Abstract
Vitamin K epoxide reductase (VKOR) is an essential enzyme for vitamin K-dependent carboxylation, while the physiological function of its paralogous enzyme VKOR-like (VKORL) is yet unknown. Although these two enzymes share approximately 50% protein sequence homology, the membrane topology of VKOR is still in debate. Here, we explored the differences in the membrane topology and disulfide-linked oligomerization of these two enzymes. Results from mutating the critical amino acid residues in the disputed transmembrane (TM) regions revealed that the second TM domain in the proposed 4-TM model of VKOR does not function as an authentic TM helix; supporting VKOR is a 3-TM protein, which is different from VKORL. Additionally, altering the loop sequence between the two conserved cysteine residues of VKORL affects its activity, supporting the notion that the conserved loop cysteines of VKORL are involved in its active site regeneration. However, a similar mutation in VKOR does not affect its enzymatic activity. Finally, our results show that although both VKOR and VKORL form disulfide-linked oligomers, the cysteine residues involved in the oligomerization appear to be different. Overall, the structural and functional differences between VKOR and VKORL shown here indicate that VKORL might have a different physiological function other than recycling vitamin K.
Highlights
Vitamin K is a family of 2-methyl-1,4-naphthoquinone derivatives that includes the naturally occurring menaquinones and phylloquinone
We have previously shown that Vitamin K epoxide reductase (VKOR) can be altered to a functional 4-TM molecule, the VKOR charge mutant (VKOR-CM), by mutating the charged residues flanking VKOR’s TMD120
The results show that the I86P mutation has only a minor effect on the activity of VKOR, but it has a dramatic effect on the activity of the VKOR-CM mutant, decreasing its activity to ~10% (Fig. 1D)
Summary
Vitamin K is a family of 2-methyl-1,4-naphthoquinone derivatives that includes the naturally occurring menaquinones and phylloquinone. KH2 is a necessary cofactor for GGCX which is responsible for the carboxylation of specific glutamic acid residues of vitamin K-dependent (VKD) proteins to γ-carboxyglutamic acid[6]. Compatible with the well-known bacterial VKOR homologue; this model proposes that both the amino terminus (N-terminus) and the carboxyl terminus (C-terminus) are located in the cytosol and that both the loop and the active site cysteines face the ER lumen. The 3-TM model proposes that the N-terminus and the active site face the ER lumen, and that the loop cysteines and the C-terminus are located in the cytosol. Previous results from our lab support the 3-TM topology model of VKOR and demonstrate that the loop cysteines are unnecessary for active site regeneration[20,21]. VKOR expression was found to be 10-fold higher than that of VKORL in liver tissue where the coagulation factors are produced; greater VKORL expression levels are observed in extrahepatic tissues where it could function to rescue VKA therapy
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