Vitamin K epoxide reductase prefers ER membrane-anchored thioredoxin-like redox partners
Vitamin K epoxide reductase prefers ER membrane-anchored thioredoxin-like redox partners
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DOI：
10.1073/pnas.1009972107
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发表时间：
2010-08-24
影响因子：
11.1
通讯作者：
Rapoport, Tom A.
中科院分区：
文献类型：
--
作者：
Schulman, Sol;Wang, Belinda;Rapoport, Tom A.
维生素K环氧化物还原酶（VKOR）通过将维生素K环氧化物还原为氢醌（许多凝血因子γ-谷氨酰羧化的重要辅因子）来维持血液凝固。VKOR的生理氧化还原伴侣仍不确定，但可能是硫氧还蛋白样蛋白。在这里，我们证明了人VKOR与聚球藻属的酶具有相同的膜拓扑结构，其晶体结构最近才被确定。我们的研究结果表明，在氧化还原反应过程中，Cys 43在人VKOR的管腔环形成一个短暂的二硫键与硫氧还蛋白（Trx）样蛋白位于内质网（ER）的内腔。我们通过测试一组这些候选者与人VKOR形成这种特异性二硫键的能力，在大量哺乳动物Trx样ER蛋白中筛选VKOR的氧化还原伴侣。我们的研究结果表明，VKOR与TMX强烈相互作用，TMX是一种具有独特CPAC活性位点的ER膜锚定Trx样蛋白。与TMX的近亲TMX 4和ER的最小Trx样蛋白ERp 18的相互作用较弱。我们用Ero 1-alpha进行了类似的筛选，Ero 1-alpha是一种氧化Trx样蛋白二硫键异构酶的ER-管腔蛋白。我们发现Ero 1-alpha与大多数测试的Trx样蛋白相互作用，尽管与该家族的膜锚定成员的相互作用很差。两者合计，我们的研究结果表明，人类VKOR采用相同的电子转移途径，其细菌同系物和VKOR一般更喜欢膜结合的Trx样氧化还原伙伴。
Vitamin K epoxide reductase (VKOR) sustains blood coagulation by reducing vitamin K epoxide to the hydroquinone, an essential cofactor for the gamma-glutamyl carboxylation of many clotting factors. The physiological redox partner of VKOR remains uncertain, but is likely a thioredoxin-like protein. Here, we demonstrate that human VKOR has the same membrane topology as the enzyme from Synechococcus sp., whose crystal structure was recently determined. Our results suggest that, during the redox reaction, Cys43 in a luminal loop of human VKOR forms a transient disulfide bond with a thioredoxin (Trx)-like protein located in the lumen of the endoplasmic reticulum (ER). We screened for redox partners of VKOR among the large number of mammalian Trx-like ER proteins by testing a panel of these candidates for their ability to form this specific disulfide bond with human VKOR. Our results show that VKOR interacts strongly with TMX, an ER membrane-anchored Trx-like protein with a unique CPAC active site. Weaker interactions were observed with TMX4, a close relative of TMX, and ERp18, the smallest Trx-like protein of the ER. We performed a similar screen with Ero1-alpha, an ER-luminal protein that oxidizes the Trx-like protein disulfide isomerase. We found that Ero1-alpha interacts with most of the tested Trx-like proteins, although only poorly with the membrane- anchored members of the family. Taken together, our results demonstrate that human VKOR employs the same electron transfer pathway as its bacterial homologs and that VKORs generally prefer membrane-bound Trx-like redox partners.