Molecular diversity of sterol 14alpha-demethylase substrates in plants, fungi and humans.
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 14alpha-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 14alpha-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 (Vmax = 0.5 nmol/min/nmol CYP51) and 24-methylene-24,25-dihydrolanosterol for C. albicans CYP51 (Vmax = 0.3 nmol/min/nmol CYP51). In contrast, S. bicolor CYP51 showed strict substrate specificity and selectivity towards its own endogenous substrate, obtusifoliol (Vmax = 5.5 nmol/min/nmol CYP51) and was inactive towards 14alpha-demethylation of lanosterol, 24-methylene-24,25-dihydrolanosterol and dihydrolanosterol. These findings confirm that the presence of the 4beta-methyl group in the sterol molecule renders the plant CYP51 incapable of 14alpha-demethylation thus revealing the strict active site conservation of plant CYP51 during evolution.
Lamb
D C
DC
Institute of Biological Sciences, University of Wales Aberystwyth, UK.
Kelly
D E
DE
Kelly
S L
SL
D002176:Candida albicans / Q000201:enzymology*; D002775:Cholestadienols / Q000737:chemistry / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D002523:Edible Grain / Q000201:enzymology*; D006801:Humans; D007810:Lanosterol / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D015394:Molecular Structure; D010088:Oxidoreductases / Q000378:metabolism*; D058886:Sterol 14-Demethylase; D013379:Substrate Specificity
C548418:CYP51A1 protein, human; D002775:Cholestadienols; D007810:Lanosterol; C008693:obtusifoliol; C014520:ebericol; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D058886:Sterol 14-Demethylase