The vitamin K-dependent γ-carboxylation system in the endoplasmic reticulum membrane responsible for γ-carboxyglutamic acid modification of vitamin K-dependent proteins includes γ-carboxylase and vitamin K 2,3-epoxide reductase (VKOR). An understanding of the mechanism by which this system works at the molecular level has been hampered by the difficulty of identifying VKOR involved in warfarin sensitive reduction of vitamin K 2,3-epoxide to reduced vitamin K1H2, the γ-carboxylase cofactor. Identification and cloning of VKORC1, a proposed subunit of a larger VKOR enzyme complex, have provided opportunities for new experimental approaches aimed at understanding the vitamin K-dependent γ-carboxylation system. In this work we have engineered stably transfected baby hamster kidney cells containing γ-carboxylase and VKORC1 cDNA constructs, respectively, and stably double transfected cells with the γ-carboxylase and the VKORC1 cDNA constructs in a bicistronic vector. All engineered cells showed increased activities of the enzymes encoded by the cDNAs. However increased activity of the γ-carboxylation system, where VKOR provides the reduced vitamin K1H2 cofactor, was measured only in cells transfected with VKORC1 and the double transfected cells. The results show that VKOR is the rate-limiting step in the γ-carboxylation system and demonstrate successful engineering of cells containing a recombinant vitamin K-dependent γ-carboxylation system with enhanced capacity for γ-carboxyglutamic acid modification. The proposed thioredoxin-like 132CXXC135 redox center in VKORC1 was tested by expressing the VKORC1 mutants Cys132/Ser and Cys135/Ser in BHK cells. Both of the expressed mutant proteins were inactive supporting the existence of a CXXC redox center in VKOR.
