Purified vitamin K epoxide reductase alone is sufficient for conversion of vitamin K epoxide to vitamin K and vitamin K to vitamin KH₂

Abstract

More than 21 million prescriptions for warfarin are written yearly in the U.S. Despite its importance, warfarin's target, vitamin K epoxide reductase (VKOR), has resisted purification since its identification in 1972. Here, we report its purification and reconstitution. HPC4, a calcium-specific antibody that recognizes a 12-aa tag, was used to purify and identify VKOR. Partial reconstitution is achieved on the column by washing with 0.4% dioleoylphosphatidylcholine/0.4% deoxycholate. Activity is completely recovered by dialysis against a buffer containing a reducing agent but lacking dioleoylphosphatidylcholine/deoxycholate. Removal of detergent from the eluted proteins apparently facilitates liposome formation. Purified recombinant VKOR with tag is ≈21 kDa, as expected; fully active; and >93% pure. The concentration of warfarin for 50% inhibition is the same for purified protein and microsomes. It has been reported that VKOR is a multisubunit enzyme. Our results, however, suggest that a single peptide can accomplish both the conversion of vitamin K epoxide to vitamin K and vitamin K to reduced vitamin K. This purification will allow further characterization of VKOR in relation to other components of the vitamin K cycle and should facilitate its structural determination.

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

“…2) are different from what was previously reported with purified VKORC1, where reduction of K to KH 2 was 50-fold lower than reduction of KO to K (20). That study monitored activity using a VKORC1 preparation that was initially inactivated during membrane solubilization and then reconstituted for activity.…”

“…Alternatively, the difference may be due to the enzyme preparation. Previously, VKORC1 was inactivated during detergent solubilization of membrane, and activity was subsequently reconstituted (20), which may have disrupted the dimer to alter the normal course of the reaction. We used an activity assay to optimize membrane solubilization, which retained activity and may have been significant in revealing that VKORC1 efficiently reduces KO to KH 2 .…”

“…Assessing the role of VKORC1 in KO to KH 2 reduction has been problematic because KH 2 is rapidly oxidized and VKORC1 has been difficult to purify. In the one reported purification of VKORC1, the efficiency of K to KH 2 production was ϳ50-fold lower than that of KO to K (20), which led to the proposal that VKORC1 only reduces KO to K (17). Our previous results suggested that VKORC1 performs both reactions (16), and we therefore developed new approaches to examine VKORC1 reactivity.…”

“…KO to K (14,15,16,18). Previously, the antioxidant ␤-hydroxytoluene was used to try to stabilize KH 2 (20); however, tests with multiple antioxidants, including ␤-hydroxytoluene, were ineffective in blocking KH 2 oxidation (e.g. Fig.…”

VKORC1L1, an Enzyme Rescuing the Vitamin K 2,3-Epoxide Reductase Activity in Some Extrahepatic Tissues during Anticoagulation Therapy

“…The recombinant VKORC1 protein expressed either in HEK293T cells (12) or in baculovirus (14) or in Pichia pastoris (15,16) effectively catalyzes the VKOR activity and is inhibited by VKAs.…”