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Literature summary for 1.1.3.6 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

Application Comment Organism
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)

Cloned (Comment) Organism
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

Protein Variants Comment Organism
E361A site-directed mutagenesis, the mutant shows no dehydrogenase activity Streptomyces sp.
F359A site-directed mutagenesis, the dehydrogenase/oxidase ratio is 12fold increased compared with the ratio for the wild-type enzyme Streptomyces sp.
F444A site-directed mutagenesis, the dehydrogenase/oxidase ratio is 4fold increased compared with the ratio for the wild-type enzyme Streptomyces sp.
M122A site-directed mutagenesis, the dehydrogenase/oxidase ratio is 26fold increased compared with the ratio for the wild-type enzyme Streptomyces sp.
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.
N485A site-directed mutagenesis, the mutant shows no dehydrogenase activity Streptomyces sp.
V124A site-directed mutagenesis, the dehydrogenase/oxidase ratio is 30fold increased compared with the ratio for the wild-type enzyme Streptomyces sp.
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.
Y446A site-directed mutagenesis, the dehydrogenase/oxidase ratio is 3fold increased compared with the ratio for the wild-type enzyme Streptomyces sp.

Inhibitors

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

KM Value [mM]

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

Natural Substrates/ Products (Substrates)

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

Organism

Organism UniProt Comment Textmining
Streptomyces sp.
-
-
-

Purification (Commentary)

Purification (Comment) Organism
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]

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

Substrates and Products (Substrate)

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

Synonyms

Synonyms Comment Organism
ChOx
-
Streptomyces sp.
type I ChOx
-
Streptomyces sp.

Temperature Optimum [°C]

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

Turnover Number [1/s]

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

pH Optimum

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

Cofactor

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

kcat/KM [mM/s]

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