title: 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.
abstract: 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.
authors.affiliation: Institute of Human Genetics, University of Würzburg, Biocenter, Würzburg, Germany.
authors.forename: Simone
authors.initials: S
authors.lastname: Rost
authors.forename: Andreas
authors.initials: A
authors.lastname: Fregin
authors.forename: Mirja
authors.initials: M
authors.lastname: Hünerberg
authors.forename: Carville G
authors.initials: CG
authors.lastname: Bevans
authors.forename: Clemens R
authors.initials: CR
authors.lastname: Müller
authors.forename: Johannes
authors.initials: J
authors.lastname: Oldenburg
mesh_terms: D000595:Amino Acid Sequence; D000925:Anticoagulants / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D003374:Coumarins / Q000378:metabolism / Q000494:pharmacology*; D004220:Disulfides / Q000378:metabolism; D004351:Drug Resistance / Q000235:genetics*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007668:Kidney / Q000166:cytology; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010837:Vitamin K 1 / Q000031:analogs & derivatives / Q000378:metabolism; D064417:Vitamin K Epoxide Reductases
chemical_list: D000925:Anticoagulants; D003374:Coumarins; D004220:Disulfides; C016186:vitamin K1 oxide; D010837:Vitamin K 1; C030123:coumarin; D006899:Mixed Function Oxygenases; C578523:VKORC1 protein, human; D064417:Vitamin K Epoxide Reductases