Isolation and reconstitution of the heme-thiolate protein obtusifoliol 14 alpha-demethylase from Sorghum bicolor (L.) Moench

The heme-thiolate (cyochrome P450) enzyme which catalyzes the 14 alpha-demethylation of obtusifoliol has been isolated from microsomes prepared from etiolated seedlings of Sorghum bicolor (L.) Moench. The obtusifoliol 14 alpha-demethylase is a key enzyme in plant sterol biosynthesis and a target for the design of phyla-specific sterol 14 alpha-demethylase inhibitors. Microsomal cytochrome P450s were solubilized by using the detergents Renex 690 and reduced Triton X-100, and the obtusifoliol 14 alpha-demethylase was isolated by DEAE ion exchange and dye affinity column chromatography. The isolated enzyme has an absorption spectrum characteristic for low spin cytochrome P450s and produces a Type I binding spectrum with obtusifoliol as substrate. Binding spectra were not obtained with lanosterol, campesterol, sitosterol, or stigmasterol. Obtusifoliol 14 alpha-demethylase has an apparent molecular mass of 53 kDa and is estimated to constitute approximately 20% of the total cytochrome P450 content of the microsomal membranes and about 0.2% of the total microsomal protein. Gas chromatography-mass spectrometry analysis of reconstitution experiments with dilauroylphosphatidylcholine micelles containing isolated obtusifoliol 14 alpha-demethylase and sorghum NADPH-cytochrome P450 oxidoreductase demonstrated the conversion of obtusifoliol(4 alpha,14 alpha-demethyl-5 alpha-ergosta-8,24-(28)-dien-3 beta-ol) to 4 alpha-methyl-5 alpha-ergosta-8,14,24(28)-trien-3 beta-ol, the 14 alpha-demethylated product of obtusifoliol with a double bond introduced at the delta 14 position. The N-terminal amino acid sequence of the protein is MDLADIPQ/KQQRLMAGXALVV. Five internal sequences were obtained after endoproteinase Lys-C and Glu-C digestion. The fragment AAGAFSYISFGGGRH aligns with the unique heme binding domain of mammalian and yeast sterol 14 alphademethylases which belong to the CYP51 family. Therefore it is conceivable that the obtusifoliol 14 alpha-demethylase from plants also belongs to the CYP51 family, the only P450 family so far known to be conserved across the phyla.