Literature summary for 1.1.3.6 extracted from

Qin, H.M.; Zhu, Z.; Ma, Z.; Xu, P.; Guo, Q.; Li, S.; Wang, J.W.; Mao, S.; Liu, F.; Lu, F.
Rational design of cholesterol oxidase for efficient bioresolution of cholestane skeleton substrates (2017), Sci. Rep., 7, 16375 .

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Protein Variants

Protein Variants	Comment	Organism
F70A	improved activities toward cholestane skeleton substrates. Improved activity toward beta-sitosterol, dehydroepiandrosterone, pregnenolone and stigmasterol	Pimelobacter simplex
F70L	the mutant enzyme shows almost the same level of activity as the wild-type enzyme	Pimelobacter simplex
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	Pimelobacter simplex
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	Pimelobacter simplex
V64I	improved activities toward cholestane skeleton substrates. Improved activity toward beta-sitosterol, dehydroepiandrosterone, pregnenolone and stigmasterol	Pimelobacter simplex
V64L	improved activities toward cholestane skeleton substrates. Improved activity toward beta-sitosterol, dehydroepiandrosterone, pregnenolone and stigmasterol	Pimelobacter simplex

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
0.15483	-	cholesterol	pH 7.5, temperature not specified in the publication, mutant enzyme F70V	Pimelobacter simplex
0.17831	-	cholesterol	pH 7.5, temperature not specified in the publication, mutant enzyme V64C	Pimelobacter simplex
0.17842	-	beta-sitosterol	pH 7.5, temperature not specified in the publication, mutant enzyme F70V	Pimelobacter simplex
0.19061	-	pregnenolone	pH 7.5, temperature not specified in the publication, mutant enzyme F70V	Pimelobacter simplex
0.20485	-	cholesterol	pH 7.5, temperature not specified in the publication, wild-type enzyme	Pimelobacter simplex
0.22076	-	beta-sitosterol	pH 7.5, temperature not specified in the publication, mutant enzyme V64C	Pimelobacter simplex
0.22842	-	pregnenolone	pH 7.5, temperature not specified in the publication, mutant enzyme V64C	Pimelobacter simplex
0.27352	-	dehydroepiandrosterone	pH 7.5, temperature not specified in the publication, mutant enzyme F70V	Pimelobacter simplex
0.29455	-	pregnenolone	pH 7.5, temperature not specified in the publication, wild-type enzyme	Pimelobacter simplex
0.31881	-	dehydroepiandrosterone	pH 7.5, temperature not specified in the publication, mutant enzyme V64C	Pimelobacter simplex
0.32861	-	beta-sitosterol	pH 7.5, temperature not specified in the publication, wild-type enzyme	Pimelobacter simplex
0.34121	-	dehydroepiandrosterone	pH 7.5, temperature not specified in the publication, wild-type enzyme	Pimelobacter simplex
0.38412	-	stigmasterol	pH 7.5, temperature not specified in the publication, mutant enzyme F70V	Pimelobacter simplex
0.52884	-	stigmasterol	pH 7.5, temperature not specified in the publication, mutant enzyme V64C	Pimelobacter simplex
0.78961	-	stigmasterol	pH 7.5, temperature not specified in the publication, wild-type enzyme	Pimelobacter simplex

Organism

Organism	UniProt	Comment	Textmining
Pimelobacter simplex	-	-	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
beta-sitosterol + O2	-	Pimelobacter simplex	? + H2O2	-	?
cholesterol + O2	-	Pimelobacter simplex	cholest-5-en-3-one + H2O2	-	?
dehydroepiandrosterone + O2	-	Pimelobacter simplex	androst-5-en-3,17-dione + H2O2	-	?
pregnenolone + O2	-	Pimelobacter simplex	5-pregnen-3,20-dione + H2O2	-	?
stigmasterol + O2	-	Pimelobacter simplex	stigmasta-5,22-dien-3-one + H2O2	-	?

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
25	-	-	Pimelobacter simplex

Temperature Stability [°C]

Temperature Stability Minimum [°C]	Temperature Stability Maximum [°C]	Comment	Organism
50	-	30 min, 25% residual activity	Pimelobacter simplex

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism	Structure
0.08	-	stigmasterol	pH 7.5, temperature not specified in the publication, mutant enzyme F70V	Pimelobacter simplex
1.58	-	stigmasterol	pH 7.5, temperature not specified in the publication, wild-type enzyme	Pimelobacter simplex
4.23	-	stigmasterol	pH 7.5, temperature not specified in the publication, mutant enzyme V64C	Pimelobacter simplex
6.82	-	dehydroepiandrosterone	pH 7.5, temperature not specified in the publication, wild-type enzyme	Pimelobacter simplex
9.56	-	dehydroepiandrosterone	pH 7.5, temperature not specified in the publication, mutant enzyme V64C	Pimelobacter simplex
9.86	-	beta-sitosterol	pH 7.5, temperature not specified in the publication, wild-type enzyme	Pimelobacter simplex
11.78	-	pregnenolone	pH 7.5, temperature not specified in the publication, wild-type enzyme	Pimelobacter simplex
12.29	-	cholesterol	pH 7.5, temperature not specified in the publication, wild-type enzyme	Pimelobacter simplex
13.25	-	beta-sitosterol	pH 7.5, temperature not specified in the publication, mutant enzyme V64C	Pimelobacter simplex
13.68	-	dehydroepiandrosterone	pH 7.5, temperature not specified in the publication, mutant enzyme F70V	Pimelobacter simplex
13.71	-	pregnenolone	pH 7.5, temperature not specified in the publication, mutant enzyme V64C	Pimelobacter simplex
15.25	-	pregnenolone	pH 7.5, temperature not specified in the publication, mutant enzyme F70V	Pimelobacter simplex
15.48	-	cholesterol	pH 7.5, temperature not specified in the publication, mutant enzyme F70V	Pimelobacter simplex
16.06	-	beta-sitosterol	pH 7.5, temperature not specified in the publication, mutant enzyme F70V	Pimelobacter simplex
23.02	-	cholesterol	pH 7.5, temperature not specified in the publication, mutant enzyme V64C	Pimelobacter simplex

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	-	Pimelobacter simplex

pH Range

pH Minimum	pH Maximum	Comment	Organism
5	9	more than 75% of the maximal activity is retained over the pH range of 5.0-9.0	Pimelobacter simplex

pH Stability

pH Stability	pH Stability Maximum	Comment	Organism
5.5	9	good pH stability over the pH range of 5.0-9.0	Pimelobacter simplex

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism	Structure
2	-	stigmasterol	pH 7.5, temperature not specified in the publication, wild-type enzyme	Pimelobacter simplex
8	-	stigmasterol	pH 7.5, temperature not specified in the publication, mutant enzyme V64C	Pimelobacter simplex
20	-	dehydroepiandrosterone	pH 7.5, temperature not specified in the publication, wild-type enzyme	Pimelobacter simplex
20	-	stigmasterol	pH 7.5, temperature not specified in the publication, mutant enzyme F70V	Pimelobacter simplex
30	-	beta-sitosterol	pH 7.5, temperature not specified in the publication, wild-type enzyme	Pimelobacter simplex
30	-	dehydroepiandrosterone	pH 7.5, temperature not specified in the publication, mutant enzyme V64C	Pimelobacter simplex
40	-	pregnenolone	pH 7.5, temperature not specified in the publication, wild-type enzyme	Pimelobacter simplex
50	-	dehydroepiandrosterone	pH 7.5, temperature not specified in the publication, mutant enzyme F70V	Pimelobacter simplex
60	-	cholesterol	pH 7.5, temperature not specified in the publication, wild-type enzyme	Pimelobacter simplex
60	-	pregnenolone	pH 7.5, temperature not specified in the publication, mutant enzyme V64C	Pimelobacter simplex
60	-	beta-sitosterol	pH 7.5, temperature not specified in the publication, mutant enzyme V64C	Pimelobacter simplex
80	-	pregnenolone	pH 7.5, temperature not specified in the publication, mutant enzyme F70V	Pimelobacter simplex
90	-	beta-sitosterol	pH 7.5, temperature not specified in the publication, mutant enzyme F70V	Pimelobacter simplex
100	-	cholesterol	pH 7.5, temperature not specified in the publication, mutant enzyme F70V	Pimelobacter simplex
130	-	cholesterol	pH 7.5, temperature not specified in the publication, mutant enzyme V64C	Pimelobacter simplex

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Release 2023.1 (January 2023)