Comment Organism
1.1.3.10 P2O
-
 Trametes ochracea
1.1.3.10 pyranose 2-Oxidase
-
 Trametes ochracea
1.1.3.12 PNO
-
 Mesorhizobium loti
1.1.3.12 PNO
-
 Microbacterium luteolum
1.1.3.12 PNO
-
 Pseudomonas sp. MA-1

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.1.3.10 25
-
 assay at Trametes ochracea

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.1.3.10 0.006
-
 D-glucose pH 6.0, 25°C, mutant H167A/H548R Trametes ochracea
1.1.3.10 0.018
-
 D-glucose pH 7.0, 25°C, mutant H167A/H548R Trametes ochracea
1.1.3.10 0.057
-
 D-glucose pH 8.0, 25°C, mutant H167A/H548R Trametes ochracea
1.1.3.10 0.3
-
 D-galactose pH 7.0, 25°C, reductive half-reaction, wild-type enzyme Trametes ochracea
1.1.3.10 0.5
-
 D-galactose pH 7.0, 25°C, reductive half-reaction, mutant T169S Trametes ochracea
1.1.3.10 0.63
-
 D-glucose pH 9.5, 25°C, mutant H167A/H548R Trametes ochracea
1.1.3.10 0.7
-
 D-glucose pH 7.0, 25°C, reductive half-reaction, mutant T169G Trametes ochracea
1.1.3.10 0.9
-
 D-galactose pH 7.0, 25°C, reductive half-reaction, mutant T169N Trametes ochracea
1.1.3.10 1.6
-
 D-glucose pH 10.25, 25°C, mutant H167A/H548R Trametes ochracea
1.1.3.10 2.13
-
 D-glucose pH 10.5, 25°C, mutant H167A/H548R Trametes ochracea
1.1.3.10 2.7
-
 D-galactose pH 7.0, 25°C, reductive half-reaction, mutant T169G Trametes ochracea
1.1.3.10 9.7
-
 D-glucose pH 7.0, 25°C, reductive half-reaction, mutant T169N Trametes ochracea
1.1.3.10 13.8
-
 D-glucose pH 7.0, 25°C, reductive half-reaction, mutant T169S Trametes ochracea
1.1.3.10 15.3
-
 D-glucose pH 7.0, 25°C, reductive half-reaction, wild-type enzyme Trametes ochracea

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.1.3.10 7
-
 assay at Trametes ochracea

Cofactor

EC Number Cofactor Comment Organism Structure
1.1.3.10 FAD the FAD cofactor in P2O is covalently linked through a histidyl linkage at His167 Trametes ochracea
1.1.3.12 FAD
-
 Mesorhizobium loti
1.1.3.12 FAD
-
 Microbacterium luteolum
1.1.3.12 FAD
-
 Pseudomonas sp. MA-1

General Information

EC Number General Information Comment Organism
1.1.3.10 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
1.1.3.12 metabolism first enzyme in the vitamin B6 degradation pathway Mesorhizobium loti
1.1.3.12 metabolism first enzyme in the vitamin B6 degradation pathway Microbacterium luteolum
1.1.3.12 metabolism first enzyme in the vitamin B6 degradation pathway Pseudomonas sp. MA-1