Abstract
Background/Purpose: Vitamin K is involved in the gamma-carboxylation of the vitamin K dependent proteins. Due to the limited intake of vitamin K, its regeneration is necessary and involves the vitamin K 2,3-epoxide reductase (VKOR) activity. This activity is catalyzed by VKORC1 and/or VKORC1L1 proteins. Warfarin is able to inhibit both enzymes, but VKORC1L1 appears to be 30-fold more resistant to warfarin than VKORC1. Methods: To predict functional peptide regions or amino acid residues important for VKOR activity or resistance to vitamin K antagonists (VKA) of human VKORC1L1, we conducted a multiple alignment of VKORC1L1 and VKORC1 sequences. The role of conserved amino acid residues between VKORC1 and VKORC1L1, but also the role of conserved amino-acid residues in VKORC1L1 but not in VKORC1 were challenged by systematic engineering of point mutations combined with in vitro functional assays. Findings: Interestingly, engineering mutants at position 130 allowed us to obtain a VKORC1L1 as susceptible to VKA as wild type VKORC1. Our results also suggested the involvement of Cys-43(+7), Cys-51(+7), Cys-132(+7) and Cys-135(+7), in the transfer of the redox power to vitamin K epoxide. Conclusion: Altogether, this study provides novel insight into VKORC1L1 active site functional domains. Glu-130 is a key residue governing the natural resistance of VKORC1L1 to VKAs.
Highlights
The vitamin K epoxide reductase (VKOR) activity has been described since the early 70s’ [1]
Glu-130 is a key residue governing the natural resistance of VKORC1L1 to vitamin K antagonist (VKA)
While VKORC1 was inhibited by all the VKAs used in this study with Ki lower than 0.5 μM, VKORC1L1 was resistant to some VKAs with Ki systematically higher than 0.5 μM
Summary
The vitamin K epoxide reductase (VKOR) activity has been described since the early 70s’ [1] This VKOR activity allows the reduction of vitamin K epoxide in order to regenerate vitamin K quinone. This activity is a key step of the vitamin cycle and is the target of vitamin K antagonist (VKA). The VKORC1 gene has only been identified in 2004 [2,3] This gene encodes for a 163 amino acid protein with, probably, four transmembrane domains and a luminal loop. This protein contains a C132XXC135 redox motif, as previously hypothesized by Silverman [4].
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