Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2005; 94(04): 780-786
DOI: 10.1160/TH05-02-0082

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

Site-directed mutagenesis of coumarin-type anticoagulant-sensitive VKORC1

Evidence that highly conserved amino acids define structural requirements for enzymatic activity and inhibition by warfarin

Simone Rost

¹Institute of Human Genetics, University of Würzburg, Biocenter, Würzburg, Germany

,

Andreas Fregin

¹Institute of Human Genetics, University of Würzburg, Biocenter, Würzburg, Germany

,

Mirja Hünerberg

¹Institute of Human Genetics, University of Würzburg, Biocenter, Würzburg, Germany

,

Carville G. Bevans

²Department of Structural Biology, Max Planck Institute of Biophysics, Frankfurt/Main, Germany

,

Clemens R. Müller

¹Institute of Human Genetics, University of Würzburg, Biocenter, Würzburg, Germany

,

Johannes Oldenburg

¹Institute of Human Genetics, University of Würzburg, Biocenter, Würzburg, Germany

³Institute of Transfusion Medicine and Immunohaematolgy, DRK Blood Donor Service Baden-Württemberg-Hessen, Frankfurt/M, Germany

⁴Institute of Experimental Haematology and Transfusion Medicine, University Clinic Bonn, Bonn, Germany

› Author Affiliations

Summary

Coumarin and homologous compounds are the most widely used anticoagulant drugs worldwide. They function as antagonists of vitamin K, an essential cofactor for the posttranslational γ-glutamyl carboxylation of the so-called vitamin K-dependent proteins. As vitamin K hydroquinone is converted to vitamin K epoxide (VKO) in every carboxylation step, the epoxide has to be recycled to the reduced form by the vitamin K epoxide reductase complex (VKOR). Recently, a single coumarin-sensitive protein of the putativeVKOR enzyme complex was identified in humans (vitamin K epoxide reductase complex subunit 1, VKORC1). Mutations inVKORC1 result in two different phenotypes: warfarin resistance (WR) and multiple coagulation factor deficiency type 2 (VKCFD2). Here, we report on the expression of site-directed VKORC1 mutants, addressing possible structural and functional roles of all seven cysteine residues (Cys16, Cys43, Cys51, Cys85, Cys96, Cys132, Cys135), the highly conserved residue Ser/Thr57, and Arg98, known to cause VKCFD2 in humans. Our results support the hypothesis that the C132-X-X-C135 motif inVKORC1 comprises part of the redox active site that catalyzes VKO reduction and also suggest a crucial role for the hydrophobicThr-Tyr-Ala motif in coumarin binding. Furthermore, our results support the concept that different structural components of VKORC1 define the binding sites for vitamin K epoxide and coumarin.

Keywords

Keywords

Vitamin K - VKOR - warfarin - CXXC motif - mutagenesis

References
1 Stenflo J, Fernlund P, Egan W. et al. Vitamin K dependent modifications of glutamic acid residues in prothrombin. Proc Natl Acad Sci U S A 1974; 71: 2730-3.
2 Hauschka PV, Reid ML. Vitamin K dependence of a calcium-binding protein containing gamma-carboxyglutamic acid in chicken bone. J Biol Chem 1978; 253: 9063-8.
3 Price PA, Urist MR, Otawara Y. Matrix Gla protein, a new gamma-carboxyglutamic acid-containing protein which is associated with the organic matrix of bone. Biochem Biophys Res Commun 1983; 117: 765-71.
4 Goruppi S, Ruaro E, Schneider C. Gas6, the ligand of Axl tyrosine kinase receptor, has mitogenic and survival activities for serum starved NIH3T3 fibroblasts. Oncogene 1996; 12: 471-80.
5 Locke D, Koreen IV, Hertzberg E L. et al. Isoelectric focusing and post-translational modifications of Cx26 and Cx32. Mol Biol Cell. 2004: 15S-266a.
6 Berkner KL. The vitamin K-dependent carboxylase. J Nutr 2000; 130: 1877-80.
7 Mukharji I, Silverman RB. Purification of a vitamin K epoxide reductase that catalyzes conversion of vitamin K 2,3-epoxide to 3-hydroxy-2-methyl-3-phytyl-2,3-dihydronaphthoquinone. Proc Natl Acad Sci U S A 1985; 82: 2713-7.
8 Cain D, Hutson SM, Wallin R. Assembly of the warfarin-sensitive vitamin K 2,3-epoxide reductase enzyme complex in the endoplasmic reticulum membrane. J Biol Chem 1997; 272: 29068-75.
9 Begent LA, Hill AP, Steventon G B. et al. Characterization and purification of the vitamin K1 2,3 epoxide reductases system from rat liver. J Pharm Pharmacol 2001; 53: 481-6.
10 Rost S, Fregin A, Ivaskevicius V. et al. Mutations in VKORC1 cause warfarin resistance and multiple coagulation factor deficiency type 2. Nature 2004; 427: 537-41.
11 Li T, Chang CY, Jin D Y. et al. Identification of the gene for vitamin K epoxide reductase. Nature 2004; 427: 541-4.
12 Oldenburg J, von Brederlow B, Fregin A. et al. Congenital deficiency of vitamin K dependent coagulation factors in two families presents as a genetic defect of the vitamin K-epoxide-reductase-complex. Thromb Haemost 2000; 84: 937-41.
13 Tie JK, Nicchitta C, von Heijne G. et al. Membrane topology mapping of vitamin K epoxide reductase by in vitro translation/cotranslocation. J Biol Chem 2005; 280: 16410-6.
14 Goodstadt L, Ponting CP. Vitamin K epoxide reductase: homology, active site and catalytic mechanism. Trends Biochem Sci 2004; 29: 289-92.
15 Powis G, Montfort WR. Properties and biological activities of thioredoxins. Annu Rev Biophys Biomol Struct 2001; 30: 421-55.
16 Soute BA, Groenen-van Dooren MM, Holmgren A. et al. Stimulation of the dithiol-dependent reductases in the vitamin K cycle by the thioredoxin system. Strong synergistic effects with protein disulphide-isomerase. Biochem J 1992; 281: 255-9.
17 Thijssen HH, Janssen YP, Vervoort LT. Microsomal lipoamide reductase provides vitamin K epoxide reductase with reducing equivalents. Biochem J 1994; 297: 277-80.
18 Wallin R, Martin LF. Vitamin K-dependent carboxylation and vitamin K metabolism in liver. Effects of warfarin. J Clin Invest 1985; 76: 1879-84.
19 Guex N, Peitsch MC. SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modeling. Electrophoresis 18: 2714-23.
20 Pelz HJ, Rost S, Hünerberg M. et al. The genetic basis of resistance to anticoagulants in rodents. Genetics. 2005 May 6; [Epub ahead of print].
21 Wajih N, Sane DC, Hutson S M. et al. Engineering of a recombinant vitamin K-dependent -carboxylation system with enhanced -carboxyglutamic acid (Gla) forming capacity: Evidence for a functional CXXC RedOx center in the system. J Biol Chem 2005; 280: 10540-7.
22 Häse CC, Fedorova ND, Galperin M Y. et al. Sodium ion cycle in bacterial pathogens: Evidence from crossgenome comparisons. Microbiol Mol Biol Rev 2001; 65: 353-70.
23 Thomsen-Zieger N, Pandini V, Caprini G. et al. A singlein vivo-selected point mutation in the active center of Toxoplasma gondii ferredoxin-NADP+ reductase leads to an inactive enzyme with greatly enhanced affinity for ferredoxin. FEBS Lett 2004; 576: 375-80.
24 Omdahl JL, Swamy N, Serda R. et al. Affinity labeling of rat cytochrome P450C24 (CYP24) and identification of Ser57 as an active site residue. J Steroid Biochem Mol Biol 2004; 89–90: 159-62.
25 Hall JM, Lind C, Golvano M P. et al. Akenson A, Ehrenberg A. Structure and Function of Oxidation Reduction Enzymes.. Oxford: Pergamon Press; 1972: 433-43.
26 Ma Q, Cui K, Xiao F. et al. Identification of a glycine-rich sequence as an NAD(P)H-binding site and tyrosine 128 as a dicumarol-binding site in rat liver NAD(P)H:quinone oxidoreductase by site-directed mutagenesis. J Biol Chem 1992; 267: 22298-304.
27 Kuijer PM, Hutten BA, Prins M H. et al. Prediction of the risk of bleeding during anticoagulant treatment for venous thromboembolism. Arch Intern Med 1999; 159: 457-60.