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

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Abstract
Summary: 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 3β‐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‐chotesten‐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.