Kahn R A, Bak S, Olsen C E, Svendsen I, Moller B L
Plant Biochemistry Laboratory, Department of Plant Biology, The Royal Veterinary and Agricultural University, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark.
J Biol Chem. 1996 Dec 20;271(51):32944-50. doi: 10.1074/jbc.271.51.32944.
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.
已从高粱（Sorghum bicolor (L.) Moench）黄化幼苗制备的微粒体中分离出催化钝叶醇14α-去甲基化反应的血红素硫醇盐（细胞色素P450）酶。钝叶醇14α-去甲基化酶是植物甾醇生物合成中的关键酶，也是设计门特异性甾醇14α-去甲基化酶抑制剂的作用靶点。使用洗涤剂Renex 690和还原型 Triton X-100使微粒体细胞色素P450溶解，然后通过DEAE离子交换和染料亲和柱色谱法分离钝叶醇14α-去甲基化酶。分离得到的酶具有低自旋细胞色素P450的吸收光谱特征，并以钝叶醇为底物产生I型结合光谱。以羊毛甾醇、菜油甾醇、谷甾醇或豆甾醇为底物未获得结合光谱。钝叶醇14α-去甲基化酶的表观分子量为53 kDa，估计约占微粒体膜细胞色素P450总含量的20%，约占微粒体总蛋白的0.2%。用含有分离的钝叶醇14α-去甲基化酶和高粱NADPH-细胞色素P450氧化还原酶的二月桂酰磷脂酰胆碱胶束进行重组实验的气相色谱-质谱分析表明，钝叶醇（4α,14α-二甲基-5α-麦角甾-8,24(28)-二烯-3β-醇）转化为4α-甲基-5α-麦角甾-8,14,24(28)-三烯-3β-醇，即钝叶醇的14α-去甲基化产物，在Δ14位引入了一个双键。该蛋白质的N端氨基酸序列为MDLADIPQ/KQQRLMAGXALVV。经内肽酶Lys-C和Glu-C消化后获得了五个内部序列。片段AAGAFSYISFGGGRH与属于CYP51家族的哺乳动物和酵母甾醇14α-去甲基化酶独特的血红素结合结构域比对。因此，可以推测植物中的钝叶醇14α-去甲基化酶也属于CYP51家族，这是目前已知的唯一一个在各门中保守的P450家族。
