Purification of a Single Peptide with Vitamin K Epoxide to Vitamin K and Vitamin K to Vitamin KH2 Activity.

Abstract More than 21 million prescriptions for warfarin are written yearly in the US. Yet, in spite of its importance, vitamin K epoxide reductase (VKOR), the target of warfarin, has resisted purification since its identification in 1972. We report the first successful purification and reconstitution of activity of a recombinant human vitamin K epoxide reductase. A series of detergents were screened to determine that best for solubilization of VKOR from microsomes. Detergents tested that were effective in solubilization of VKOR also led to loss of measurable activity. This loss of activity supports our previous prediction that VKOR is embedded in and requires a membrane environment for enzymatic activity. The short-chain phospholipid, DHPC (1,2-Dihexanoyl-sn-Glycero-3-Phosphocholine) was the detergent of choice to efficiently extract VKOR from the microsomes, even though this reagent completely inhibited enzyme activity. Partial reconstitution was achieved on-column by washing with 0.4 % dioleoylphosphatidylcholine/0.4% deoxycholate. Complete recovery of activity was achieved by removing the deoxycholate through dialysis in the presence of the reducing reagent, THP (Tris(hydroxypropyl)phosphine). During dialysis, the solution became cloudy indicating the formation of membrane-like structure. Purified recombinant VKOR is ~21 kDa (~18.5 kDa + tag); fully active; and over 93% pure. The concentration of warfarin for 50% inhibition is the same for purified protein and microsomes. It has been reported and assumed that VKOR is a multi-subunit enzyme. Our results, however, suggest that a single peptide can accomplish the reaction. The trace amounts of contaminating proteins were identified by mass spectrometry; however, none are apparently relevant to the VKOR reaction. Moreover, the turn-over number of purified VKOR (0.25 sec-1 is approximately two-fold higher than microsomes and about 10 fold higher than the turnover number of gamma-glutamyl carboxylase for CO2 addition. In addition to the vitamin K epoxide to vitamin K reaction, our results also indicate that VKOR can efficiently convert vitamin K to vitamin K epoxide. Our results suggest that ancillary proteins (other than a thioredoxin-like enzyme) are not necessary for full VKOR activity. This purification will allow further characterization of VKOR in relation to other components of the vitamin K cycle and should facilitate its structural determination.

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Microsomal lipoamide reductase provides vitamin K epoxide reductase with reducing equivalents

Biochemical Journal ◽

10.1042/bj2970277 ◽

1994 ◽

Vol 297 (2) ◽

pp. 277-280 ◽

Cited By ~ 21

Author(s):

H H W Thijssen ◽

Y P G Janssen ◽

L T M Vervoort

Keyword(s):

Vitamin K ◽

Lipoic Acid ◽

Reductase Activity ◽

Vitamin K Epoxide Reductase ◽

Vitamin K Cycle ◽

Redox Active ◽

Starting Point ◽

Epoxide Reductase ◽

Triton X ◽

Reducing Equivalents

This study was undertaken to search for the endogenous dithiol cofactor of the reductases of the vitamin K cycle. As a starting point, the redox-active lipophilic endogenous compounds lipoic acid and lipoamide were looked at. The study shows that microsomes contain NADH-dependent lipoamide reductase activity. Reduced lipoamide stimulates microsomal vitamin K epoxide reduction with kinetics comparable with those for the synthetic dithiol dithiothreitol (DTT). Reduced lipoic acid shows higher (4-fold) Km values. No reductase activity with lipoic acid was found to be present in microsomes or cytosol. The reduced-lipoamide-stimulated vitamin K epoxide reductase is as sensitive to warfarin and salicylate inhibition as is the DTT-stimulated one. Both vitamin K epoxide reductase and lipoamide reductase activity are recovered in the rough microsomes. NADH/lipoamide-stimulated vitamin K epoxide reduction is uncoupled by traces of Triton X-100, suggesting that microsomal lipoamide reductase and vitamin K epoxide reductase are associated. The results suggest that the vitamin K cycle obtains reducing equivalents from NADH through microsomal lipoamide reductase.

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Solubilization and characterization of vitamin K epoxide reductase from normal and warfarin-resistant rat liver microsomes

Archives of Biochemistry and Biophysics ◽

10.1016/0003-9861(84)90014-6 ◽

1984 ◽

Vol 228 (2) ◽

pp. 480-492 ◽

Cited By ~ 45

Author(s):

E.F. Hildebrandt ◽

P.C. Preusch ◽

J.L. Patterson ◽

J.W. Suttie

Keyword(s):

Rat Liver ◽

Vitamin K ◽

Liver Microsomes ◽

Rat Liver Microsomes ◽

Vitamin K Epoxide Reductase ◽

Epoxide Reductase

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Overexpression of protein disulfide isomerase enhances vitamin K epoxide reductase activity

Biochemistry and Cell Biology ◽

10.1139/bcb-2021-0441 ◽

2022 ◽

Author(s):

Thomas Chetot ◽

Etienne Benoit ◽

Véronique Lambert ◽

Virginie Lattard

Keyword(s):

Vitamin K ◽

Protein Disulfide Isomerase ◽

Reductase Activity ◽

Functional Characterization ◽

Hek293 Cells ◽

Vitamin K Epoxide Reductase ◽

Disulfide Isomerase ◽

Vitamin K Cycle ◽

Epoxide Reductase ◽

Protein Disulfide

Vitamin K epoxide reductase (VKOR) activity is catalyzed by the VKORC1 enzyme. It is the target of vitamin K antagonists (VKA). Numerous mutations of VKORC1 have been reported and have been suspected to confer resistance to VKA and/or affect its velocity. Nevertheless, the results between studies have been conflicting, the functional characterization of these mutations in a cell system being complex due to the interweaving of VKOR activity in the vitamin K cycle. In this study, a new cellular approach was implemented to globally evaluate the vitamin K cycle in the HEK293 cells. This global approach was based on the vitamin K quinone/vitamin K epoxide (K/KO) balance. In the presence of VKA or when the VKORC1/VKORC1L1 were knocked out, the K/KO balance decreased significantly due to an accumulation of vitamin KO. On the contrary, when VKORC1 was overexpressed, the balance remained unchanged, demonstrating a limitation of the VKOR activity. This limitation was shown to be due to an insufficient expression of the activation partner of VKORC1, as overexpressing the protein disulfide isomerase (PDI) overcomes the limitation. This study is the first to demonstrate a functional interaction between VKORC1 and the PDI enzyme.

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Key Pathways and Regulators of Vitamin K Function and Intermediary Metabolism

Annual Review of Nutrition ◽

10.1146/annurev-nutr-082117-051741 ◽

2018 ◽

Vol 38 (1) ◽

pp. 127-151 ◽

Cited By ~ 24

Author(s):

Martin J. Shearer ◽

Toshio Okano

Keyword(s):

Intestinal Absorption ◽

Vitamin K ◽

Intermediary Metabolism ◽

Biological Functions ◽

Vitamin K Epoxide Reductase ◽

Pathophysiological Mechanisms ◽

Vitamin K Cycle ◽

Rare Mutations ◽

Epoxide Reductase ◽

Key Pathways

Vitamin K (VK) is an essential cofactor for the post-translational conversion of peptide-bound glutamate to γ-carboxyglutamate. The resultant vitamin K–dependent proteins are known or postulated to possess a variety of biological functions, chiefly in the maintenance of hemostasis. The vitamin K cycle is a cellular pathway that drives γ-carboxylation and recycling of VK via γ-carboxyglutamyl carboxylase (GGCX) and vitamin K epoxide reductase (VKOR), respectively. In this review, we show how novel molecular biological approaches are providing new insights into the pathophysiological mechanisms caused by rare mutations of both GGCX and VKOR. We also discuss how other protein regulators influence the intermediary metabolism of VK, first through intestinal absorption and second through a pathway that converts some dietary phylloquinone to menadione, which is prenylated to menaquinone-4 (MK-4) in target tissues by UBIAD1. The contribution of MK-4 synthesis to VK functions is yet to be revealed.

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Lack of Association Between Type 2 Diabetes and the 3673G / A and 9041G / A Gene Variants of Vitamin K Epoxide Reductase Complex Subunit 1 (VKORC1)

International Journal for Vitamin and Nutrition Research ◽

10.1024/0300-9831/a000302 ◽

2016 ◽

Vol 86 (3-4) ◽

pp. 133-139

Author(s):

Cansu Ozbayer ◽

Hulyam Kurt ◽

Medine Nur Kebapci ◽

Didem Turgut Cosan ◽

Ertugrul Colak ◽

...

Keyword(s):

Type 2 Diabetes ◽

Vitamin K ◽

Control Group ◽

Gene Variants ◽

Vitamin K Epoxide Reductase ◽

Vitamin K Cycle ◽

Genotype Frequencies ◽

Epoxide Reductase ◽

Vkorc1 Gene

The vitamin K epoxide reductase complex subunit 1 (VKORC1) gene is expressed in many tissue types, and encodes the VKORC1 protein, which is a key enzyme in the vitamin K cycle. Although researchers have focused on the effects of vitamin K on glucose metabolism, and on its role in the development of type 2 diabetes (T2DM), no consensus has yet been reached. Therefore, here we aimed to investigate the association between VKORC1 variants and the risk of T2DM. The 3673G / A (rs9923231) and 9041G / A (rs7294) VKORC1 variants were investigated by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 100 control individuals and 100 patients with T2DM. The genomic regions were amplified by PCR; amplicons were digested using the AciI and NciI enzymes and visualized by agarose gel electrophoresis. The genotype frequencies of the 3673G / A variants were GG (22%), GA (56%), and AA (22%) in the control group and GG (19%), GA (52%), and AA (29%) in patients with T2DM (p > 0.05). The genotype frequencies of the 9041G / A variants were GG (37%), GA (53%), and AA (10%) in the control group and GG (46%), GA (45%), and AA (9%) in patients with T2DM (p > 0.05). In conclusion, we found no significant correlation between the control group and patients with T2DM, with regard to the different genetic models of the 3673G / A and 9041G / A variants. These data suggest that these VKORC1 gene variants may not be linked to T2DM.

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Presence of a T383G Mutation in the Vitamin K Epoxide Reductase Gene (VKORC1) in a Patient Resistant to Four Different Vitamin K Antagonists.

Blood ◽

10.1182/blood.v104.11.4068.4068 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4068-4068

Author(s):

Michel-Meyer M.M. Samama ◽

Laurent L. Bodin ◽

Marie Helene M.H. Horellou ◽

Florence F. Parent ◽

Anne A. Kereveur ◽

...

Keyword(s):

Molecular Weight ◽

Vitamin K ◽

Low Molecular Weight Heparin ◽

Vitamin K Antagonists ◽

Low Molecular Weight ◽

Clotting Factors ◽

Vitamin K Epoxide Reductase ◽

Vitamin K Cycle ◽

Epoxide Reductase ◽

Warfarin Resistance

Abstract Resistance to vitamin K antagonists is a rare disorder which until recently has not been associated to a genetic factor. The existence of inherited warfarin resistant rat strains has been related to mutations of the genes involved in the vitamin K cycle. Vitamin K dependent carboxylase was cloned in 19911, while human vitamin K epoxide reductase complex 1 (VKORC1), another enzyme implicated in vitamin K cycle, has been cloned more recently2,3. Mutations in this gene are responsible for both human and rat warfarin resistance 3. In 1997, we reported 4 a 63-year old patient with recurrent pulmonary embolism and deep vein thrombosis without known hereditary or acquired thrombophilia who was found resistant to warfarin (up to 45mg/day), fluindione (up to 80mg/day), acenocoumarol (up to 12 mg/day) and phenprocoumon (up to 30 mg/day). With phenprocoumon, long-acting vitamin K antagonist, drug concentration reached 85 mg/L (usual range 1–5 mg/L) but the INR remained around 1. Daily low molecular weight heparin (LMWH) was continued 3 months. Treatment was discontinued on 2 occasions and a recurrent thrombotic episode was observed. The patient is now receiving a single daily dose of low molecular weight heparin (80 mg Enoxaparin, body weight 120 kg, 67 IU/kg once a day) for the past 10 years without thrombotic or bleeding episodes. Osteodensitometry remains normal after 9 years of treatment. Careful biochemichal investigation had demonstrated a deficiency in vitamin K dependent carboxylation and absence of blockade of vitamin K epoxide reductase by phenprocoumon. This year, we sequenced the three exons of VKORC1 in this patient and detected a heterozygous T383G transversion resulting in a leucine to arginine substitution (L128R). This mutation has been recently identified by Rost3 et al. in an individual with warfarin resistance. It was not found in 259 control subjects that we had tested. Interestingly, another gene mutation of the VKORC1 can be responsible for a combined deficiency of vitamin K dependent clotting factors 3. In conclusion, a resistance to all vitamin K antagonists, including warfarin up to 45 mg/day is extremely rare and the mutation T383G of the VKORC gene has been reported in only one patient before the case reported here. Testing for mutation in VKORC1 will help in explaining some cases of anti vitamin K resistance.

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Mutational Analysis of VKOR.

Blood ◽

10.1182/blood.v108.11.1628.1628 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1628-1628

Author(s):

Da-Yun Jin ◽

Jian-Ke Tie ◽

Darrel W. Stafford

Keyword(s):

Vitamin K ◽

Disulfide Bond ◽

Active Sites ◽

Mutational Analysis ◽

Residual Activity ◽

Wild Type ◽

Vitamin K Epoxide Reductase ◽

The Us ◽

Epoxide Reductase ◽

Inhibition Studies

Abstract More than 21 million prescriptions for warfarin are written yearly in the US for Vitamin K epoxide reductase (VKOR), the target of warfarin The vitamin K epoxide reductase, VKOR, apparently uses cysteines, 132 and 135 as active sites. In addition to cysteines 132 and 135, cysteines 43 and 51 are conserved throughout evolution. Rost et al. have mutated each of the cysteines in VKOR and found that, in whole cell lysates, mutations in of C43 result in less than 20% of wild-type activity while mutation of C53 eliminates activity. We have repeated these experiments and mutated all of the cysteine residues in VKOR. Our results in microsomes are similar to the results of Rost et. al. (2005) Thromb. Haemost. 94, 780–786. However, when the mutated enzymes are purified we find that the activity of C43 has 30% residual activity while C51 has 60% residual activity. Mutation of both residues in the same molecule results in an enzyme that is similar to the C51 mutation. In addition, we find that a portion of purified VKOR has a disulfide bond between residues 43 and 51. This suggested that we might be able to remove the loop between C43 and C51 and retain activity. Indeed, the mutated enzyme with this loop removed also has substantial activity. Warfarin inhibition studies suggest that these mutations do not materially affect warfarin sensitivity. Our results stress the importance of utilizing purified enzyme for interpreting the results of mutations in VKOR.

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