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 14␣-demethylation of obtusifoliol has been isolated from microsomes prepared from etiolated seedlings of Sorghum bicolor (L.) Moench. The obtusifoliol 14␣-demethylase is a key enzyme in plant sterol biosynthesis and a target for the design of phyla-specific sterol 14␣-demethylase inhibitors. Microsomal cytochrome P450s were solubilized by using the detergents Renex 690 and reduced Triton X-100, and the obtusifoliol 14␣-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␣-demethylase has an apparent molecular mass of 53 kDa and is estimated to constitute Ϸ20% 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␣-demethylase and sorghum NADPHcytochrome P450 oxidoreductase demonstrated the conversion of obtusifoliol (4␣,14␣-dimethyl-5␣-ergosta-8,24-(28)-dien-3␤-ol) to 4␣-methyl-5␣-ergosta-8,14,24(28)-trien-3␤-ol, the 14␣-demethylated product of obtusifoliol with a double bond introduced at the ⌬ 14 position. The Nterminal 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 14␣-demethylases which belong to the CYP51 family. Therefore it is conceivable that the obtusifoliol 14␣-demethylase from plants also belongs to the CYP51 family, the only P450 family so far known to be conserved across the phyla.

Sterol 14α-demethylase cytochrome P450 (CYP51), a P450 in all biological kingdoms

“…The activity of the single characterized CYP51 from filamentous fungi (U. maydis) was reconstituted with its physiological substrate (24, 25-methylenedihydrolanosterol) only [35]. CYP51 from the flowering plant S. bicolor is highly specific toward the C4-monomethylated sterols, the turnover number with the physiological substrate obtusifoliol being the highest (36), which is in good agreement with the results of the earlier work on characterization of this CYP51, yet in this earlier work norlanosterol was not tested [12]. Strict preference toward obtusifoliol was also reported for membrane fractions containing CYP51 orthologs from maize [39] and wheat [40] and thus it was considered a feature general for all plant orthologs until recently, when the ability of CYP51 from S. chacoense (chaco potato) to metabolize lanosterol (though at a rate considerably lower than that with obtusifoliol) was reported [41].…”

“…Amongst them 9 are microsomal, eukaryotic enzymes (including 3 mammalian: rat [7], pig [32], and human [33]; 1 plant: S. bicolor [12], 3 fungal: 2 from yeasts (S. cerevisiae [8], Candida albicans [34]), and one from filamentous fungus Ustilago maydis [35] and 2 trypanosomal (Trypanosoma brucei [27] and T. cruzi [36]) while the other 3 are water soluble bacterial orthologs (from M. tuberculosis [14], M. smegmatis [37], and a fusion CYP51-ferredoxin protein from Methylococcus capsulatus [38]). …”

Isolation and Reconstitution of Cytochrome P450ox and in Vitro Reconstitution of the Entire Biosynthetic Pathway of the Cyanogenic Glucoside Dhurrin from Sorghum

“…Under the experimental conditions used, CYP79 and CYP51 (obtusifoliol 14a-demethylase) had previously been shown to bind to the ion-exchange column. (Sibbesen et al, 1994;Kahn et al, 1996). Unexpectedly, P450ox was not retained on the column, but co-eluted with the yellow pigments in the run-through and wash fractions.…”

“…The protein band corresponding to the P450 isolated from the Cibacron blue 3GA-agarose column was excised from 8 to 25% SDS-polyacrylamide gels and electroeluted as previously described (Kahn et al, 1996). The electroeluted protein was digested with endoproteinase Glu-C (protease V8 sequencing grade, 18 h, 23°C) according to the manufacturer (Boehringer Mannheim) using an approximately 1:lOO weight ratio between proteinase and protein.…”

“…The electroeluted protein and the digested protein sample were subjected to SDS-PAGE, and the protein and fragments were transferred to ProBlott membranes (Applied Biosystems) as described previously (Kahn et al, 1996). Coomassie blue-stained regions of the membrane were excised and subjected to N-terminal amino acid sequencing on a sequenator (model 470A, Applied Biosystems) equipped with an online phenylthiohydantoin amino acid analyzer (model 120A, Applied Biosystems).…”