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

Abstract

The heme-thiolate (cytochrome P450) enzyme which catalyzes the 14α- demethylation of obtusifoliol has been isolated from microsomes prepared from etiolated seedlings of Sorghum bicolor (L.) Moench. The obtusifoliol 14α- demethylase is a key enzyme in plant sterol biosynthesis and a target for the design of phyla-specific sterol 14α-demethylase inhibitors. Microsomal cytochrome P450s were solubilized by using the detergents Renex 690 and reduced Triton X-100, and the obtusifoliol 14α-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 14α-demethylase has an apparent molecular mass of 53 kDa and is estimated to constitute ≃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 14α-demethylase and sorghum NADPH-cytochrome P450 oxidoreductase demonstrated the conversion of obtusifoliol (4α, 14α- dimethyl-5α-ergosta-8,24(28)-dien-3β-ol) to 4α-methyl-5α-ergosta- 8,14,24(28)-trien-3β-ol, the 14α-demethylated product of obtusifoliol with a double bond introduced at the Δ14 position. The N-terminal amino acid sequence of the protein is MDLAD-IPQ/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 14α-demethylases which belong to the CYP51 family. Therefore it is conceivable that the obtusifoliol 14α-demethylase from plants also belongs to the CYP51 family, the only P450 family so far known to be conserved across the phyla.