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Literature summary extracted from

  • Kojima, K.; Kobayashi, T.; Tsugawa, W.; Ferri, S.; Sode, K.
    Mutational analysis of the oxygen-binding site of cholesterol oxidase and its impact on dye-mediated dehydrogenase activity (2013), J. Mol. Catal. B, 88, 41-46.
No PubMed abstract available

Application

EC Number Application Comment Organism
1.1.3.6 analysis the development of an enzyme-based sensor employing the enzyme ChOx has great potential as a simple and economical sensor system, engineering of the enzyme for electrochemical monitoring of cholesterol, overview Streptomyces sp.

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.1.3.6 expression of wild-type and mutant enzymes with with His-tags both at the N-terminus and at the C-terminus in Escherichia coli strain BL21 (DE3) Streptomyces sp.

Protein Variants

EC Number Protein Variants Comment Organism
1.1.3.6 E361A site-directed mutagenesis, the mutant shows no dehydrogenase activity Streptomyces sp.
1.1.3.6 F359A site-directed mutagenesis, the dehydrogenase/oxidase ratio is 12fold increased compared with the ratio for the wild-type enzyme Streptomyces sp.
1.1.3.6 F444A site-directed mutagenesis, the dehydrogenase/oxidase ratio is 4fold increased compared with the ratio for the wild-type enzyme Streptomyces sp.
1.1.3.6 M122A site-directed mutagenesis, the dehydrogenase/oxidase ratio is 26fold increased compared with the ratio for the wild-type enzyme Streptomyces sp.
1.1.3.6 additional information site-directed mutagenesis on oxygen-binding residues, which are observed in the high-resolution crystal structure, in order to elucidate the amino acid residues responsible for the oxidase activity, overview. Engineering of the enzyme for electrochemical monitoring of cholesterol Streptomyces sp.
1.1.3.6 N485A site-directed mutagenesis, the mutant shows no dehydrogenase activity Streptomyces sp.
1.1.3.6 V124A site-directed mutagenesis, the dehydrogenase/oxidase ratio is 30fold increased compared with the ratio for the wild-type enzyme Streptomyces sp.
1.1.3.6 V191A site-directed mutagenesis, the mutant enzymes shows a significant decrease in its oxidase activity, but shows increased dehydrogenase activity. The dehydrogenase/oxidase ratio of Val191Ala is more than 150%, which is a 408fold increase compared with the ratio for the wild-type enzyme, substrate inhibition with cholesterol Streptomyces sp.
1.1.3.6 Y446A site-directed mutagenesis, the dehydrogenase/oxidase ratio is 3fold increased compared with the ratio for the wild-type enzyme Streptomyces sp.

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.1.3.6 cholesterol substrate inhibition of mutant V191A Streptomyces sp.

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.1.3.6 0.00245
-
 cholesterol recombinant mutant V191A, pH 7.0, 22°C Streptomyces sp.
1.1.3.6 0.12
-
 cholesterol recombinant wild-type enzyme, pH 7.0, 22°C Streptomyces sp.

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.1.3.6 cholesterol + O2 Streptomyces sp.
-
 cholest-5-en-3-one + H2O2
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.1.3.6 Streptomyces sp.
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.1.3.6 recombinant wild-type and mutant enzymes with with His-tags both at the N-terminus and at the C-terminus from Escherichia coli strain BL21 (DE3) by nickel affinity chromatography Streptomyces sp.

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
1.1.3.6 0.049
-
 purified recombinant mutant V191A, oxidase activity, pH 7.0, 22°C Streptomyces sp.
1.1.3.6 0.061
-
 purified recombinant mutant M122A, oxidase activity, pH 7.0, 22°C Streptomyces sp.
1.1.3.6 0.074
-
 purified recombinant mutant F359A, oxidase activity, pH 7.0, 22°C Streptomyces sp.
1.1.3.6 0.15
-
 purified recombinant mutant V124A, oxidase activity, pH 7.0, 22°C Streptomyces sp.
1.1.3.6 0.22
-
 purified recombinant mutant F444A, oxidase activity, pH 7.0, 22°C Streptomyces sp.
1.1.3.6 0.28
-
 purified recombinant mutant Y446A, oxidase activity, pH 7.0, 22°C Streptomyces sp.
1.1.3.6 2.7
-
 purified recombinant wild-type enzyme, oxidase activity, pH 7.0, 22°C Streptomyces sp.

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.1.3.6 cholesterol + O2
-
 Streptomyces sp. cholest-5-en-3-one + H2O2
-
 ?
1.1.3.6 cholesterol + O2 assay with phenazine sulfate and dichlorophenylindophenol as electron acceptors Streptomyces sp. cholest-5-en-3-one + H2O2
-
 ?
1.1.3.6 additional information the wild-type enzyme also shows cholesterol dehydrogenase activity with 0.001 U/mg Streptomyces sp. ?
-
 ?

Synonyms

EC Number Synonyms Comment Organism
1.1.3.6 ChOx
-
 Streptomyces sp.
1.1.3.6 type I ChOx
-
 Streptomyces sp.

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.1.3.6 22
-
 assay at room temperature Streptomyces sp.

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.1.3.6 2.21
-
 cholesterol recombinant mutant V191A, pH 7.0, 22°C Streptomyces sp.
1.1.3.6 47.3
-
 cholesterol recombinant wild-type enzyme, pH 7.0, 22°C Streptomyces sp.

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.1.3.6 7
-
 assay at Streptomyces sp.

Cofactor

EC Number Cofactor Comment Organism Structure
1.1.3.6 FAD responsible for electron transfer to O2 Streptomyces sp.

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.1.3.6 394
-
 cholesterol recombinant wild-type enzyme, pH 7.0, 22°C Streptomyces sp.
1.1.3.6 902
-
 cholesterol recombinant mutant V191A, pH 7.0, 22°C Streptomyces sp.