Characterization and purification of the vitamin K1 2,3 epoxide reductase system from rat liver

Begent, Louise A.; Hill, Anthony; Steventon, Glyn B.; Hutt, Andrew J.; Pallister, C. J.; Cowell, David C.

doi:10.1211/0022357011775776

Cited by 22 publications

(15 citation statements)

References 23 publications

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“…The CXXC motif is the active site in a number of thioredoxin and protein disulfide isomerase enzymes (32). In keeping with this idea, there is extensive literature detailing the importance of cysteines in the activity of VKOR (11,12,14,(33)(34)(35)(36)(37)(38) and that the active site cysteines reside in a hydrophobic environment (33). Recently, Wajih et al (39) reported that mutation of Cys-132 or Cys-135 (the CXXC motif in VKOR) to alanine abolished VKOR activity.…”

Section: Discussionmentioning

confidence: 99%

“…Warfarin inhibition of VKOR reduces the availability of reduced vitamin K, which reduces the rate of carboxylation and leads to partially carboxylated, inactive vitamin K-dependent proteins. Since its discovery in 1970 (6), numerous futile attempts to purify the enzyme were reported (7)(8)(9)(10)(11). Attempts to understand the mechanism underlying warfarinsensitive vitamin K epoxide reduction have been somewhat more successful (8,(12)(13)(14)(15).…”

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confidence: 99%

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Membrane Topology Mapping of Vitamin K Epoxide Reductase by in Vitro Translation/Cotranslocation

Tie

Nicchitta

Heijne

et al. 2005

Journal of Biological Chemistry

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Vitamin K epoxide reductase (VKOR) catalyzes the conversion of vitamin K 2,3-epoxide into vitamin K in the vitamin K redox cycle. Recently, the gene encoding the catalytic subunit of VKOR was identified as a 163-amino acid integral membrane protein. In this study we report the experimentally derived membrane topology of VKOR. Our results show that four hydrophobic regions predicted as the potential transmembrane domains in VKOR can individually insert across the endoplasmic reticulum membrane in vitro. However, in the intact enzyme there are only three transmembrane domains, residues 10 -29, 101-123, and 127-149, and membrane-integration of residues 75-97 appears to be suppressed by the surrounding sequence. Results of N-linked glycosylation-tagged full-length VKOR shows that the N terminus of VKOR is located in the endoplasmic reticulum lumen, and the C terminus is located in the cytoplasm. Further evidence for this topological model of VKOR was obtained with freshly prepared intact microsomes from insect cells expressing HPC4-tagged full-length VKOR. In these experiments an HPC4 tag at the N terminus was protected from proteinase K digestion, whereas an HPC4 tag at the C terminus was susceptible. Altogether, our results suggest that VKOR is a type III membrane protein with three transmembrane domains, which agrees well with the prediction by the topology prediction program TMHMM.The K vitamins, phylloquinone (K1), menaquinones (K2), and menadione (K3), are a family of structurally similar, fatsoluble, 2-methyl-1,4-naphthoquinones. The main function of vitamin K is to act as a co-factor for the ␥-glutamyl carboxylase that catalyzes the post-translational carboxylation of specific glutamic acid to ␥-carboxyglutamic acid (Gla) 1 of variety of vitamin K-dependent proteins (1). Members of the vitamin K-dependent protein family include coagulation factors (factor II, VII, IX, X) as well as several other proteins that function in bone metabolism (2) and signal transduction (3). Concomitant with ␥-glutamyl carboxylation, the reduced form of vitamin K (vitamin K hydroquinone) is converted to vitamin K 2,3-epoxide, which must be converted back to vitamin K hydroquinone for the reaction to continue because of limited vitamin K amounts in vivo (4). This cyclic conversion of vitamin K establishes a redox cycle known as the vitamin K cycle (5).VKOR is responsible for the conversion of vitamin K 2,3-epoxide into vitamin K and is highly sensitive to inhibition by coumarin drugs, such as R,S-warfarin (4-hydroxy-3-(3-oxo-1-phenylbutyl-coumarin)), the most commonly prescribed oral anticoagulant. Warfarin inhibition of VKOR reduces the availability of reduced vitamin K, which reduces the rate of carboxylation and leads to partially carboxylated, inactive vitamin K-dependent proteins. Since its discovery in 1970 (6), numerous futile attempts to purify the enzyme were reported (7-11). Attempts to understand the mechanism underlying warfarinsensitive vitamin K epoxide reduction have been somewhat more successful (8,(12)(13)(14)(15)....

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Membrane Topology Mapping of Vitamin K Epoxide Reductase by in Vitro Translation/Cotranslocation

Tie

Nicchitta

Heijne

et al. 2005

Journal of Biological Chemistry

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“…Finally if we consider that the catalytic center is localized in the two transmembrane helix alpha (amino acid 100-120 and AA 128-148, [32,33]), the importance of AA59 (the mutation observed in our resistant strain is Trp59Gly), which is in a cytoplasmic loop of the protein, should be more in the interaction with the other proteins of the VKOR complex [34] or in the protein conformation. In such an hypothesis, the VKOR-C1 protein should be in equilibrium within two conformations (high affinity, low capacity, high K i corresponding to component B and low affinity, high capacity and low K i , corresponding to component A) and the mutation should allow the protein to be in the first conformation only.…”

Section: Discussionmentioning

confidence: 99%

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

Lasseur¹,

Grandemange²,

Longin-Sauvageon³

et al. 2006

J Biochem & Molecular Tox

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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.

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“…26 The enzyme vitamin K epoxide reductase (VKOR) was identified as warfarin's target 30 years ago, 27,28 but the actual enzyme proved difficult to purify. 29 The gene of the major protein component of VKOR was eventually mapped to human chromosome 16p12-q21, 30 and it was recently identified as VKOR complex subunit 1 (VKORC1). 31,32 VKORC1 spans 4 kilobases (kb), consists of three exons (NM_024006), and encodes a 163 amino-acid enzyme located in the endoplasmic reticulum (NP_076869).…”

Section: Introductionmentioning

confidence: 99%

Common VKORC1 and GGCX polymorphisms associated with warfarin dose

et al. 2005

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We report a novel combination of factors that explains almost 60% of variable response to warfarin. Warfarin is a widely used anticoagulant, which acts through interference with vitamin K epoxide reductase that is encoded by VKORC1. In the next step of the vitamin K cycle, gamma-glutamyl carboxylase encoded by GGCX uses reduced vitamin K to activate clotting factors. We genotyped 201 warfarin-treated patients for common polymorphisms in VKORC1 and GGCX. All the five VKORC1 single-nucleotide polymorphisms covary significantly with warfarin dose, and explain 29-30% of variance in dose. Thus, VKORC1 has a larger impact than cytochrome P450 2C9, which explains 12% of variance in dose. In addition, one GGCX SNP showed a small but significant effect on warfarin dose. Incorrect dosage, especially during the initial phase of treatment, carries a high risk of either severe bleeding or failure to prevent thromboembolism. Genotype-based dose predictions may in future enable personalised drug treatment from the start of warfarin therapy.

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Characterization and purification of the vitamin K1 2,3 epoxide reductase system from rat liver

Cited by 22 publications

References 23 publications

Membrane Topology Mapping of Vitamin K Epoxide Reductase by in Vitro Translation/Cotranslocation

Membrane Topology Mapping of Vitamin K Epoxide Reductase by in Vitro Translation/Cotranslocation

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

Common VKORC1 and GGCX polymorphisms associated with warfarin dose

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