Novel Insight into the Mechanism of the Vitamin K Oxidoreductase (VKOR)

Abstract

The vitamin K oxidoreductase (VKOR) reduces vitamin K to support the carboxylation and consequent activation of vitamin K-dependent proteins, but the mechanism of reduction is poorly understood. VKOR is an integral membrane protein that reduces vitamin K using membrane-embedded thiols (Cys-132 and Cys-135), which become oxidized with concomitant VKOR inactivation. VKOR is subsequently reactivated by an unknown redox protein that is currently thought to act directly on the Cys132-Cys135 residues. However, VKOR contains evolutionarily conserved Cys residues (Cys-43 and Cys-51) that reside in a loop outside of the membrane, raising the question of whether they mediate electron transfer from a redox protein to Cys-132/Cys-135. To assess a possible role, the activities of mutants with Ala substituted for Cys (C43A and C51A) were analyzed in intact membranes using reductants that were either membrane-permeable or -impermeable. Both reductants resulted in wild type VKOR reduction of vitamin K epoxide; however, the C43A and C51A mutants only showed activity with the membrane-permeant reductant. We obtained similar results when testing the ability of wild type and mutant VKORs to support carboxylation, using intact membranes from cells coexpressing VKOR and carboxylase. These results indicate a role for Cys-43 and Cys-51 in catalysis, suggesting a relay mechanism in which a redox protein transfers electrons to these loop residues, which in turn reduce the membraneembedded Cys132-Cys135 disulfide bond to activate VKOR. The results have implications for the mechanism of warfarin resistance, the topology of VKOR in the membrane, and the interaction of VKOR with the carboxylase.

Intramembrane Thiol Oxidoreductases: Evolutionary Convergence and Structural Controversy

“…For different bacterial VKORs and a human VKOR-like protein, the shuttle cysteines are clearly required for their in vitro and in vivo activities 15,24,36,49 . Consistently, it was later showed for hVKOR that, with the shuttle cysteines mutated, its reductase activity cannot be maintained in vitro 50 . However, in a cell-based assay, hVKOR with the Cys51Ala mutation, the Cys43/Cys51 double mutation, or a deletion from Cys43 to Cys51 retains most of the activity, although Cys43Ala alone is nearly inactive 51 .…”

Adaptative evolution of the Vkorc1 gene in Mus musculus domesticus is influenced by the selective pressure of anticoagulant rodenticides

“…This variability being partly due to genetic polymorphisms in the Vkorc1 gene, numerous studies have been performed to detect new Vkorc1 mutations in patients resistant to AVK. These studies allowed to detect in humans 27 mutations in the coding sequence of the Vkorc1 gene (Bodin, Horellou, Flaujac, Loriot, & Samama, 2005; Bodin, Perdu, Diry, Horellou, & Loriot, 2008; D'Andrea et al., 2005; Harrington, Siddiq, Allford, Shearer, & Mumford, 2011; Harrington et al., 2008; Loebstein et al., 2007; Osman, Enström, Arbring, Söderkvist, & Lindahl, 2006; Peoc'h, Pruvot, Gourmel, Dit Sollier, & Drouet, 2009; Rieder et al., 2005; Rishavy, Usubalieva, Hallgren, & Berkner, 2011; Rost et al., 2004; Schmeits et al., 2010; Watzka et al., 2011; Wilms, Touw, Conemans, Veldkamp, & Hermans, 2008). In rats, 15 missense mutations in the Vkorc1 gene of Rattus norvegicus have been described in Europe (Grandemange, Lasseur, Longin‐Sauvageon, Benoit, & Berny, 2010; Haniza et al., 2015; Pelz et al., 2005, 2012; Rost et al., 2004).…”

Disulfide bond formation in prokaryotes: History, diversity and design

“…DsbB and VKORs from bacteria, archaea and vertebrates share in common their overall mechanism of action as well as their architecture (Figure 2) [48–50]. These similarities include the presence of two pairs of redox-active cysteines, one of them the redox active motif CXXC.…”

“…Several of the experimental approaches aimed at probing the activity of VKORC1 in microsomal preparations use the dithiol reducing agent DTT [56]. This membrane diffusible chemical can directly reduce the CXXC motif, bypassing thereby the need for the second pair of active cysteines to provide reducing equivalents [50]. It is worth mentioning that in some prokaryotes (such as Natronomonas pharaonis , Halobiforma nitratireducens , Bacillus cellulosilyticus and Bacillus pseudofirmus ), homologues of both DsbB and VKOR can be identified [18].…”