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

我们已经确定eIF5A是唯一一种含有不寻常氨基酸的细胞蛋白，hypusine [neplsilon -（4-氨基-2-羟基丁基）赖氨酸]，并确定了hypusine的生物合成发生在两个连续的酶促反应中，脱氧hypusine合成和脱氧hypusine羟基化。它在eIF5A前体中一个赖氨酸残基上的合成代表了迄今为止已知的最特异性的翻译后修饰。Hypusine是eIF-5A活性和真核细胞增殖所必需的。抑制hypusine生物合成导致细胞增殖阻滞。脱氧hypusine合成酶催化多胺亚精胺的丁胺部分转移到eIF-5A前体蛋白中的特定赖氨酸残基上，形成脱氧hypusine。我们从大鼠睾丸中纯化了该酶，在酿酒酵母中鉴定了其基因，并克隆了人类cDNA。酵母中脱氧hypusine合酶基因的失活导致细胞活力的丧失，这直接证明了hypusine修饰对酵母细胞的生长至关重要。我们对酶的物理性质和催化性质进行了表征，并对其反应机理进行了研究。我们已经证明，脱氧亚精氨酸的合成通过四个步骤进行：1)亚精胺依赖于nadh的脱氢，2)将去氢亚精胺的丁胺部分转移到酶的特定赖氨酸上，形成酶-亚胺中间体，3)将丁胺部分从酶中间体转移到eIF5A前体，4)将eIF5A亚胺中间体还原为含有脱氧亚精氨酸的形式。参与酶中间体形成的活性位点残基已被鉴定为：人酶的lys -329和酵母酶的Lys-350；因此，脱氧hypusine的合成机制似乎在真核生物中是保守的。人脱氧hypusine合酶与NAD复合物的x射线晶体结构揭示了NAD结合位点和一个推测亚精胺结合的活性位点袋。一些氨基酸的作用被预测参与NAD的结合，亚精胺，和那些对催化至关重要的，通过位点定向诱变评估。亚精胺结合位点的分子模型应该有助于开发脱氧hypusine合成酶的特异性抑制剂，这些抑制剂可能作为抗增殖剂有用。

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 studied 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 reveals NAD binding sites and an active site pocket where spermidine is presumed to bind. The role of a number of amino acids predicted to be involved in the binding of NAD, of spermidine, and those critical for the catalysis, was assessed 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.