title: 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 (KH2). The vitamin K epoxide (KO) product is recycled by two reactions, i.e. KO reduction to vitamin K quinone (K) and then to KH2, and recent studies have called into question whether VKORC1 reduces K to KH2. Analysis in insect cells lacking endogenous carboxylation components showed that r-VKORC1 reduces KO to efficiently drive carboxylation, indicating KH2 production. Direct detection of the vitamin K reaction products is confounded by KH2 oxidation, and we therefore developed a new assay that stabilized KH2 and allowed quantitation. Purified VKORC1 analyzed in this assay showed efficient KO to KH2 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.
authors.affiliation: From the Departments of Molecular Cardiology and.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Rishavy
authors.forename: Kevin W
authors.initials: KW
authors.lastname: Hallgren
authors.forename: Lee A
authors.initials: LA
authors.lastname: Wilson
authors.forename: Aisulu
authors.initials: A
authors.lastname: Usubalieva
authors.forename: Kurt W
authors.initials: KW
authors.lastname: Runge
authors.forename: Kathleen L
authors.initials: KL
authors.lastname: Berkner
mesh_terms: D019943:Amino Acid Substitution; D000925:Anticoagulants / Q000627:therapeutic use; D004351:Drug Resistance / Q000187:drug effects / Q000502:physiology; D057809:HEK293 Cells; D006801:Humans; D006873:Hydroquinones / Q000737:chemistry / Q000378:metabolism*; D020125:Mutation, Missense; D010084:Oxidation-Reduction; D055503:Protein Multimerization / Q000502:physiology*; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D014812:Vitamin K / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D064417:Vitamin K Epoxide Reductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014859:Warfarin / Q000627:therapeutic use
chemical_list: D000925:Anticoagulants; D006873:Hydroquinones; D014812:Vitamin K; D014859:Warfarin; C578523:VKORC1 protein, human; D064417:Vitamin K Epoxide Reductases; C031927:hydroquinone
keywords: Enzyme Mechanisms; Membrane Enzymes; Protein Carboxylation; Reductase; Vitamins and Cofactors
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