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首页> 外文期刊>The FEBS journal >Purification, kinetics, inhibitors and CD for recombinant p-amyrin synthase from Euphorbia tirucalli L and functional analysis of the DCTA motif, which is highly conserved among oxidosqualene cyclases
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Purification, kinetics, inhibitors and CD for recombinant p-amyrin synthase from Euphorbia tirucalli L and functional analysis of the DCTA motif, which is highly conserved among oxidosqualene cyclases

机译：大戟大戟重组p-amyrin合酶的纯化，动力学，抑制剂和CD以及DCTA基序的功能分析，该结构在氧化角鲨烯环化酶中高度保守

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摘要

Beta-Amyrin, a natural tritcrpcnc, is widely distributed in the plant kingdom, and its pcntacyclic skeleton is produced by oxidosqualcnc cyclase (OSC). OSC enzymes arc classified as membrane proteins, and they catalyze the polycyclization reaction of(3S)-2,3-oxidosqualcnc to yield nearly 150 different cyclic tritcrpcnc skeletons. To date, no report has described the successful purification and characterization of plant fi-amyrin synthase. The fi-amyrin synthase from Euphorbia lirucalli (EtAS) was expressed as a poly-histidinc-tagged protein in Saccharomyc.es cerevisiae GIL77, which lacks the lanostcrol synthase gene. The expression yield, determined by western blotting analysis, was 5-7 mg. By Ni~(21) -nitrilotriacctic acid affinity column chromatography and careful selection of the proper imidazole concentration during the purification processes of washing and clution, 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 mu m and 46.4 ± 0.68 min~(-1), respectively. To the best of our knowledge, there arc no reports describing both K_m and k_(cat) for OSCs except for two examples of rat and bovine lanostcrol synthases. The fi-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 lanostcrol synthases. Gel-filtration HPLC indicated that the OSC exists as a monomer, and the clutcd OSC retained its activity. Furthermore, the inhibition constants K_i and IC_(50) and types of inhibition by iminosqua-lcnc, Ro48-8071 and U18666A were determined, and indicated that imino-squalcnc and Ro48-8071 arc potent inhibitors. Additionally, this is the first report of the kinetic data of the mutated enzymes targeted forthe 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 theCD spectra of the wild-type and the mutated EtASs were almost the same, indicating that the protein architecture was not altered by these mutations.
机译：Beta-Amyrin是一种天然的Tritcrpcnc，广泛分布于植物界，其全环骨架是由氧化丝氨酸环化酶（OSC）产生的。 OSC酶被归类为膜蛋白，它们催化（3S）-2,3-oxidosqualcnc的多环化反应，产生近150个不同的环状tritcrpcnc骨架。迄今为止，尚无报道描述植物fi-amyrin合酶的成功纯化和鉴定。大戟大戟（EtAS）的fi-amyrin合酶在酿酒酵母GIL77中表达为带有多组氨酸标签的蛋白，该蛋白缺少羊毛甾醇合酶基因。通过蛋白质印迹分析确定的表达产量为5-7mg。通过Ni〜（21）-亚硝基三乙酸亲和柱色谱，并在洗涤和洗脱纯化过程中仔细选择合适的咪唑浓度，在SDS / PAGE上成功获得了一条条带。然后，我们测试了四种洗涤剂对酶活性的影响。浓度为0.05％的Triton X-100的补充产生最高的活性。最佳pH和温度分别为7.0和30°C。动力学参数K_m和k_（cat）分别确定为33.8±0.53μm和46.4±0.68 min〜（-1）。据我们所知，除大鼠和牛羊毛甾醇合酶的两个例子外，没有关于OSC的描述K_m和k_（cat）的报道。在这项研究中纯化的fi-amyrin合酶显示出比两种报道的羊毛甾醇合酶明显更高的催化效率（k_（cat）/ K_m）（-10〜3倍）。凝胶过滤HPLC表明OSC作为单体存在，并且成块的OSC保留了其活性。此外，测定了抑制常数K_i和IC_（50）以及亚氨基quanc，Ro48-8071和U18666A的抑制类型，表明亚氨基角鲨烯和Ro48-8071是有效的抑制剂。此外，这是针对DCTAE（485-489）主题的突变酶的动力学数据的首次报道，该DCTAE（485-489）主题是多环化反应的假定起始位点。没有发现D485N变体的活性，也没有发现C564A变体的活性明显降低，这表明酸性羧基残基Asp485充当质子供体来引发多环化反应，并且Cys564与羧基残基Asp458参与氢键形成。增强酸度。 CD光谱是OSCs的第一个报道，野生型和突变的EtAS的CD光谱几乎相同，表明这些突变不会改变蛋白质的结构。

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