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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
1   Institute of Human Genetics, University of Würzburg, Biocenter, Würzburg, Germany
,
Andreas Fregin
1   Institute of Human Genetics, University of Würzburg, Biocenter, Würzburg, Germany
,
Mirja Hünerberg
1   Institute of Human Genetics, University of Würzburg, Biocenter, Würzburg, Germany
,
Carville G. Bevans
2   Department of Structural Biology, Max Planck Institute of Biophysics, Frankfurt/Main, Germany
,
Clemens R. Müller
1   Institute of Human Genetics, University of Würzburg, Biocenter, Würzburg, Germany
,
Johannes Oldenburg
1   Institute of Human Genetics, University of Würzburg, Biocenter, Würzburg, Germany
3   Institute of Transfusion Medicine and Immunohaematolgy, DRK Blood Donor Service Baden-Württemberg-Hessen, Frankfurt/M, Germany
4   Institute of Experimental Haematology and Transfusion Medicine, University Clinic Bonn, Bonn, Germany
› Author AffiliationsGrant support:The study was supported by grants from the DFG (01-100/3), the National Genome Research Net Cardiovascular Diseases (BMBF-DLR-01GS 0424) and Baxter to J.O.
Further Information

Publication History

Received02 February 2005

Accepted after resubmission16 July 2005

Publication Date:
07 December 2017 (online)

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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

Vitamin K - VKOR - warfarin - CXXC motif - mutagenesis
 

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