Isolation and reconstitution of the heme-thiolate protein obtusifoliol 14alpha-demethylase from Sorghum bicolor (L.) Moench.
The heme-thiolate (cytochrome P450) enzyme which catalyzes the 14alpha-demethylation of obtusifoliol has been isolated from microsomes prepared from etiolated seedlings of Sorghum bicolor (L.) Moench. The obtusifoliol 14alpha-demethylase is a key enzyme in plant sterol biosynthesis and a target for the design of phyla-specific sterol 14alpha-demethylase inhibitors. Microsomal cytochrome P450s were solubilized by using the detergents Renex 690 and reduced Triton X-100, and the obtusifoliol 14alpha-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 14alpha-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 14alpha-demethylase and sorghum NADPHcytochrome P450 oxidoreductase demonstrated the conversion of obtusifoliol (4alpha,14alpha-dimethyl-5alpha-ergosta-8, 24(28)-dien-3beta-ol) to 4alpha-methyl-5alpha-ergosta-8,14, 24(28)-trien3beta-ol, the 14alpha-demethylated product of obtusifoliol with a double bond introduced at the Delta14 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 14alpha-demethylases which belong to the CYP51 family. Therefore it is conceivable that the obtusifoliol 14alpha-demethylase from plants also belongs to the CYP51 family, the only P450 family so far known to be conserved across the phyla.
Kahn
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Plant Biochemistry Laboratory, Department of Plant Biology, The Royal Veterinary and Agricultural University, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark.
Bak
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Olsen
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Svendsen
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Moller
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D002248:Carbon Monoxide; D002775:Cholestadienols / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification* / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D006420:Hemeproteins / Q000302:isolation & purification / Q000378:metabolism; D008861:Microsomes / Q000201:enzymology; D010088:Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D010940:Plant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013025:Glycine max / Q000201:enzymology*; D013057:Spectrum Analysis; D058886:Sterol 14-Demethylase
D002775:Cholestadienols; D006420:Hemeproteins; D010940:Plant Proteins; C008693:obtusifoliol; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D058886:Sterol 14-Demethylase