title: Vitamin K epoxide reductase prefers ER membrane-anchored thioredoxin-like redox partners.
abstract: 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.
authors.affiliation: Department of Cell Biology, Harvard Medical School, Howard Hughes Medical Institute, 240 Longwood Avenue, Boston, MA 02115, USA.
authors.forename: Sol
authors.initials: S
authors.lastname: Schulman
authors.forename: Belinda
authors.initials: B
authors.lastname: Wang
authors.forename: Weikai
authors.initials: W
authors.lastname: Li
authors.forename: Tom A
authors.initials: TA
authors.lastname: Rapoport
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D019556:COS Cells; D020134:Catalytic Domain / Q000235:genetics; D002522:Chlorocebus aethiops; D004579:Electron Transport; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity; D040681:Structural Homology, Protein; D046939:Synechocystis / Q000201:enzymology; D013879:Thioredoxins / Q000378:metabolism*; D064417:Vitamin K Epoxide Reductases
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D013879:Thioredoxins; D006899:Mixed Function Oxygenases; D064417:Vitamin K Epoxide Reductases