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发生 通过两个连续的翻译后修饰反应：i） 通过脱氧羟腐胺赖氨酸合成酶合成脱氧羟腐胺赖氨酸，和ii）脱氧羟腐胺赖氨酸 通过脱氧羟腐胺赖氨酸羟化酶进行羟基化。 我们已经纯化了第一种酶，脱氧羟腐胺赖氨酸合成酶， 从大鼠睾丸中富集约100，000倍。纯化的酶 对底物显示出非常窄的特异性， 亚精胺，NAD和eIF-5A前体蛋白，并催化 在完全反应混合物中的脱氧羟腐胺赖氨酸合成。在没有 然而，它进行部分反应， NAD依赖的亚精胺裂解。该酶以四聚体形式存在， 42 kDa亚基，pI为4.75。 使用大鼠睾丸酶的部分氨基酸序列，我们 能够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。我们还克隆了人类编码 通过HeLa细胞的免疫筛选获得全长脱氧羟腐胺赖氨酸合酶 cDNA文库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. coli中，我们纯化了重组蛋白 酶，并表征其物理和酶性质。 缺失、插入和定点突变研究正在进行中， 深入了解活性位点结构和结构-功能 这一重要的酶。

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, to homogeneity after about 100,000-fold enrichment 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 were able to identify YHRO68W of Saccharomyces cerevisiae chromosome VIII as a gene for deoxyhypusine synthase. 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 characterized their physical and enzymatic properties. Deletion, insertion and site-directed mutagenesis studies are underway to gain insights into the active site structure and the structure-function relationship of this important enzyme.