The Vitamin K Oxidoreductase Is a Multimer That Efficiently Reduces Vitamin K Epoxide to Hydroquinone to Allow Vitamin K-dependent Protein Carboxylation

Abstract

The vitamin K oxidoreductase (VKORC1) recycles vitamin K to support the activation of vitamin K-dependent (VKD) proteins, which have diverse functions that include hemostasis and calcification. VKD proteins are activated by Glu carboxylation, which depends upon the oxygenation of vitamin K hydroquinone (KH 2 ). The vitamin K epoxide (KO) product is recycled by two reactions, i.e. KO reduction to vitamin K quinone (K) and then to KH 2 , and recent studies have called into question whether VKORC1 reduces K to KH 2 . Analysis in insect cells lacking endogenous carboxylation components showed that r-VKORC1 reduces KO to efficiently drive carboxylation, indicating KH 2 production. Direct detection of the vitamin K reaction products is confounded by KH 2 oxidation, and we therefore developed a new assay that stabilized KH 2 and allowed quantitation. Purified VKORC1 analyzed in this assay showed efficient KO to KH 2 reduction. Studies in 293 cells expressing tagged r-VKORC1 revealed that VKORC1 is a multimer, most likely a dimer. A monomer can only perform one reaction, and a dimer is therefore interesting in explaining how VKORC1 accomplishes both reactions. An inactive mutant (VKORC1(C132A/C135A)) was dominant negative in heterodimers with wild type VKORC1, resulting in decreased KO reduction in cells and carboxylation in vitro . The results are significant regarding human VKORC1 mutations, as warfarin-resistant patients have mutant and wild type VKORC1 alleles. A VKORC1 dimer indicates a mixed population of homodimers and heterodimers that may have different functional properties, and VKORC1 reduction may therefore be more complex in these patients than appreciated previously. Background: How the vitamin K oxidoreductase (VKORC1) supports vitamin K-dependent protein carboxylation is poorly understood. Results: VKORC1 multimers efficiently perform both reactions that reduce vitamin K, and the inactive monomer in wild type mutant heteromers suppresses reduction. Conclusion: VKORC1 fully reduces vitamin K required for carboxylation. Significance: Multimers are important in VKORC1 mechanism and wild type mutant heteromers impact patients with warfarin resistance.