Graduate School of Science and Technology, Niigata University, Nishi-ku, Niigata, Japan.
FEBS J. 2013 Mar;280(5):1267-80. doi: 10.1111/febs.12119. Epub 2013 Feb 13.
β-Amyrin, a natural triterpene, is widely distributed in the plant kingdom, and its pentacyclic skeleton is produced by oxidosqualene cyclase (OSC). OSC enzymes are classified as membrane proteins, and they catalyze the polycyclization reaction of (3S)-2,3-oxidosqualene to yield nearly 150 different cyclic triterpene skeletons. To date, no report has described the successful purification and characterization of plant β-amyrin synthase. The β-amyrin synthase from Euphorbia tirucalli (EtAS) was expressed as a polyhistidine-tagged protein in Saccharomyces cerevisiae GIL77, which lacks the lanosterol synthase gene. The expression yield, determined by western blotting analysis, was 5-7 mg. By Ni(2+) -nitrilotriacetic acid affinity column chromatography and careful selection of the proper imidazole concentration during the purification processes of washing and elution, a single band was successfully obtained on SDS/PAGE. We then tested the effects of four detergents on the enzyme activity. Supplementation with Triton X-100 at a concentration of 0.05% yielded the highest activity. The optimal pH and temperature were 7.0 and 30 °C, respectively. The kinetic parameters, K(m) and k(cat) , were determined to be 33.8 ± 0.53 μm and 46.4 ± 0.68 min(-1), respectively. To the best of our knowledge, there are no reports describing both K(m) and k(cat) for OSCs except for two examples of rat and bovine lanosterol synthases. The β-amyrin synthase purified in this study showed a significantly higher catalytic efficiency (k(cat)/K(m)) (~ 10(3)-fold) than those of the two reported lanosterol synthases. Gel-filtration HPLC indicated that the OSC exists as a monomer, and the eluted OSC retained its activity. Furthermore, the inhibition constants K(i) and IC(50) and types of inhibition by iminosqualene, Ro48-8071 and U18666A were determined, and indicated that iminosqualene and Ro48-8071 are potent inhibitors. Additionally, this is the first report of the kinetic data of the mutated enzymes targeted for the DCTAE(485-489) motif, which is a putative initiation site for the polycyclization reaction. No activity of the D485N variant and significantly decreased activity of the C564A variant were found, definitively demonstrating that the acidic carboxyl residue Asp485 serves as a proton donor to initiate the polycyclization reaction, and that Cys564 is involved in hydrogen bond formation with the carboxyl residue Asp458 to enhance the acidity. The CD spectrum is the first to be reported for OSCs, and the CD spectra of the wild-type and the mutated EtASs were almost the same, indicating that the protein architecture was not altered by these mutations.
β-香树脂醇，一种天然的三萜烯，广泛分布于植物界，其五环骨架由角鲨烯环化酶（OSC）产生。OSC 酶被归类为膜蛋白，它们催化（3S）-2,3-氧化鲨烯的多环化反应，生成近 150 种不同的环状三萜烯骨架。迄今为止，尚无报道描述植物 β-香树脂醇合酶的成功纯化和表征。从Euphorbia tirucalli（EtAS）表达的β-香树脂醇合酶作为聚组氨酸标记蛋白在缺乏羊毛甾醇合酶基因的酿酒酵母 GIL77 中表达。通过 Western blot 分析确定表达产量为 5-7mg。通过 Ni(2+)-次氮基三乙酸亲和柱层析和在纯化过程中仔细选择适当的咪唑浓度进行洗涤和洗脱，在 SDS/PAGE 上成功获得了单一条带。然后我们测试了四种去污剂对酶活性的影响。在 0.05%的 Triton X-100 浓度下添加可获得最高的活性。最佳 pH 值和温度分别为 7.0 和 30°C。确定动力学参数 K(m)和 k(cat)分别为 33.8±0.53μm 和 46.4±0.68min(-1)。据我们所知，除了大鼠和牛羊毛甾醇合酶的两个例子外，没有关于 OSC 的 K(m)和 k(cat)的报道。本研究中纯化的β-香树脂醇合酶显示出明显更高的催化效率（k(cat)/K(m)）（~10(3)-倍），高于报道的两种羊毛甾醇合酶。凝胶过滤 HPLC 表明 OSC 以单体形式存在，洗脱的 OSC 保持其活性。此外，还确定了异牟替莫烷、Ro48-8071 和 U18666A 的抑制常数 K(i)和 IC(50)以及抑制类型，并表明异牟替莫烷和 Ro48-8071 是有效的抑制剂。此外，这是针对 DCTAE(485-489)基序的突变酶的动力学数据的首次报道，该基序是多环化反应的潜在起始位点。未发现 D485N 变体的活性，C564A 变体的活性显著降低，这明确表明酸性羧基残基天冬氨酸 485 作为质子供体启动多环化反应，半胱氨酸 564 参与与羧基残基天冬氨酸 458 的氢键形成，以增强酸性。CD 光谱是首次报道的 OSC，野生型和突变型 EtASs 的 CD 光谱几乎相同，表明这些突变未改变蛋白质结构。
