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1.1.3.6: cholesterol oxidase

This is an abbreviated version!
For detailed information about cholesterol oxidase, go to the full flat file.

Word Map on EC 1.1.3.6

Reaction

cholesterol
+
O2
=
cholest-5-en-3-one
+
H2O2

Synonyms

3beta-hydroxy steroid oxidoreductase, 3beta-hydroxysteroid: oxygen oxidoreductase, 3beta-hydroxysteroid:oxygen oxidoreductase, 3beta-hydroxysterol oxidase, BsChOx, CgChoA, CHO, CHO-U, ChO3, ChoA, choBb, CHOD, ChoG, ChoL, cholesterol oxidase, cholesterol oxidase I, cholesterol oxidase II, cholesterol-O2 oxidoreductase, CHOLOX, choM, ChoM1, ChoM2, choP, ChoRI, ChoRII, ChoS, ChOx, CO, CO1, COase, COD, COD-B, COX, HCEO-forming enzyme, HMPREF0204_11499, oxidase, cholesterol, PimE, ShChOx, type I ChOx

ECTree

     1 Oxidoreductases
         1.1 Acting on the CH-OH group of donors
             1.1.3 With oxygen as acceptor
                1.1.3.6 cholesterol oxidase

Engineering

Engineering on EC 1.1.3.6 - cholesterol oxidase

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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E311D
-
mutations of Glu311 cause a switch in the basic kinetic mechanism of the reaction of reduced cholesterol oxidase with dioxygen: wild-type cholesterol oxidase shows a saturation behavior with increasing oxygen concentration, while for Glu311 mutants a linear dependence is found that is assumed to reflect a simple second order process
E311L
-
mutations of Glu311 cause a switch in the basic kinetic mechanism of the reaction of reduced cholesterol oxidase with dioxygen: wild-type cholesterol oxidase shows a saturation behavior with increasing oxygen concentration, while for Glu311 mutants a linear dependence is found that is assumed to reflect a simple second order process
E311Q
-
mutations of Glu311 cause a switch in the basic kinetic mechanism of the reaction of reduced cholesterol oxidase with dioxygen: wild-type cholesterol oxidase shows a saturation behavior with increasing oxygen concentration, while for Glu311 mutants a linear dependence is found that is assumed to reflect a simple second order process
F70A
-
improved activities toward cholestane skeleton substrates. Improved activity toward beta-sitosterol, dehydroepiandrosterone, pregnenolone and stigmasterol
F70L
-
the mutant enzyme shows almost the same level of activity as the wild-type enzyme
F70V
-
mutation increases the binding free energy between the mutant enzyme (PsChO) and cholesterol and might cause the movement of loops L56-P77, K45-P49 and L350-E354 at active site. Improved activities toward cholestane skeleton substrates. 50% higher activities toward cholesterol when compared with that of the wild-type enzyme
V64C
-
mutation increases the binding free energy between the mutant enzyme (PsChO) and cholesterol and might cause the movement of loops L56-P77, K45-P49 and L350-E354 at active site. Improved activities toward cholestane skeleton substrates. 50% higher activities toward cholesterol when compared with that of the wild-type enzyme
V64I
-
improved activities toward cholestane skeleton substrates. Improved activity toward beta-sitosterol, dehydroepiandrosterone, pregnenolone and stigmasterol
V64L
-
improved activities toward cholestane skeleton substrates. Improved activity toward beta-sitosterol, dehydroepiandrosterone, pregnenolone and stigmasterol
E361A
-
site-directed mutagenesis, the mutant shows no dehydrogenase activity
E361Q
F359A
-
site-directed mutagenesis, the dehydrogenase/oxidase ratio is 12fold increased compared with the ratio for the wild-type enzyme
F359W
kcat for the F359W mutant-catalyzed reaction decreases 13fold relative to that of the wild-type-catalyzed reaction. Transfer of hydride from the sterol to the flavin prosthetic group is no longer rate-limiting for the mutant enzyme. Kinetic cooperativity with respect to molecular oxygen is observed with the tunnel mutant, but not with the wild-type enzyme. In the atomic-resolution structure of F359W, the indole ring of the tryptophan completely fills the tunnel and is observed in only a single conformation. The size of the indole is proposed to limit conformational rearrangement of residue 359 that leads to tunnel opening in the wild-type enzyme
F444A
-
site-directed mutagenesis, the dehydrogenase/oxidase ratio is 4fold increased compared with the ratio for the wild-type enzyme
G347N
mutant can not be saturated with oxygen. Transfer of hydride from the sterol to the flavin prosthetic group is no longer rate-limiting for the mutant enzyme. Kinetic cooperativity with respect to molecular oxygen is observed with the tunnel mutant, but not with the wild-type enzyme
H447E
-
turnover number for cholesterol is decreased about 2600fold compared to wild-type enzyme
H447E/E361Q
-
turnover number for cholesterol is decreased about 31500fold compared to wild-type enzyme. Mutant enzyme is not able to isomerize cholest-5-en-3-one. Mutant enzyme is folded like native enzyme and still associates with model membranes
H447N
H447Q mutant shows wild-type activity in isomerization, kcat for oxidation reaction is reduced 120fold, Km increases 2fold compared to wild-type, E361Q mutant has no isomerization activity, kcat is 30fold slower than for wild-type, Glu361 may therefore act as general base in oxidation reaction
H447Q
H447Q/E361Q
-
turnover number for cholesterol is decreased 473fold compared to wild-type enzyme, 1.7fold increase in Km-value for cholesterol compared to wild-type enzyme. Mutant enzyme is not able to isomerize cholest-5-en-3-one
L117P
-
mutant enzyme without activity
L119A
-
specific activity is similar to that of the wild-type enzyme
L119F
-
specific activity is similar to that of the wild-type enzyme
M122A
-
site-directed mutagenesis, the dehydrogenase/oxidase ratio is 26fold increased compared with the ratio for the wild-type enzyme
N485A
-
site-directed mutagenesis, the mutant shows no dehydrogenase activity
N485D
N485L
the kcat of N485D is diminished about 1110times compared with that of wild type, while the apparent Km value is minimally affected
P357N
-
specific activity is similar to that of the wild-type enzyme
Q286R
-
specific activity is similar to that of the wild-type enzyme
R135H
-
random mutagenesis
S103T
-
random mutagenesis
S279R
-
mutant enzyme without activity on cholesterol and pregnenolone
S379D
-
mutant enzyme without activity on cholesterol and pregnenolone
S379T
-
specific activity is similar to that of the wild-type enzyme
V121A
-
random mutagenesis
V124A
-
site-directed mutagenesis, the dehydrogenase/oxidase ratio is 30fold increased compared with the ratio for the wild-type enzyme
V145E
-
random mutagenesis
V145Q
-
specific activity is similar to that of the wild-type enzyme
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
Y446A
-
site-directed mutagenesis, the dehydrogenase/oxidase ratio is 3fold increased compared with the ratio for the wild-type enzyme
A184C/A301C/T394C
kcat/KM is comparable to wild-type value
A184C/T239C/A407C/A465C
kcat/KM is 1.1fold lower than wild-type value
A32C/S129C/T371C/A423C
kcat/KM is 2.55fold lower than wild-type value
A32C/T168C/S312C/A465C
kcat/KM is 2.1fold lower than wild-type value
His447Gln/H447N
mutations cause notable decreases in the catalytic activity
L274C
kcat/KM is 1.13fold lower than wild-type value
L80C
kcat/KM is 1.6fold higher than wild-type value
S153C/A205C/S312C/T435C
kcat/KM is 1.4fold higher than wild-type value
T168C/A276C
kcat/KM is 1.5fold lower than wild-type value
additional information