Human Vitamin K Epoxide Reductase and Its Bacterial Homologue Have Different Membrane Topologies and Reaction Mechanisms

• Published in: The Journal of biological chemistry 2012-10, Vol.287 (41), p.33945-33955
• Main Authors: Tie, Jian-Ke, Jin, Da-Yun, Stafford, Darrel W.
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Carboxylation; Cell Membrane - enzymology; Cell Membrane - genetics; Coagulation Factors; Crystallography, X-Ray; Cysteine Modification; Cytoplasm - enzymology; Cytoplasm - genetics; Endoplasmic Reticulum - enzymology; Endoplasmic Reticulum - genetics; HEK293 Cells; Humans; Membrane Biology; Membrane Proteins; Membrane Topology; Mixed Function Oxygenases - chemistry; Mixed Function Oxygenases - genetics; Mixed Function Oxygenases - metabolism; Models, Molecular; Protein Carboxylation; Structural Homology, Protein; Synechococcus; Vitamin K; Vitamin K Epoxide Reductase; Vitamin K Epoxide Reductases
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.402941

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. Background: The membrane topology and the role of certain cysteines in human vitamin K epoxide reductase (VKOR) are disputed. Results: VKOR has three transmembrane domains, and only the active site cysteines are required for function. Conclusion: Despite similarities, VKOR and its bacterial homologues (VKORHs) have different topologies and reaction mechanisms. Significance: The VKOR does not employ the intramolecular electron transfer pathway proposed for VKORH.