The first step in the catabolism of
cholesterol, i.e. the transformation of
cholesterol into cholestenone, has been investigated in Mycobacterium smegmatis. In silico analysis identified the MSMEG_1604 gene encoding a putative
protein similar to the ChoD
cholesterol oxidase of M.
tuberculosis H37Rv (Rv3409c) and the MSMEG_5228 gene coding for a
protein similar to the
NAD(P)-dependent
cholesterol dehydrogenase/
isomerase of Nocardia sp. The expression of the MSMEG_5228 gene was inducible by
cholesterol whereas the expression of MSMEG_1604 gene was constitutive. When both genes were expressed in Escherichia coli only the MSMEG_5228
protein was active on
cholesterol. The function of ChoD-like MSMEG_1604
protein remains to be elucidated, but it does not appear to play a critical role in the mineralization of
cholesterol as a MSMEG_1604(-) mutant was not affected in the production of cholestenone. However, a MSMEG_5228(-) mutant showed a drastic reduction in the synthesis of cholestenone. The finding that this mutant was still able to grow in
cholesterol, allowed us to demonstrate that the
cholesterol-inducible MSMEG_5233 gene encodes an additional
cholesterol dehydrogenase/
isomerase similar to the AcmA
dehydrogenase of Sterolibacterium denitrificans. The observation that the double MSMEG_5228-5233(-) mutant was able to grow in
cholesterol suggests that in addition to these
enzymes other
dehydrogenase/
isomerases can also catalyse the first reaction of the
cholesterol degradation pathway in M. smegmatis, which is not the limiting step of the process.