Literature summary for 1.1.3.6 extracted from

Golden, E.; Karton, A.; Vrielink, A.
High-resolution structures of cholesterol oxidase in the reduced state provide insights into redox stabilization (2014), Acta Crystallogr. Sect. D, 70, 3155-3166 .

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of His6-tagged enzyme in Escherichia coli train BL21(DE3)	Streptomyces sp. SA-COO

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified nontagged and His6-tagged enzyme reduced with dithionite under aerobic conditions and in the presence of the substrate 2-propanol under both aerobic and anaerobic conditions, vapour diffusion using the hanging-drop method, mixing of 7 mg/ml protein in 50 mM HEPES pH 7.0 with reservoir solution containing 7% PEG 8000, 100 mM cacodylate, pH 5.2, 125 mM MnSO4, followed by microseeding in 12% PEG 8000, 100 mM sodium cacodylate, pH 5.2, 125 mM MnSO4, X-ray diffraction structure determination and analysis at 1.12-1.34 A resolution	Streptomyces sp. SA-COO

Crystallization (Comment)

Organism

purified nontagged and His6-tagged enzyme reduced with dithionite under aerobic conditions and in the presence of the substrate 2-propanol under both aerobic and anaerobic conditions, vapour diffusion using the hanging-drop method, mixing of 7 mg/ml protein in 50 mM HEPES pH 7.0 with reservoir solution containing 7% PEG 8000, 100 mM cacodylate, pH 5.2, 125 mM MnSO4, followed by microseeding in 12% PEG 8000, 100 mM sodium cacodylate, pH 5.2, 125 mM MnSO4, X-ray diffraction structure determination and analysis at 1.12-1.34 A resolution

Streptomyces sp. SA-COO

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
cholesterol + O2	Streptomyces sp. SA-COO	via cholest-5-en-3-one	cholest-4-en-3-one + H2O2	-	?
additional information	Streptomyces sp. SA-COO	cholesterol oxidase is a bifunctional flavoenzyme that catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one	?	-	?

Organism

Organism	UniProt	Comment	Textmining
Streptomyces sp. SA-COO	P12676	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His6-tagged enzyme from Escherichia coli train BL21(DE3) by nickel affinity chromatography, dialysis and ultrafiltration	Streptomyces sp. SA-COO

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
cholesterol + O2	via cholest-5-en-3-one	Streptomyces sp. SA-COO	cholest-4-en-3-one + H2O2	-	?
additional information	cholesterol oxidase is a bifunctional flavoenzyme that catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one	Streptomyces sp. SA-COO	?	-	?
additional information	the enzyme catalyzes the oxidation and isomerization of steroids containing a 3-hydroxyl group, with the cholestane group conferring binding specificity in the active site. The enzyme is also able to oxidize small-molecule alcohols such as 2-propanol and methanol, with a preference for those containing aromatic ring, but with lower activity. Substrate oxidation results in reduction of the cofactor during the reductive half reaction. An oxidative half reaction occurs with re-oxidation of the flavin cofactor by molecular oxygen to form hydrogen peroxide. Structure-function relationship and analysis overview	Streptomyces sp. SA-COO	?	-	?

Cofactor

Cofactor	Comment	Organism	Structure
FAD	the structure of the dithionite-reduced enzyme reveals a sulfite molecule covalently bound to the FAD cofactor. The hydride transfer generates a tetrahedral geometry about the flavin N5 atom	Streptomyces sp. SA-COO

General Information

General Information	Comment	Organism
evolution	the type I CO from Streptomyces sp. SA-COO is a member of the glucosemethanolcholine (GMC) oxidoreductase family and contains a single molecule of flavin adenine dinucleotide (FAD) noncovalently but tightly bound to the protein	Streptomyces sp. SA-COO
additional information	the structure of the dithionite-reduced enzyme reveals a sulfite molecule covalently bound to the FAD cofactor. The isoalloxazine ring system displays a bent structure relative to that of the oxidized enzyme, and alternate conformations of a triad of aromatic residues near to the cofactor are evident. The strutcure of anaerobically trapped reduced enzyme structure in the presence of 2-propanol does not show a similar bending of the flavin ring system, but does show alternate conformations of the aromatic triad. The hydride transfer generates a tetrahedral geometry about the flavin N5 atom	Streptomyces sp. SA-COO