Description:
<jats:p>Vitamin K epoxide reductase (VKOR) sustains blood coagulation by reducing vitamin K epoxide to the hydroquinone, an essential cofactor for the γ-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<jats:italic>Synechococcus</jats:italic>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-α, an ER-luminal protein that oxidizes the Trx-like protein disulfide isomerase. We found that Ero1-α 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.</jats:p>