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.