DEOXYHYPUSINE SYNTHASE--PURIFICATION, CHARACTERIZATION, AND CDNA CLONING

一种不常见的氨基酸，羟腐胺赖氨酸，只存在于一种细胞中， 真核生物翻译分离因子5A（eIF-5A）， 与细胞增殖密切相关。 羟腐胺赖氨酸生物合成 通过两个连续的翻译后修饰发生 反应：i）通过脱氧羟腐胺赖氨酸合成酶合成脱氧羟腐胺赖氨酸， ii）通过脱氧羟腐胺赖氨酸羟化酶的脱氧羟腐胺赖氨酸羟化。 我们已经从大鼠中纯化了第一种酶，脱氧羟腐胺赖氨酸合成酶， 睾丸纯化的酶显示出非常窄的特异性 其底物亚精胺、NAD和eIF-5A前体 蛋白质和催化脱氧羟腐胺赖氨酸合成的完整 反应混合物。然而，在缺乏底物蛋白的情况下， 它进行部分反应，NAD依赖的切割， 亚精胺该酶以42 kDa亚基的四聚体形式存在， pI为4.75。 利用大鼠睾丸酶的部分氨基酸序列， 鉴定酿酒酵母染色体VIII的YHRO 68 W为 脱氧羟腐胺赖氨酸合酶的基因。 脱氧羟腐胺赖氨酸的灭活 合成酶基因导致酵母菌活力丧失，这证明， 羟腐胺赖氨酸修饰对于真核细胞是必需 增殖 我们还克隆了编码一个完整的- 免疫筛选HeLa细胞脱氧羟腐胺赖氨酸合成酶cDNA 图书馆在人脱氧羟腐胺赖氨酸合酶过表达后 YHRO 68 W的cDNA或酵母cDNA在E.大肠杆菌，我们纯化了 重组酶，并表征其物理和 酶学性质 正在进行合作，以确定 人和酵母脱氧羟腐胺赖氨酸的三维结构 合成酶 缺失、插入和定点突变研究 是为了深入了解活性位点的结构 以及这种重要酶的结构-功能关系。

An unusual amino acid, hypusine, which occurs in only one cellular protein, eukaryotic translation isolation factor 5A (eIF-5A), is intimately involved in cell proliferation. Hypusine biosynthesis occurs by way of two sequential post-translational modification reactions: i) deoxyhypusine synthesis by deoxyhypusine synthase and ii) deoxyhypusine hydroxylation by deoxyhypusine hydroxylase. We have purified the first enzyme, deoxyhypusine synthase, from rat testis. The purified enzyme displays a remarkably narrow specificity toward its substrates, spermidine, NAD, and the eIF-5A precursor protein and catalyzes deoxyhypusine synthesis in the complete reaction mixture. In the absence of the substrate protein, however, it carries out a partial reaction, the NAD-dependent cleavage of spermidine. The enzyme exists as a tetramer of 42 kDa subunits, with a pI of 4.75. Using partial amino acid sequences from the rat testis enzyme, we identified YHRO68W of Saccharomyces cerevisiae chromosome VIII as the gene for deoxyhypusine synthase. Inactivation of the deoxyhypusine synthase gene causes loss of viability in yeast, providing proof that the hypusine modification is essential for eukaryotic cell proliferation. We have also cloned human cDNAs encoding a full- length deoxyhypusine synthase by immunoscreening of a HeLa cell cDNA library. After overexpression of the human deoxyhypusine synthase cDNA or the yeast cDNA of YHRO68W in E. coli, we purified the recombinant enzymes, and are characterizing their physical and enzymatic properties. Collaborations are underway to determine the three-dimensional structures of human and yeast deoxyhypusine synthase. Deletion, insertion and site-directed mutagenesis studies are being carried out to gain insights into the active site structure and the structure-function relationship of this important enzyme.