Disulfide transfer between two conserved cysteine pairs imparts selectivity to protein oxidation by Ero1
Disulfide transfer between two conserved cysteine pairs imparts selectivity to protein oxidation by Ero1
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DOI：
10.1091/mbc.e05-05-0417
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发表时间：
2006-05-01
影响因子：
3.3
通讯作者：
Kaiser, CA
中科院分区：
文献类型：
--
作者：
Sevier, CS;Kaiser, CA
膜相关黄蛋白Ero1p通过选择性氧化可溶性氧化还原酶蛋白二硫异构酶（Pdi1p）促进内质网（ER）中二硫键的形成，而Pdi1p又可以直接氧化分泌蛋白。两个具有氧化还原活性的二硫键是Ero1p氧化酶活性所必需的：Cys100-Cys105和Cys352-Cys355。遗传和结构数据表明，一个二硫键从Cys100-Cys105直接转移到Pdi1p上，而一个Cys352-Cys355二硫键通过内部硫醇转移反应将还原的Cys100-Cys105对再氧化。电子通过黄素辅助因子从Cys352-Cys355转移到分子氧，使Cys352-Cys355保持氧化状态。在此，我们鉴定了一种混合二硫化合物，在体内证实了Ero1p半胱氨酸间硫醇转移中继，并鉴定了Cys105和Cys352是介导硫醇-二硫交换的半胱氨酸。此外，我们描述了在缺乏Cys100-Cys105的情况下具有令人惊讶的氧化底物能力的Ero1p突变体。我们发现这些突变体的氧化酶活性是由Ero1p的结构变化引起的，这使得底物增加了对Cys352-Cys355的接触，而Cys352-Cys355通常埋在蛋白质表面之下。这些Ero1p突变体对选定底物的活性改变使我们提出了催化机制，包括半胱氨酸对之间的转移，从而赋予Ero1p底物特异性。
The membrane-associated flavoprotein Ero1p promotes disulfide bond formation in the endoplasmic reticulum (ER) by selectively oxidizing the soluble oxidoreductase protein disulfide isomerase (Pdi1p), which in turn can directly oxidize secretory proteins. Two redox-active disulfide bonds are essential for Ero1p oxidase activity: Cys100-Cys105 and Cys352-Cys355. Genetic and structural data indicate a disulfide bond is transferred from Cys100-Cys105 directly to Pdi1p, whereas a Cys352-Cys355 disulfide bond is used to reoxidize the reduced Cys100-Cys105 pair through an internal thiol-transfer reaction. Electron transfer from Cys352-Cys355 to molecular oxygen, by way of a flavin cofactor, maintains Cys352-Cys355 in an oxidized form. Herein, we identify a mixed disulfide species that confirms the Ero1p intercysteine thiol-transfer relay in vivo and identify Cys105 and Cys352 as the cysteines that mediate thiol-disulfide exchange. Moreover, we describe Ero1p mutants that have the surprising ability to oxidize substrates in the absence of Cys100-Cys105. We show the oxidase activity of these mutants results from structural changes in Ero1p that allow substrates increased access to Cys352-Cys355, which are normally buried beneath the protein surface. The altered activity of these Ero1p mutants toward selected substrates leads us to propose the catalytic mechanism involving transfer between cysteine pairs evolved to impart substrate specificity to Ero1p.