Structural requirements for substrate recognition of Mycobacterium tuberculosis 14 alpha-demethylase: implications for sterol biosynthesis

Sterol 14 alpha -demethylase (14DM) is a cytochrome P-450 involved in stero l biosynthesis in eukaryotes, It was reported that Mycobacterium smegmatis also makes cholesterol and that cholesterol is essential to Mycobacterium t uberculosis (MT) infection, although the origin of the cholesterol is unkno wn. A protein product from MT having about 30% sequence identity with eukar yotic 14 alpha -demethylases has been found to convert sterols to their 14- demethyl products indicating that a sterol pathway might exist in MT: To de termine the optimal sterol structure recognized by MT 14DM, binding of 28 s terol and sterol-like (triterpenoids) molecules to the purified recombinant 14 alpha -demethylase was examined, Like eukaryotic forms, a 3 beta -hydro xy group and a 14 alpha -methyl group are essential for substrate acceptabi lity by the bacterial 14 alpha -demethylase. The high affinity binding of 3 1-norcycloartenol without detectable activity indicates that the Delta (8)- bond is required for activity but not for binding. As for plant 14 alpha -d emethylases, 31-nor-sterols show a binding preference for MT 14DM, Similar to enzymes from mammals and yeast, a C24-alkyl group is not required for MT 14DM binding and activity, whereas it is for plant 14 alpha -demethylases. Thus, substrate binding to MT 14DM seems to share common features with all eukaryotic 14 alpha -demethylases, the MT form seemingly having the broade st substrate recognition of all forms of 14 alpha -demethylase studied so f ar.