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Literature summary extracted from

  • Finnegan, S.; Agniswamy, J.; Weber, I.T.; Gadda, G.
    Role of valine 464 in the flavin oxidation reaction catalyzed by choline oxidase (2010), Biochemistry, 49, 2952-2961.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.1.3.17 expression of wild-type and mutants in Escherichia coli strain Rosetta(DE3)pLysS Arthrobacter globiformis

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.1.3.17 purified recombinnat wild-type enzyme and mutant V464A, hanging drop vapor diffusion, room temperature, from 10-15% v/v PEG 6000, 50-200 mM magnesium acetate, 200 mM trimethylamine, and 0.08 M sodium cacodylate, pH 6.0, X-ray diffraction structure determination and analysis at 2.2 A resolution Arthrobacter globiformis

Protein Variants

EC Number Protein Variants Comment Organism
1.1.3.17 V464A site-directed mutagenesis, mutation of the residue near the flavin C(4a) atom and the hydrophobic cavity, replacement of Val464 with alanine or threonine does not affect the reductive half-reaction, but it reduces the oxidative half-reaction by about 50fold, and the 3D structure of the Val464Ala enzyme is essentially identical to that of the wild-type enzyme Arthrobacter globiformis
1.1.3.17 V464T site-directed mutagenesis, mutation of the residue near the flavin C(4a) atom and the hydrophobic cavity, replacement of Val464 with alanine or threonine does not affect the reductive half-reaction, but it reduces the oxidative half-reaction by about 50fold Arthrobacter globiformis

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.1.3.17 additional information
-
 additional information minimal kinetic mechanism for reductive half-reaction of the V464A and V464T mutant enzymes, oxidative half-reactions' steady-state kinetic mechanisms, overview Arthrobacter globiformis

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.1.3.17 choline + 2 O2 + H2O2 Arthrobacter globiformis
-
 betaine + 2 H2O2
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.1.3.17 Arthrobacter globiformis Q7X2H8
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.1.3.17 recombinant wild-type and mutants from Escherichia coli strain Rosetta(DE3)pLysS Arthrobacter globiformis

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.1.3.17 3,3-dimethylbutan-1-ol + 2 O2 + H2O2
-
 Arthrobacter globiformis ?
-
 ?
1.1.3.17 choline + 2 O2 + H2O2
-
 Arthrobacter globiformis betaine + 2 H2O2
-
 ?
1.1.3.17 choline + 2 O2 + H2O2 two-step oxidation of choline with formation of betaine aldehyde as intermediate, the overall reaction consists of oxidative and reductive half-reactions Arthrobacter globiformis betaine + 2 H2O2
-
 ?

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.1.3.17 25
-
 assay at Arthrobacter globiformis

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.1.3.17 10
-
 assay at Arthrobacter globiformis

Cofactor

EC Number Cofactor Comment Organism Structure
1.1.3.17 FAD required for catalysis, covalently bound to the Nepsilon2 atom of His99 of the wild-type enzyme Arthrobacter globiformis

General Information

EC Number General Information Comment Organism
1.1.3.17 additional information the side chain of Val464 in the active site cavity in choline oxidase provides a nonpolar site that is required to guide oxygen in proximity of the C(4a) atom of the flavin, where it will subsequently react via electrostatic catalysis Arthrobacter globiformis

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.1.3.17 additional information
-
 additional information kcat/Koxygen values are independent of the pH between pH 5.0 and 10.0, average value is 1.7 mM Arthrobacter globiformis
1.1.3.17 0.031
-
 3,3-dimethylbutan-1-ol pH 8.0, 25°C, wild-type enzyme Arthrobacter globiformis
1.1.3.17 0.031
-
 3,3-dimethylbutan-1-ol pH 8.0, 25°C, mutant V464A Arthrobacter globiformis
1.1.3.17 0.1
-
 O2 kcat/Kox, pH 10.0, 25°C, mutant V464A Arthrobacter globiformis
1.1.3.17 0.125
-
 O2 kcat/Kox, pH 10.0, 25°C, mutant V464T Arthrobacter globiformis
1.1.3.17 1.9
-
 O2 kcat/Kox, pH 10.0, 25°C, wild-type enzyme Arthrobacter globiformis
1.1.3.17 2
-
 3,3-dimethylbutan-1-ol pH 8.0, 25°C, wild-type enzyme Arthrobacter globiformis