DEOXYHYPUSINE SYNTHASE--PURIFICATION, CHARACTERIZATION, AND CDNA CLONING

一种不寻常的氨基酸，苏氨酸，只存在于一种细胞中 真核细胞翻译分离因子5A(eIF-5A)，是 与细胞增殖密切相关。Hypusine生物合成 以两个顺序的翻译后修改的方式发生 反应：I)脱氧亚硫氨酸合成酶合成脱氧亚硫氨酸 Ii)通过脱氧硫胺羟基酶进行脱氧硫胺羟基化。 我们已经从大鼠体内提纯了第一种酶--脱氧亚硫氨酸合成酶 睾丸。纯化的酶表现出极窄的特异性。 其底物亚精胺、NAD和eIF-5A前体 蛋白质和催化脱氧亚硫氨酸的合成 反应混合物。然而，在没有底物蛋白质的情况下， 它进行部分反应，依赖于NAD的切割 亚精胺。该酶以42 kDa亚基的四聚体形式存在， 圆周率为4.75。 利用大鼠睾丸酶的部分氨基酸序列，我们 酿酒酵母第八号染色体YHRO68W的鉴定 脱氧亚精氨酸合酶基因。脱氧亚硫氨酸的失活 合酶基因导致酵母失去活力，提供了证据 亚精氨酸修饰是真核细胞所必需的 扩散。我们还克隆了编码一个完整的- HeLa细胞全长脱氧硫氨酸合成酶的免疫筛选 图书馆。人脱氧亚硫氨酸合成酶过表达后 YHRO68W的酵母基因在大肠杆菌中的表达，我们纯化了YHRO68W的酵母基因。 重组酶，并正在表征它们的物理和 酶性质。目前正在进行合作，以确定 人和酵母菌脱氧硫氨酸的三维结构 合成酶。缺失、插入和定点突变研究 正在进行中，以深入了解活跃的站点结构 以及这种重要酶的结构与功能的关系。

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.