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Sterol 14 ␣ -demethylase (14DM) is a cytochrome P-450 involved in sterol biosynthesis in eukaryotes. It was reported that Mycobacterium smegmatis also makes cholesterol and that cholesterol is essential to Mycobacterium tuberculosis (MT) infection, although the origin of the cholesterol is unknown. A protein product from MT having about 30% sequence identity with eukaryotic 14 ␣ -demethylases has been found to convert sterols to their 14-demethyl products indicating that a sterol pathway might exist in MT. To determine the optimal sterol structure recognized by MT 14DM, binding of 28 sterol and sterol-like (triterpenoids) molecules to the purified recombinant 14 ␣ -demethylase was examined. Like eukaryotic forms, a 3 ␤ -hydroxy group and a 14 ␣ -methyl group are essential for substrate acceptability by the bacterial 14 ␣ -demethylase. The high affinity binding of 31-norcycloartenol without detectable activity indicates that the ⌬ 8 -bond is required for activity but not for binding. As for plant 14 ␣ -demethylases, 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 ␣demethylases.Thus, substrate binding to MT 14DM seems to share common features with all eukaryotic 14 ␣demethylases, the MT form seemingly having the broadest substrate recognition of all forms of 14 ␣ -demethylase studied so far. -Bellamine, A., A. T. Mangla, A. L. Dennis, W. D. Nes, and M. R. Waterman. Structural requirements for substrate recognition of Mycobacterium tuberculosis 14 ␣ -demethylase: implications for sterol biosynthesis.

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