The post-translational synthesis of hypusine in eIF5A: deoxyhypusine synthase

在以前的研究中，我们已经确定eIF5A是 只有含有一种不寻常的氨基酸--苏氨酸的细胞蛋白质 [海参-(4-氨基-2-羟基丁基)赖氨酸]，并已建立 亚精氨酸的生物合成是由两种连续的酶进行的 反应、脱氧亚硫氨酸合成和脱氧亚硫氨酸 羟化作用。它在eIF5A前体中以单一赖氨酸合成 残基代表最具体的翻译后修饰 迄今为止已知的。Hypusine对eIF-5A的活性是必需的，并且 用于真核细胞的增殖。亚精氨酸生物合成的抑制剂 导致细胞增殖受阻。脱氧亚精氨酸合酶催化 多胺亚精胺中丁胺部分的转移 与eIF-5A前体蛋白中的特定赖氨酸残基形成 脱氧亚硫氨酸。我们已经从大鼠睾丸中提纯了这种酶， 在酿酒酵母中鉴定其基因并克隆 人类的基因。脱氧亚精氨酸合酶基因失活 会导致细胞活力丧失提供了直接证据 亚硫氨酸修饰对酵母细胞的生长至关重要。我们有 表征了物理和催化性能以及反应 酶的作用机制。我们已经证明了脱氧亚硫氨酸 合成通过四个步骤进行：i)NAD依赖 亚精胺的脱氢，II)丁胺的转移 从脱氢亚精胺到酶的一种特定赖氨酸 形成酶-亚胺中间体，iii)转移 从酶中间到eIF5A的丁胺部分 前体，iv)将eIF5A亚胺还原为 含有脱氧亚硫氨酸的形式。活性中心残留物，即 参与酶中间体的形成已被确定 人酶的AS Lys-329和酵母的Lys-350 因此，脱氧亚硫氨酸的合成机制似乎是 在真核生物中是保守的。人体的X射线晶体结构 与NAD形成的复合体中的脱氧硫氨酸合酶(测定于 与廖东一博士和戴维斯博士合作) 显示NAD结合部位和活性部位口袋，其中 亚精胺被认为是结合的。几种氨基的作用 参与NAD、亚精胺和那些的结合的酸 对催化起关键作用的基因，通过定点定位得到证实 诱变。亚精胺结合部位的分子模拟 应协助开发脱氧亚硫氨酸的特异性抑制剂 可能作为抗增殖剂有用的合成酶。

In previous studies we have identified eIF5A as the only cellular protein that contains an unusual amino acid, hypusine [Nepsilon-(4-amino-2-hydroxybutyl)lysine], and have established that hypusine biosynthesis occurs by two sequential enzymatic reactions, deoxyhypusine synthesis and deoxyhypusine hydroxylation. Its synthesis in the eIF5A precursor at a single lysine residue represents the most specific post-translational modification known to date. Hypusine is essential for the activity of eIF-5A and for eukaryotic cell proliferation. Inhibitors of hypusine biosynthesis cause arrest in cell proliferation. Deoxyhypusine synthase catalyzes the transfer of the butylamine moiety of the polyamine spermidine to a specific lysine residue in the eIF-5A precursor protein to form deoxyhypusine. We have purified this enzyme from rat testis, identified its gene in the yeast Saccharomyces cerevisiae and cloned the human cDNA. Inactivation of the deoxyhypusine synthase gene in yeast causes loss of cell viability providing direct evidence that the hypusine modification is vital for growth of yeast cells. We have characterized the physical and catalytic properties and the reaction mechanism of the enzyme. We have shown that deoxyhypusine synthesis occurs by way of four steps: i) NAD-dependent dehydrogenation of spermidine, ii) transfer of the butyl amine moiety from dehydrospermidine to a specific lysine of the enzyme to form an enzyme-imine intermediate, iii) transfer of the butylamine moiety from the enzyme intermediate to the eIF5A precursor, iv) reduction of the eIF5A imine intermediate to the deoxyhypusine-containing form. The active site residue that is involved in enzyme-intermediate formation has been identified asLys-329 for the human enzyme and Lys-350 for the yeast enzyme; thus, the mechanism of deoxyhypusine synthesis appears to be conserved in eukaryotes. The X-ray crystal structure of human deoxyhypusine synthase in a complex with NAD (determined in collaboration with Drs. D.-I. Liao and D. R. Davies, NIDDK) reveals NAD binding sites and an active site pocket where spermidine is presumed to bind. The role of a number of amino acids involved in the binding of NAD, of spermidine, and those critical for the catalysis, was confirmed by site-directed mutagenesis. Molecular modeling of the spermidine binding site should aid development of specific inhibitors of deoxyhypusine synthase that may be useful as anti-proliferative agents.