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首页> 外文期刊>The FEBS journal >The 3-ureidopropionase of Caenorhabditis elegans, an enzyme involved in pyrimidine degradation.
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The 3-ureidopropionase of Caenorhabditis elegans, an enzyme involved in pyrimidine degradation.

机译：秀丽隐杆线虫的3-脲基丙酸酶，一种参与嘧啶降解的酶。

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摘要

Pyrimidines are important metabolites in all cells. Levels of cellular pyrimidines are controlled by multiple mechanisms, with one of these comprising the reductive degradation pathway. In the model invertebrate Caenorhabditis elegans, two of the three enzymes of reductive pyrimidine degradation have previously been characterized. The enzyme catalysing the final step of pyrimidine breakdown, 3-ureidopropionase (-alanine synthase), had only been identified based on homology. We therefore cloned and functionally expressed the 3-ureidopropionase of C. elegans as hexahistidine fusion protein. The purified recombinant enzyme readily converted the two pyrimidine degradation products: 3-ureidopropionate and 2-methyl-3-ureidopropionate. The enzyme showed a broad pH optimum between pH 7.0 and 8.0. Activity was highest at approximately 40 C, although the half-life of activity was only 65 s at that temperature. The enzyme showed clear Michaelis-Menten kinetics, with a K(m) of 147 +/- 26 M and a V(max) of 1.1 +/- 0.1 Umg protein(-1). The quaternary structure of the recombinant enzyme was shown to correspond to a dodecamer by 'blue native' gel electrophoresis and gel filtration. The organ specific and subcellular localization of the enzyme was determined using a translational fusion to green fluorescent protein and high expression was observed in striated muscle cells. With the characterization of the 3-ureidopropionase, the reductive pyrimidine degradation pathway in C. elegans has been functionally characterized. [copyright sign] 2010 The Authors Journal compilation [copyright sign] 2010 FEBS.
机译：嘧啶是所有细胞中重要的代谢产物。细胞嘧啶的水平由多种机制控制，其中一种机制包括还原降解途径。在模型无脊椎动物秀丽隐杆线虫中，先前已鉴定了还原性嘧啶降解的三种酶中的两种。仅基于同源性才鉴定出催化嘧啶分解的最终步骤的酶3-脲基丙酸酶（-丙氨酸合酶）。因此，我们克隆和功能表达秀丽隐杆线虫的3-脲基丙酸酶为六组氨酸融合蛋白。纯化的重组酶易于转化两个嘧啶降解产物：3-脲基丙酸酯和2-甲基-3-脲基丙酸酯。该酶显示出最适的pH值，介于7.0和8.0之间。尽管在该温度下活性的半衰期仅为65 s，但其活性最高约为40℃。该酶显示出清晰的Michaelis-Menten动力学，K（m）为147 +/- 26 M，V（max）为1.1 +/- 0.1 Umg蛋白（-1）。通过“蓝色天然”凝胶电泳和凝胶过滤显示重组酶的四级结构对应于十二聚体。使用绿色荧光蛋白的翻译融合确定酶的器官特异性和亚细胞定位，并在横纹肌细胞中观察到高表达。通过3-脲基丙酸酶的表征，已对秀丽隐杆线虫中的还原嘧啶降解途径进行了功能表征。 [版权符号] 2010作者期刊汇编[版权符号] 2010 FEBS。

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