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

• Published in: Thrombosis and haemostasis Vol. 94; no. 4; p. 780
• Main Authors: Rost, Simone, Fregin, Andreas, Hünerberg, Mirja, Bevans, Carville G, Müller, Clemens R, Oldenburg, Johannes
• Format: Journal Article
• Language: English
• Published: Germany 01.10.2005
• Subjects: Amino Acid Sequence; Anticoagulants - metabolism; Anticoagulants - pharmacology; Binding Sites - genetics; Cell Line; Coumarins - metabolism; Coumarins - pharmacology; Disulfides - metabolism; Drug Resistance - genetics; Humans; Hydrophobic and Hydrophilic Interactions; Kidney - cytology; Mixed Function Oxygenases - chemistry; Mixed Function Oxygenases - genetics; Mixed Function Oxygenases - metabolism; Molecular Sequence Data; Mutagenesis, Site-Directed; Vitamin K 1 - analogs & derivatives; Vitamin K 1 - metabolism; Vitamin K Epoxide Reductases
• ISSN: 0340-6245
• DOI: 10.1160/TH05-02-0082

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 gamma-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 putative VKOR enzyme complex was identified in humans (vitamin K epoxide reductase complex subunit 1, VKORC1). Mutations in VKORC1 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 in VKORC1 comprises part of the redox active site that catalyzes VKO reduction and also suggest a crucial role for the hydrophobic Thr-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.