Cholesterol to cholestenone oxidation by ChoG, the main extracellular cholesterol oxidase of Rhodococcus ruber strain Chol-4

• Published in: The Journal of steroid biochemistry and molecular biology Vol. 139; pp. 33 - 44
• Main Authors: Fernández de las Heras, Laura, Perera, Julián, Navarro Llorens, Juana María
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.01.2014
• Subjects: Amino Acid Sequence; Bacterial Proteins - biosynthesis; Bacterial Proteins - genetics; Biocatalysis; Cholestenone; Cholestenones - metabolism; Cholesterol - metabolism; Cholesterol oxidase; Cholesterol Oxidase - biosynthesis; Cholesterol Oxidase - genetics; Cloning, Molecular; Enzyme Induction; Gene Expression; Gene Expression Regulation, Bacterial; Molecular Sequence Data; Oxidation-Reduction; Phylogeny; Promoter Regions, Genetic; Rhodococcus; Rhodococcus - enzymology; Rhodococcus - genetics; Rhodococcus - growth & development; Rhodococcus ruber; Sequence Deletion; Steroids; Rhodococcus; Cholesterol oxidase; Cholestenone; Steroids
• ISSN: 0960-0760; 1879-1220
• DOI: 10.1016/j.jsbmb.2013.10.001

•Several putative cholesterol oxidase genes have been found in the genome of R. ruber.•ChoG is the only cholesterol oxidase that transforms cholesterol to cholestenone outside the cell.•The choG basal transcription levels are increased after induction with cholesterol.•The choG mutant strain displays a slower growth rate in cholesterol compared to the WT.•Cholestenone accumulation observed in cultures of R. ruber WT does not occur in the choG mutant. The choG ORF of Rhodococcus ruber strain Chol-4 (referred from now as Chol-4) encodes a putative extracellular cholesterol oxidase. In the Chol-4 genome this ORF is located in a gene cluster that includes kstD3 and hsd4B, showing the same genomic context as that found in other Rhodococcus species. The putative ChoG protein is grouped into the class II of cholesterol oxidases, close to the Rhodococcus sp. CECT3014 ChoG homolog. The Chol-4 choG was cloned and expressed in a CECT3014 ΔchoG host strain in order to assess its ability to convert cholesterol into cholestenone. The RT-PCR analysis showed that choG gene was constitutively expressed in all the conditions assayed, but a higher induction could be inferred when cells were growing in the presence of cholesterol. A Chol-4 ΔchoG mutant strain was still able to grow in minimal medium supplemented with cholesterol, although at a slower rate. A comparative study of the removal of both cholesterol and cholestenone from the culture medium of either the wild type Chol-4 or its choG deletion mutant revealed a major role of ChoG in the extracellular production of cholestenone from cholesterol and, therefore, this enzyme may be related with the maintenance of a convenient supply of cholestenone for the succeeding steps of the catabolic pathway.