Molecular diversity of sterol 14α‐demethylase substrates in plants, fungi and humans

Abstract

Metabolism of lanosterol (LAN), 24-methylene-24,25-dihydrolanosterol (24-methyleneDHL), dihydrolanosterol (DHL) and obtusifoliol (OBT) by purified human, plant ( Sorghum bicolor ) and fungal ( Candida albicans ) sterol 14α-demethylase (CYP51; P450 14DM ) reconstituted with NADPH cytochrome P450 reductases was studied in order to elucidate the substrate specificity and sterol stereo- and regio-structural requirements for optimal CYP51 activity. Both human and C. albicans CYP51 could catalyse 14α-demethylation of each substrate with varying levels of activity, but having slightly higher activity for their respective endogenous substrates in vivo, dihydrolanosterol for human CYP51 ( V max =0.5 nmol/min/nmol CYP51) and 24-methylene-24,25-dihydrolanosterol for C. albicans CYP51 ( V max =0.3 nmol/min/nmol CYP51). In contrast, S. bicolor CYP51 showed strict substrate specificity and selectivity towards its own endogenous substrate, obtusifoliol ( V max =5.5 nmol/min/nmol CYP51) and was inactive towards 14α-demethylation of lanosterol, 24-methylene-24,25-dihydrolanosterol and dihydrolanosterol. These findings confirm that the presence of the 4β-methyl group in the sterol molecule renders the plant CYP51 incapable of 14α-demethylation thus revealing the strict active site conservation of plant CYP51 during evolution.