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

eIF5A 中的马尿苷翻译后合成：脱氧马尿苷合成酶

• 批准号: 6432029
• 负责人: MYUNG HEE PARK
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6432029
• 关键词: Saccharomyces cerevisiae; X ray crystallography; active sites; amine oxidoreductase; aminoacid analog; aminoacid biosynthesis; cell cycle; cell growth regulation; cell proliferation; enzyme mechanism; enzyme structure; human genetic material tag; laboratory rat; lysine; molecular cloning; oxidoreductase inhibitor; oxygenases; posttranslational modifications; protein purification; recombinant proteins; spermidine; translation factor

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.

我们已经鉴定出 eIF5A 是唯一含有一种不寻常的氨基酸——山尿苷 [Nepsilon-(4-氨基-2-羟丁基)赖氨酸] 的细胞蛋白，并确定山尿苷生物合成是通过两个连续的酶促反应，即脱氧山尿苷合成和脱氧山尿苷羟基化而发生的。它在 eIF5A 前体中单个赖氨酸残基处的合成代表了迄今为止已知的最特异的翻译后修饰。 Hypusine 对于 eIF-5A 的活性和真核细胞增殖至关重要。山楂碱生物合成抑制剂会导致细胞增殖停滞。脱氧马尿苷合酶催化聚胺亚精胺的丁胺部分转移至 eIF-5A 前体蛋白中的特定赖氨酸残基，形成脱氧马尿苷。我们从大鼠睾丸中纯化了这种酶，在酿酒酵母中鉴定了其基因，并克隆了人类 cDNA。酵母中脱氧马尿苷合酶基因的失活会导致细胞活力丧失，这提供了马尿苷修饰对于酵母细胞生长至关重要的直接证据。我们表征了酶的物理和催化特性，并研究了酶的反应机理。我们已经证明，脱氧马尿苷合成通过四个步骤发生：i) 亚精胺的 NAD 依赖性脱氢，ii) 丁胺部分从脱氢亚精胺转移到酶的特定赖氨酸以形成酶-亚胺中间体，iii) 丁胺部分从酶中间体转移到 eIF5A 前体， iv)将eIF5A亚胺中间体还原为含有脱氧马匹碱的形式。参与酶中间体形成的活性位点残基已被鉴定为人类酶的Lys-329和酵母酶的Lys-350；因此，脱氧马尿苷合成机制似乎在真核生物中是保守的。人脱氧马尿苷合成酶与 NAD 复合物的 X 射线晶体结构揭示了 NAD 结合位点和推测亚精胺结合的活性位点袋。通过定点诱变评估了预测参与 NAD、亚精胺以及催化关键氨基酸结合的许多氨基酸的作用。亚精胺结合位点的分子建模应有助于开发脱氧马尿苷合成酶的特异性抑制剂，该抑制剂可用作抗增殖剂。