Bacterial cholesterol oxidases are able to act as flavoprotein-linked ketosteroid monooxygenases that catalyse the hydroxylation of cholesterol to 4-cholesten-6-ol-3-one.
A new metabolite of cholesterol was found in reaction mixtures containing cholesterol or 4-cholesten-3-one as a substrate and extra- or intracellular protein extracts from recombinant Streptomyces lividans and Escherichia coli strains carrying cloned DNA fragments of Streptomyces sp. SA-COO, the producer of Streptomyces cholesterol oxidase. The new metabolite was identified as 4-cholesten-6-ol-3-one based on comparisons of its high-performance liquid chromatography, gas chromatography/mass spectrometry, infrared and proton-nuclear magnetic resonance spectra with those of an authentic standard. Genetic analyses showed that the enzyme responsible for the production of 4-cholesten-6-ol-3-one is cholesterol oxidase encoded by the choA gene. Commercially purified cholesterol oxidase (EC 1.1.3.6.) of a Streptomyces sp., as well as of Brevibacterium sterolicum and a Pseudomonas sp., and a highly purified recombinant Streptomyces cholesterol oxidase were also able to catalyse the 6-hydroxylation reaction. Hydrogen peroxide accumulating in the reaction mixtures as a consequence of the 3 beta-hydroxysteroid oxidase activity of the enzyme was shown to have no role in the formation of the 6-hydroxylated derivative. We propose a possible scheme of a branched reaction pathway for the concurrent formation of 4-cholesten-3-one and 4-cholesten-6-ol-3-one by cholesterol oxidase, and the observed differences in the rate of formation of the 6-hydroxy-ketosteroid by the enzymes of different bacterial sources are also discussed.
Molnár
I
I
Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, Japan.
Hayashi
N
N
Choi
K P
KP
Yamamoto
H
H
Yamashita
M
M
Murooka
Y
Y
D001673:Biodegradation, Environmental; D002384:Catalysis; D002783:Cholestenones / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism*; D002789:Cholesterol Oxidase / Q000302:isolation & purification / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000201:enzymology; D005420:Flavoproteins / Q000302:isolation & purification / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D006900:Hydroxylation; D007664:Ketosteroids / Q000378:metabolism; D010105:Oxygenases / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity
D002783:Cholestenones; D005420:Flavoproteins; D007664:Ketosteroids; D011994:Recombinant Proteins; C080484:4-cholesten-6-ol-3-one; C000475:cholest-4-en-3-one; D002784:Cholesterol; D006861:Hydrogen Peroxide; D002789:Cholesterol Oxidase; D010105:Oxygenases