Abstract
Oxidization of cholesterol by cholesterol oxidase (ChOx) is thought to the initiation step in cholesterol translocation and degradation. However, the mechanism of cholesterol translocation in mycobacteria have not been thoroughly investigated. Previously, we analyzed mycobacteria phytosterol metabolism pathway in Mycobacterium HGMS2 strain we identified three enzymes, i.e., ChoM1, ChoM2 and HsD, which could oxidized cholesterol oxidization. In this work, we evaluated the activities of the three enzymes in vitro. The three enzymes exhibited different abilities to oxidize cholesterol as ranking by ChoM1≫HsD>ChoM2. Furthermore, we employed gene knockout approach to verify the functional role of the three enzymes. Unexpectedly, a triple mutant that deleted these three genes still enabled to oxidized cholesterol as the wild-type HGMS2 strain, implying that the mycobacterial stain contained unexplored enzymes for cholesterol oxidization. As such, we used structural biology, bioinformatic and enzymatic strategies to search for new cholesterol oxidases. The results and discussion on mechanism of cholesterol translocation in mycobacteria will be presented in this report.
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