Human vitamin K 2,3-epoxide reductase complex subunit 1-like 1 (VKORC1L1) mediates vitamin K-dependent intracellular antioxidant function

Human vitamin K 2,3-epoxide reductase complex subunit 1-like 1 (VKORC1L1), expressed in HEK 293T cells and localized exclusively to membranes of the endoplasmic reticulum, was found to support both vitamin K 2,3-epoxide reductase (VKOR) and vitamin K reductase enzymatic activities. Michaelis-Menten kinetic parameters for dithiothreitol-driven VKOR activity were: K(m) (μM) = 4.15 (vitamin K(1) epoxide) and 11.24 (vitamin K(2) epoxide); V(max) (nmol·mg(-1)·hr(-1)) = 2.57 (vitamin K(1) epoxide) and 13.46 (vitamin K(2) epoxide). Oxidative stress induced by H(2)O(2) applied to cultured cells up-regulated VKORC1L1 expression and VKOR activity. Cell viability under conditions of no induced oxidative stress was increased by the presence of vitamins K(1) and K(2) but not ubinquinone-10 and was specifically dependent on VKORC1L1 expression. Intracellular reactive oxygen species levels in cells treated with 2,3-dimethoxy-1,4-naphthoquinone were mitigated in a VKORC1L1 expression-dependent manner. Intracellular oxidative damage to membrane intrinsic proteins was inversely dependent on VKORC1L1 expression and the presence of vitamin K(1). Taken together, our results suggest that VKORC1L1 is responsible for driving vitamin K-mediated intracellular antioxidation pathways critical to cell survival.     

Conserved Loop Cysteines of Vitamin K Epoxide Reductase Complex Subunit 1-like 1 (VKORC1L1) Are Involved in Its Active Site Regeneration

Vitamin K epoxide reductase complex subunit 1 (VKORC1) reduces vitamin K epoxide in the vitamin K cycle for post-translational modification of proteins that are involved in a variety of biological functions. However, the physiological function of VKORC1-like 1 (VKORC1L1), a paralogous enzyme sharing about 50% protein identity with VKORC1, is unknown. Here we determined the structural and functional differences of these two enzymes using fluorescence protease protection (FPP) assay and an in vivo cell-based activity assay. We show that in vivo VKORC1L1 reduces vitamin K epoxide to support vitamin K-dependent carboxylation as efficiently as does VKORC1. However, FPP assays show that unlike VKORC1, VKORC1L1 is a four-transmembrane domain protein with both its termini located in the cytoplasm. Moreover, the conserved loop cysteines, which are not required for VKORC1 activity, are essential for VKORC1L1''s active site regeneration. Results from domain exchanges between VKORC1L1 and VKORC1 suggest that it is VKORC1L1''s overall structure that uniquely allows for active site regeneration by the conserved loop cysteines. Intermediate disulfide trapping results confirmed an intra-molecular electron transfer pathway for VKORC1L1''s active site reduction. Our results allow us to propose a concerted action of the four conserved cysteines of VKORC1L1 for active site regeneration; the second loop cysteine, Cys-58, attacks the active site disulfide, forming an intermediate disulfide with Cys-139; the first loop cysteine, Cys-50, attacks the intermediate disulfide resulting in active site reduction. The different membrane topologies and reaction mechanisms between VKORC1L1 and VKORC1 suggest that these two proteins might have different physiological functions.     

Determination of the warfarin inhibition constant Ki for vitamin K 2,3-epoxide reductase complex subunit-1 (VKORC1) using an in vitro DTT-driven assay

Background

Warfarin directly inhibits vitamin K 2,3-epoxide reductase (VKOR) enzymes. Since the early 1970s, warfarin inhibition of vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1), an essential enzyme for proper function of blood coagulation in higher vertebrates, has been studied using an in vitro dithiothreitol (DTT) driven enzymatic assay. However, various studies based on this assay have reported warfarin dose–response data, usually summarized as half-maximal inhibitory concentration (IC₅₀), that vary over orders of magnitude and reflect the broad range of conditions used to obtain VKOR assay data.

Methods

We standardized the implementation of the DTT-driven VKOR activity assay to measure enzymatic Michaelis constants (K_m) and warfarin IC₅₀ for human VKORC1. A data transformation is defined, based on the previously confirmed bi bi ping-pong mechanism for VKORC1, that relates assay condition-dependent IC₅₀ to condition-independent K_i.

Results

Determination of the warfarin K_i specifically depends on measuring both substrate concentrations, both Michaelis constants for the VKORC1 enzyme, and pH in the assay.

Conclusion

The K_i is not equal to the IC₅₀ value directly measured using the DTT-driven VKOR assay.

General significance

In contrast to warfarin IC₅₀ values determined in previous studies, warfarin inhibition expressed as K_i can now be compared between studies, even when the specific DTT-driven VKOR assay conditions differ. This implies that warfarin inhibition reported for wild-type and variant VKORC1 enzymes from previous reports should be reassessed and new determinations of K_i are required to accurately report and compare in vitro warfarin inhibition results.     

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

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.     

Engineering of a recombinant vitamin K-dependent gamma-carboxylation system with enhanced gamma-carboxyglutamic acid forming capacity: evidence for a functional CXXC redox center in the system

The vitamin K-dependent gamma-carboxylation system in the endoplasmic reticulum membrane responsible for gamma-carboxyglutamic acid modification of vitamin K-dependent proteins includes gamma-carboxylase and vitamin K 2,3-epoxide reductase (VKOR). An understanding of the mechanism by which this system works at the molecular level has been hampered by the difficulty of identifying VKOR involved in warfarin sensitive reduction of vitamin K 2,3-epoxide to reduced vitamin K(1)H(2), the gamma-carboxylase cofactor. Identification and cloning of VKORC1, a proposed subunit of a larger VKOR enzyme complex, have provided opportunities for new experimental approaches aimed at understanding the vitamin K-dependent gamma-carboxylation system. In this work we have engineered stably transfected baby hamster kidney cells containing gamma-carboxylase and VKORC1 cDNA constructs, respectively, and stably double transfected cells with the gamma-carboxylase and the VKORC1 cDNA constructs in a bicistronic vector. All engineered cells showed increased activities of the enzymes encoded by the cDNAs. However increased activity of the gamma-carboxylation system, where VKOR provides the reduced vitamin K(1)H(2) cofactor, was measured only in cells transfected with VKORC1 and the double transfected cells. The results show that VKOR is the rate-limiting step in the gamma-carboxylation system and demonstrate successful engineering of cells containing a recombinant vitamin K-dependent gamma-carboxylation system with enhanced capacity for gamma-carboxyglutamic acid modification. The proposed thioredoxin-like (132)CXXC(135) redox center in VKORC1 was tested by expressing the VKORC1 mutants Cys(132)/Ser and Cys(135)/Ser in BHK cells. Both of the expressed mutant proteins were inactive supporting the existence of a CXXC redox center in VKOR.     

Novel insight into the mechanism of the vitamin K oxidoreductase (VKOR): electron relay through Cys43 and Cys51 reduces VKOR to allow vitamin K reduction and facilitation of vitamin K-dependent protein carboxylation

The vitamin K oxidoreductase (VKOR) reduces vitamin K to support the carboxylation and consequent activation of vitamin K-dependent proteins, but the mechanism of reduction is poorly understood. VKOR is an integral membrane protein that reduces vitamin K using membrane-embedded thiols (Cys-132 and Cys-135), which become oxidized with concomitant VKOR inactivation. VKOR is subsequently reactivated by an unknown redox protein that is currently thought to act directly on the Cys132-Cys135 residues. However, VKOR contains evolutionarily conserved Cys residues (Cys-43 and Cys-51) that reside in a loop outside of the membrane, raising the question of whether they mediate electron transfer from a redox protein to Cys-132/Cys-135. To assess a possible role, the activities of mutants with Ala substituted for Cys (C43A and C51A) were analyzed in intact membranes using reductants that were either membrane-permeable or -impermeable. Both reductants resulted in wild type VKOR reduction of vitamin K epoxide; however, the C43A and C51A mutants only showed activity with the membrane-permeant reductant. We obtained similar results when testing the ability of wild type and mutant VKORs to support carboxylation, using intact membranes from cells coexpressing VKOR and carboxylase. These results indicate a role for Cys-43 and Cys-51 in catalysis, suggesting a relay mechanism in which a redox protein transfers electrons to these loop residues, which in turn reduce the membrane-embedded Cys132-Cys135 disulfide bond to activate VKOR. The results have implications for the mechanism of warfarin resistance, the topology of VKOR in the membrane, and the interaction of VKOR with the carboxylase.     

A hetero-dimer model for concerted action of vitamin K carboxylase and vitamin K reductase in vitamin K cycle

Vitamin K carboxylase (VKC) is believed to convert vitamin K, in the vitamin K cycle, to an alkoxide-epoxide form which then reacts with CO₂ and glutamate to generate γ-carboxyglutamic acid (Gla). Subsequently, vitamin K epoxide reductase (VKOR) is thought to convert the alkoxide-epoxide to a hydroquinone form. By recycling vitamin K, the two integral-membrane proteins, VKC and VKOR, maintain vitamin K levels and sustain the blood coagulation cascade. Unfortunately, NMR or X-ray crystal structures of the two proteins have not been characterized. Thus, our understanding of the vitamin K cycle is only partial at the molecular level. In this study, based on prior biochemical experiments on VKC and VKOR, we propose a hetero-dimeric form of VKC and VKOR that may explain the efficient oxidation and reduction of vitamin K during the vitamin K cycle.     

Warfarin resistance in a French strain of rats

A warfarin‐resistant strain and a warfarin‐susceptible strain of wild rats (Rattus norvegicus) maintained in enclosures of the National Veterinary School of Lyon (France) were studied to determine the mechanism of the resistance to anticoagulant rodenticides. A low vitamin K epoxide reductase (VKOR) activity has been reported for many resistant rat strains. As recently suggested, mutations in the vitamin K epoxide reductase subunit 1 (VKORC1) gene are the genetic basis of anticoagulant resistance in wild populations of rats from various locations in Europe. Here we report, for our strain, one of the seven described mutations (Tyr139Phe) for VKORC1 in rats. In addition, a low expression of mRNA encoding VKORC1 gene is observed in resistant rats, which could explain their low VKOR activity. We calculated kinetic parameters of VKOR in the warfarin‐resistant and warfarin‐susceptible rats. The V_max and the K_m of the VKOR obtained in resistant rats were lowered by 57 and 77%, respectively, compared to those obtained in susceptible rats. As a consequence, the enzymatic efficiency (V_m/K_m) of the VKOR was similar between resistant and susceptible rats. This result could be a good explanation to the observation that no clinical signs of vitamin K deficiency was observed in the warfarin‐resistant strain, while a low VKOR activity was found. VKOR activity in warfarin‐resistant rats was poorly inhibited by warfarin (K_i for warfarin is 29 μM and 0.72 μM for resistant and susceptible rats, respectively). © 2005 Wiley Periodicals, Inc. J Biochem Mol Toxicol 19:379‐385, 2005; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20104     

Characterization of bromadiolone resistance in a danish strain of Norway rats, Rattus norvegicus, by hepatic gene expression profiling of genes involved in vitamin K-dependent gamma-carboxylation

The present study characterizes the anticoagulant resistance mechanism in a Danish bromadiolone-resistant strain of Norway rats (Rattus norvegicus), with a Y139C VKORC1 mutation. We compared liver expression of the VKORC1 gene, which encodes a protein of the vitamin K 2,3-epoxide reductase complex, the NQO1 gene, which encodes a NAD(P)H quinone dehydrogenase and the Calumenin gene between bromadiolone-resistant and anticoagulant-susceptible rats upon saline and bromadiolone administration. Additionally, we established the effect of bromadiolone on the gene expression in the resistant and susceptible phenotype. Bromadiolone had no effect on VKORC1 and NQO1 expression in resistant rats, but induced significantly Calumenin expression in the susceptible rats. Calumenin expression was similar between the resistant and the susceptible rats upon saline administration but twofold lower in resistant rats after bromadiolone treatment. These results indicate that Danish bromadiolone resistance does not involve an overexpression of calumenin. Independent of the treatment, we observed a low VKORC1 expression in resistant rats, which in conjugation with the Y139C polymorphism most likely explains the low VKOR activity and the enhanced need for vitamin K observed in Danish resistant rats. Furthermore the bromadiolone resistance was found to be associated with a low expression of the NQO1 gene.     

Disulfide-dependent protein folding is linked to operation of the vitamin K cycle in the endoplasmic reticulum. A protein disulfide isomerase-VKORC1 redox enzyme complex appears to be responsible for vitamin K1 2,3-epoxide reduction

Gamma-carboxylation of vitamin K-dependent proteins is dependent on formation of reduced vitamin K1 (Vit.K1H2) in the endoplasmic reticulum (ER), where it works as an essential cofactor for gamma-carboxylase in post-translational gamma-carboxylation of vitamin K-dependent proteins. Vit.K1H2 is produced by the warfarin-sensitive enzyme vitamin K 2,3-epoxide reductase (VKOR) of the vitamin K cycle that has been shown to harbor a thioredoxin-like CXXC center involved in reduction of vitamin K1 2,3-epoxide (Vit.K>O). However, the cellular system providing electrons to the center is unknown. Here data are presented that demonstrate that reduction is linked to dithiol-dependent oxidative folding of proteins in the ER by protein disulfide isomerase (PDI). Oxidative folding of reduced RNase is shown to trigger reduction of Vit.K>O and gamma-carboxylation of the synthetic gamma-carboxylase peptide substrate FLEEL. In liver microsomes, reduced RNase-triggered gamma-carboxylation is inhibited by the PDI inhibitor bacitracin and also by small interfering RNA silencing of PDI in HEK 293 cells. Immunoprecipitation and two-dimensional SDS-PAGE of microsomal membrane proteins demonstrate the existence of a VKOR enzyme complex where PDI and VKORC1 appear to be tightly associated subunits. We propose that the PDI subunit of the complex provides electrons for reduction of the thioredoxin-like CXXC center in VKORC1. We can conclude that the energy required for gamma-carboxylation of proteins is provided by dithiol-dependent oxidative protein folding in the ER and thus is linked to de novo protein synthesis.     

Vitamin K epoxide reductase: homology, active site and catalytic mechanism

Vitamin K epoxide reductase (VKOR) recycles reduced vitamin K, which is used subsequently as a co-factor in the gamma-carboxylation of glutamic acid residues in blood coagulation enzymes. VKORC1, a subunit of the VKOR complex, has recently been shown to possess this activity. Here, we show that VKORC1 is a member of a large family of predicted enzymes that are present in vertebrates, Drosophila, plants, bacteria and archaea. Four cysteine residues and one residue, which is either serine or threonine, are identified as likely active-site residues. In some plant and bacterial homologues the VKORC1 homologous domain is fused with domains of the thioredoxin family of oxidoreductases. These might reduce disulfide bonds of VKORC1-like enzymes as a prerequisite for their catalytic activities.     

Tris(3-hydroxypropyl)phosphine is superior to dithiothreitol for in vitro assessment of vitamin K 2,3-epoxide reductase activity

Use of the reductant dithiothreitol (DTT) as a substrate for measuring vitamin K 2,3-epoxide reductase (VKOR) activity in vitro has been reported to be problematic because it enables side reactions involving the vitamin K₁ 2,3-epoxide (K₁>O) substrate. Here we characterize specific problems when using DTT and show that tris(3-hydroxypropyl)phosphine (THPP) is a reliable alternative to DTT for in vitro assessment of VKOR enzymatic activity. In addition, the pH buffering compound imidazole was found to be problematic in enhancing DTT-dependent non-enzymatic side reactions. Using THPP and phosphate-based pH buffering, we measured apparent Michaelis–Menten constants of 1.20 μM for K₁>O and 260 μM for the active neutral form of THPP. The K_m value for K₁>O is in agreement with the value that we previously obtained using DTT (1.24 μM). Using THPP, we successfully eliminated non-enzymatic production of 3-hydroxyvitamin K₁ and its previously reported base-catalyzed conversion to K₁, both of which were shown to occur when DTT and imidazole are used as the reductant and pH buffer, respectively, in the in vitro VKOR assay. Accordingly, substitution of THPP for DTT in the in vitro VKOR assay will ensure more accurate enzymatic measurements and assessment of warfarin and other 4-hydroxycoumarin inhibition constants.     

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

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.     

The inhibitory effect of calumenin on the vitamin K-dependent gamma-carboxylation system. Characterization of the system in normal and warfarin-resistant rats

The vitamin K-dependent gamma-carboxylation system is responsible for post-translational modification of vitamin K-dependent proteins, converting them to Gla-containing proteins. The system consists of integral membrane proteins located in the endoplasmic reticulum membrane and includes the gamma-carboxylase and the warfarin-sensitive enzyme vitamin K(1) 2,3-epoxide reductase (VKOR), which provides gamma-carboxylase with reduced vitamin K(1) cofactor. In this work, an in vitro gamma-carboxylation system was designed and used to understand how VKOR and gamma-carboxylase work together as a system and to identify factors that can regulate the activity of the system. Results are presented that demonstrate that the endoplasmic reticulum chaperone protein calumenin is associated with gamma-carboxylase and inhibits its activity. Silencing of the calumenin gene with siRNA resulted in a 5-fold increase in gamma-carboxylase activity. The results provide the first identification of a protein that can regulate the activity of the gamma-carboxylation system. The propeptides of vitamin K-dependent proteins stimulate gamma-carboxylase activity. Here we show that the factor X and prothrombin propeptides do not increase reduced vitamin K(1) cofactor production by VKOR in the system where VKOR is the rate-limiting step for gamma-carboxylation. These findings put calumenin in a central position concerning regulation of gamma-carboxylation of vitamin K-dependent proteins. Reduced vitamin K(1) cofactor transfer between VKOR and gamma-carboxylase is shown to be significantly impaired in the in vitro gamma-carboxylation system prepared from warfarin-resistant rats. Furthermore, the sequence of the 18-kDa subunit 1 of the VKOR enzyme complex was found to be identical in the two rat strains. This finding supports the notion that different forms of genetic warfarin resistance exist.     

Heterogeneity of the coumarin anticoagulant targeted vitamin K epoxide reduction system. Study of kinetic parameters in susceptible and resistant mice (Mus musculus domesticus)

Vitamin K epoxide reductase (VKOR) activity in liver microsomes from a susceptible and a genetically warfarin-resistant strain of mice (Mus Musculus domesticus) was analyzed to determine the mechanism of resistance to this 4-hydroxycoumarin derivative. Kinetic parameters for VKOR were calculated for each strain by incubating liver microsomes with vitamin K epoxide +/- warfarin. In susceptible mice, an Eadie-Hofstee plot of the data was not linear and suggested the involvement of at least two different components. Apparent kinetic parameters were obtained by nonlinear regression using a Michaelis--Menten model, which takes into account two enzymatic components. Component A presents a high Km and a high Vm, and as a consequence only an enzymatic efficiency Vm/Km was obtained (0.0024 mL/min/mg). Estimated warfarin Ki was 0.17 microM. Component B presented an apparent Km of 12.73 microM, an apparent Vm of 0.32 nmol/min/mg, and an apparent Ki for warfarin of 6.0 microM. In resistant mice, the enzymatic efficiency corresponding to component A was highly decreased (0.0003-0.00066 mL/min/mg) while the Ki for warfarin was not modified. The apparent Vm of component B was poorly modified between susceptible and resistant mice. The apparent Km of component B observed in resistant mice was similar to the Km observed in susceptible mice. These modifications of the catalytic properties are associated with a single nucleotide polymorphism (T175G) in the VKOR-C1 gene, which corresponds to a Trp59Gly mutation in the protein.     

Vitamin K-dependent carboxylation of glutamic acid residues to γ-carboxyglutamic acid in lung microsomes

Vitamin K-dependent carboxylation of glutamic acid residues to γ-carboxyglutamic acid was demonstrated in proteins of lung microsomes. The carboxylation was 12% of that in liver microsomes per milligram of mierosomal protein. Carboxylation was very low with microsomes of untreated rats but increased with time up to 42 h after warfarin administration. Carboxylation was highest with microsomes from rats fed a vitamin K-deficient diet. This suggests that a protein(s) accumulates which can be carboxylated in vitro/J. Lung microsomes also catalyzed the vitamin K-dependent carboxylation of the peptide Phe-Leu-Glu-Glu-Leu. The peptide carboxylase activity was 9% of that obtained with liver microsomes. Vitamin K-dependent protein carboxylation required NADH or dithioerythritol, suggesting that vitamin K had to be reduced to the hydroquinone. Accordingly, vitamin K₁ hydroquinone had carboxylating activity without added reducing agents. Menaquinone-3 was considerably more active than phylloquinone. The temperature optimum for carboxylation was around 27 °C.     

Direct measurement of vitamin K-dependent enzymes in various isolated and cultured tumor and non-tumor cells

A modification of the assay for vitamin K-dependent carboxylase is described with which the enzyme could be detected in relatively low amounts of cells (n = 10⁶). Using this assay, we could demonstrate vitamin K-dependent carboxylase activity in hepatocytes, renal tubular cells, osteoblasts, endothelial cells and macrophages, but not in lymphocytes or platelets. The cultured tumor cells UMR-106, B16 and 5583 also contained vitamin K-dependent carboxylase activity. Vitamin K epoxide reductase activity was demonstrated only in cells where vitamin K-dependent carboxylase activity was present. The tumor cells possessed remarkably less K epoxide reductase activity than the normal cells. When cells were cultured in medium containing warfarin, the K epoxide reductase activity was found to be decreased and the amount of non-carboxylated precursor protein and increased, suggesting an analogous vitamin K mechanism as in liver.     

Structural and functional insights into human vitamin K epoxide reductase and vitamin K epoxide reductase-like1

Abstract

Human vitamin K epoxide reductase (hVKOR) is a small integral membrane protein involved in recycling vitamin K. hVKOR produces vitamin K hydroquinone, a crucial cofactor for γ-glutamyl carboxylation of vitamin K dependent proteins, which are necessary for blood coagulation. Because of this, hVKOR is the target of a common anticoagulant, warfarin. Spurred by the identification of the hVKOR gene less than a decade ago, there have been a number of new insights related to this protein. Nonetheless, there are a number of key issues that have not been resolved; such as where warfarin binds hVKOR, or if human VKOR shares the topology of the structurally characterized but distantly related prokaryotic VKOR. The pharmacogenetics and single nucleotide polymorphisms of hVKOR used in personalized medicine strategies for warfarin dosing should be carefully considered to inform the debate. The biochemical and cell biological evidence suggests that hVKOR has a distinct fold from its ancestral protein, though the controversy will likely remain until structural studies of hVKOR are accomplished. Resolving these issues should impact development of new anticoagulants. The paralogous human protein, VKOR-like1 (VKORL1) was recently shown to also participate in vitamin K recycling. VKORL1 was also recently characterized and assigned a functional role as a housekeeping protein involved in redox homeostasis and oxidative stress with a potential role in cancer regulation. As the physiological interplay between these two human paralogs emerge, the impacts could be significant in a number of diverse fields from coagulation to cancer.