title: Human vitamin K epoxide reductase and its bacterial homologue have different membrane topologies and reaction mechanisms.
abstract: Vitamin K epoxide reductase (VKOR) is essential for the production of reduced vitamin K that is required for modification of vitamin K-dependent proteins. Three- and four-transmembrane domain (TMD) topology models have been proposed for VKOR. They are based on in vitro glycosylation mapping of the human enzyme and the crystal structure of a bacterial (Synechococcus) homologue, respectively. These two models place the functionally disputed conserved loop cysteines, Cys-43 and Cys-51, on different sides of the endoplasmic reticulum (ER) membrane. In this study, we fused green fluorescent protein to the N or C terminus of human VKOR, expressed these fusions in HEK293 cells, and examined their topologies by fluorescence protease protection assays. Our results show that the N terminus of VKOR resides in the ER lumen, whereas its C terminus is in the cytoplasm. Selective modification of cysteines by polyethylene glycol maleimide confirms the cytoplasmic location of the conserved loop cysteines. Both results support a three-TMD model of VKOR. Interestingly, human VKOR can be changed to a four-TMD molecule by mutating the charged residues flanking the first TMD. Cell-based activity assays show that this four-TMD molecule is fully active. Furthermore, the conserved loop cysteines, which are essential for intramolecular electron transfer in the bacterial VKOR homologue, are not required for human VKOR whether they are located in the cytoplasm (three-TMD molecule) or the ER lumen (four-TMD molecule). Our results confirm that human VKOR is a three-TMD protein. Moreover, the conserved loop cysteines apparently play different roles in human VKOR and in its bacterial homologues.
authors.affiliation: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3280, USA. jktie@email.unc.edu
authors.forename: Jian-Ke
authors.initials: JK
authors.lastname: Tie
authors.forename: Da-Yun
authors.initials: DY
authors.lastname: Jin
authors.forename: Darrel W
authors.initials: DW
authors.lastname: Stafford
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology* / Q000235:genetics; D018360:Crystallography, X-Ray; D003593:Cytoplasm / Q000201:enzymology* / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000201:enzymology* / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D040681:Structural Homology, Protein; D046940:Synechococcus; D064417:Vitamin K Epoxide Reductases
chemical_list: D001426:Bacterial Proteins; D006899:Mixed Function Oxygenases; D064417:Vitamin K Epoxide Reductases