Engineering of a Recombinant Vitamin K-dependent γ-Carboxylation System with Enhanced γ-Carboxyglutamic Acid Forming Capacity

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. 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. The family of vitamin K-dependent proteins include the liver produced blood coagulation factors II, VII, IX, X, protein S, protein C, and protein Z (1Furie B. Furie C. Cell. 1988; 53: 505-518Abstract Full Text PDF PubMed Scopus (991) Google Scholar) and several proteins made in extrahepatic tissues. These proteins are osteocalcin (2Hauscha P.V. Lian J.B. Cole D.E. Gundberg C.M. Physiol. Rev. 1989; 69: 990-1047Crossref PubMed Scopus (1041) Google Scholar), matrix γ-carboxyglutamic acid (Gla) protein (2Hauscha P.V. Lian J.B. Cole D.E. Gundberg C.M. Physiol. Rev. 1989; 69: 990-1047Crossref PubMed Scopus (1041) Google Scholar), protein S (3Lei Shen X.H. Bjartell A. Dhalbäck B. J. Histochem. Cytochem. 1995; 43: 85-96Crossref PubMed Scopus (35) Google Scholar), growth arrest gene 6 (Gas6) (4Funakoshi H. Yonemasu T. Nakano T. Matumoto K. Nakamura T. J. Neurosci. Res. 2002; 68: 150-160Crossref PubMed Scopus (65) Google Scholar), and four putative trans-membrane proteins, PRGP1, PRGP2, TmG3, and TmG4, some of which are located in the brain (5Kulman J.D. Harris J.E. Haldeman B.A. Davie E.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9058-9062Crossref PubMed Scopus (94) Google Scholar, 6Kulman J.V. Harris J.E. Xie L. Davie E.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1370-1375Crossref PubMed Scopus (91) Google Scholar). The vitamin K-dependent proteins are modified post-translationally to contain Gla, Ca2+ binding residues. The modification is carried out by γ-carboxylase, an integral protein of the endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; Vit.KH2, chemically reduced vitamin K1H2; Vit.K>O, vitamin K 2,3-epoxide; BHK, baby hamster kidney; CHAPS, 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonic acid. membrane, which requires the reduced form of vitamin K (Vit.KH2) as cofactor (7Wallin R. Hutson S.M. Trends Mol. Med. 2004; 10: 299-302Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Concomitant with formation of Gla residues, Vit.KH2 is converted to vitamin K 2,3-epoxide (Vit.K>O) (7Wallin R. Hutson S.M. Trends Mol. Med. 2004; 10: 299-302Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The epoxide can be reduced and recycled to the Vit.KH2 cofactor by the warfarin sensitive enzyme vitamin K 2,3-epoxide reductase (VKOR) (7Wallin R. Hutson S.M. Trends Mol. Med. 2004; 10: 299-302Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), which also is an integral protein of the ER membrane (8Wajih N. Sane D.C. Hutson S.M. Wallin R. J. Biol. Chem. 2004; 279: 25276-25283Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). This redox cycling of vitamin K is known as that vitamin K cycle (9Suttie J.W. Deluca H.F. Handbook of Lipid Research. Plenum Press, New York1978: 211-277Google Scholar). The vitamin K-dependent γ-carboxylation system in the ER membrane consists of the vitamin K cycle enzymes plus additional proteins involved in regulation and formation of the Gla modification in vitamin K-dependent proteins (7Wallin R. Hutson S.M. Trends Mol. Med. 2004; 10: 299-302Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Purification of γ-carboxylase from the ER membrane and cloning of the enzyme (10Wu S.M. Cheung W.F. Frazier D. Stafford D.W. Science. 1991; 254: 1634-1636Crossref PubMed Scopus (171) Google Scholar) has resulted in a broad understanding of this membrane enzyme and the mechanism by which the enzyme carries out vitamin K-dependent modification of proteins (11Dowd P. Hershline R. Ham S.W. Naganathan S. Science. 1995; 269: 1684-1691Crossref PubMed Scopus (112) Google Scholar). On the other hand, very little is known about the enzymology of VKOR and the mechanism by which the vitamin K-dependent γ-carboxylation system operates in the ER membrane. This lack of insight into the mechanism of operation of the system stems from the difficulty of obtaining purified preparations of VKOR (12Cain D. Hutson S.M. Wallin R. J. Biol. Chem. 1998; 273: 4982-4989Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) for identification of the protein(s) responsible for VKOR activity. In our attempts to identify protein components of VKOR by photo affinity labeling with azido derivatives of warfarin, we identified a regulatory protein of the vitamin K-dependent γ-carboxylation system (12Cain D. Hutson S.M. Wallin R. J. Biol. Chem. 1998; 273: 4982-4989Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The protein was shown to be calumenin, a known ER chaperone belonging to the calcium binding, EF hand motif containing family of proteins (7Wallin R. Hutson S.M. Trends Mol. Med. 2004; 10: 299-302Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 8Wajih N. Sane D.C. Hutson S.M. Wallin R. J. Biol. Chem. 2004; 279: 25276-25283Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). The azido derivative of warfarin did not label the catalytic protein responsible for warfarin sensitive VKOR activity (12Cain D. Hutson S.M. Wallin R. J. Biol. Chem. 1998; 273: 4982-4989Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Recently the location of genetic warfarin resistance in man was mapped to the 16p12-q21 region on chromosome 16, and the effected gene was identified (13Fregin A. Rost S. Wolz W. Krebsova A. Muller C.R. Oldenburg J. Blood. 2002; 100: 3229-3232Crossref PubMed Scopus (58) Google Scholar). Rost et al. (14Rost S. Fregin A. Ivaskevicius V. Conzelmann E. Hortnagel K. Pelz H.J. Lappegard K. Seifried E. Scharrer I. Tuddenham E.G. Muller C.R. Strom T.M. Oldenburg J. Nature. 2004; 427: 537-541Crossref PubMed Scopus (987) Google Scholar) and Li et al. (15Li T. Chang C.Y. Jin D.Y. Lin P.J. Khvorova A. Stafford D.W. Nature. 2004; 427: 493-494Crossref PubMed Scopus (57) Google Scholar) showed that the gene encodes an 18-kDa transmembrane protein that exhibits warfarin sensitive VKOR activity when expressed in cell lines. The protein is believed to be a subunit (VKORC1) of a larger VKOR lipidenzyme complex in the ER membrane that we have shown is destroyed upon purification (12Cain D. Hutson S.M. Wallin R. J. Biol. Chem. 1998; 273: 4982-4989Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). In our efforts to understand how the vitamin K-dependent γ-carboxylation system functions at the molecular level, we have generated stably transfected BHK cells containing VKORC1 and γ-carboxylase cDNAs constructs, respectively, and stably double transfected BHK cells with VKORC1 and γ-carboxylase cDNA constructs in a bicistronic vector. The activities of VKOR and γ-carboxylase are shown to be significantly enhanced in all transfected cell lines with the highest activities measured in the double transfected cells. We present evidence that VKOR is the rate-limiting step in an in vitro vitamin K-dependent γ-carboxylation system where VKOR produces the Vit.KH2 cofactor for γ-carboxylase and show that the double transfected cells harbor a recombinant vitamin K-dependent γ-carboxylation system with enhanced capacity for post-translational Gla modification. Furthermore we show by mutagenesis that changing either of the two Cys residues in the proposed CXXC redox center in VKORC1 (16Goodstadt L. Ponting C.P. Trends Biochem. Sci. 2004; 29: 289-292Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) to Ser residues results in inactivation of VKOR activity. This finding confirms earlier data (17Lee J.J. Fasco M.J. Biochemistry. 1984; 23: 2246-2252Crossref PubMed Scopus (48) Google Scholar) that suggest that a Cys containing redox center in VKOR is essential for reduction of Vit.K>O to the Vit.KH2 γ-carboxylase cofactor. Materials—The γ-carboxylase peptide substrate FLEEL was from Sigma. The factor was by The VKORC1 and were by and used for in by the to recombinant were in as by the and at of All were affinity purified by our as N. T. W. Hutson S.M. Wallin R. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar) and in at CHAPS, vitamin warfarin, and the for with cells and were from Sigma. Vit.KH2 was as by et al. J.W. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and Vit.K>O was as by et al. J. Chem. Scopus Google Scholar). was from were from were and used for as by the and at of were as by our R. Hutson S.M. D. A. Sane D.C. J. 2001; PubMed Scopus Google Scholar). of the of and membrane proteins, from of liver were in of with a and at for R. Sane D.C. Hutson S.M. Res. Full Text Full Text PDF Scopus Google Scholar). The was in of with the and a at for were at were used for of VKORC1 and of an of from liver was used for cDNA to the The were used in for VKORC1 The were purified purification and on a The purified was into cloning were identified by and on the and and were generated at the and of VKORC1 cDNA to the cDNA into the for in cell lines. was also generated at the of VKOR The modified cDNA for VKORC1 was into the and of the the of with cDNA for VKORC1 was on to The new with VKORC1 cDNA is of and of an The containing γ-carboxylase was a from of of and were generated at the and to the cDNA into the vector. was also generated at the The modified cDNA for γ-carboxylase was into the and on the of the with cDNA for γ-carboxylase was on the to The new with γ-carboxylase cDNA is of VKORC1 and is a of expressing two recombinant has two cloning the of two and The γ-carboxylase was from and The γ-carboxylase was and into and on the The VKORC1 and γ-carboxylase cDNAs. The new was and of the putative CXXC redox center in VKORC1 (16Goodstadt L. Ponting C.P. Trends Biochem. Sci. 2004; 29: 289-292Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) was to Ser residues the mutagenesis to the the Cys132/Ser mutant protein the were and was the Cys135/Ser mutant protein the were and was mutagenesis the modified VKOR cDNAs were into the as for cloning of the VKORC1 cDNA into the vector. was used to BHK cells were transfected the 6 to the cells were at into growth modified containing with for containing was for 6 the of cell lines expressing VKORC1, cells were at of in modified containing were was used to BHK cells. were transfected and stably transfected of cells as were VKORC1 and was used to BHK cells. were transfected and stably transfected of cells as were VKORC1 containing the Cys132/Ser and the Cys135/Ser respectively, were used to BHK cells. were transfected and stably transfected of cells as were in for with the K-dependent were and in and in a in CHAPS, containing of the protein was of VKORC1 from liver were in a in CHAPS, containing of the The was with VKORC1 affinity purified were The was at An the peptide as were to the and were by VKOR activities in the and the were and of in were in containing for as by J. Biol. Chem. Full Text PDF PubMed Google Scholar) and modified by our R. Proc. Natl. Acad. Sci. U. S. A. 1984; PubMed Scopus Google Scholar). was by the was to the was carried out and in the in as (12Cain D. Hutson S.M. Wallin R. J. Biol. Chem. 1998; 273: 4982-4989Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). were with protein were from the and to for protein The was at the by on a proteins were to and with to peptide and as N. T. W. Hutson S.M. Wallin R. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar) the plus system from and of in were and in and with the modified containing of the proteins in were with to of cell proteins to was carried out as sensitive VKOR activity was measured as R. J. PubMed Scopus Google Scholar) by the of Vit.K>O to vitamin The vitamin and the epoxide was on a in and activity was as R. Sane D.C. Hutson S.M. Res. Full Text Full Text PDF Scopus Google Scholar) as into the peptide The was either by chemically reduced vitamin K1H2 (Vit.KH2) to the by reduced Vit.KH2 in a containing Vit.K>O and Both were carried out as R. Sane D.C. Hutson S.M. Res. Full Text Full Text PDF Scopus Google Scholar) with FLEEL for the The factor was to the to a of of a of the acid of The the on the of transmembrane at a at The and this protein we from two of the in the VKORC1 and acid are shown in the VKORC1 residues the Cys was to the of peptide for to and for affinity purification of peptide of proteins in and with the two peptide The peptide a the peptide a protein and a a of proteins present in the protein on the identified by the peptide and the peptide shown in C, two proteins a and were identified with the peptide the peptide only the protein in The protein in was to and identified as I. D. K. PubMed Scopus Google Scholar). the and we that the peptide an on the that peptide on we on the peptide as for VKORC1 which as a protein in and not as an 18-kDa protein by acid that the peptide VKORC1, we tested the of to VKOR from were with The of the by VKOR activity the which was that did not contain the peptide peptide of VKORC1, the were to all enzymes VKOR activity in a of and and of proteins in from liver were as of proteins present in VKORC1 and VKORC1 show of the proteins in with the VKORC1 of the proteins shown in with the peptide and peptide The to the protein and by the shown in was from the and to for protein The in BHK the enzyme activities of the γ-carboxylation system measured in BHK cells stably transfected with VKORC1 (VKOR) and γ-carboxylase cDNA constructs, respectively, and BHK cells stably double transfected with cDNA constructs VKOR activities measured in all cell lines were sensitive to warfarin not shown in γ-carboxylase activity with was not enhanced in cells transfected with the VKORC1 (VKOR) the On the other hand, Vit.KH2 γ-carboxylase activity was significantly in γ-carboxylase transfected and the double transfected cells γ-carboxylase activity was measured in the double transfected cells. VKOR activity measured in the This activity measured in VKOR transfected (VKOR) and the double transfected cells was enhanced significantly the On the other hand VKOR activity measured in the γ-carboxylase transfected cells was not significantly from the The results shown in and demonstrate that of VKORC1 and γ-carboxylase in BHK cells not of in the γ-carboxylation system when on enzyme activity The results shown in about the γ-carboxylation system in the stably transfected cell lines. In which were carried out with the used for the shown in and C, VKOR is the reduced Vit.KH2 cofactor for In the double transfected BHK cells the highest activity measured as into the FLEEL γ-carboxylase This activity was also enhanced in the VKORC1 transfected cells (VKOR). in the γ-carboxylase transfected cells the activity was not significantly from the These results that of γ-carboxylase not post-translational γ-carboxylation of proteins, VKOR is the rate-limiting step in the γ-carboxylation system, and the double transfected BHK cells to be cells containing a recombinant vitamin K-dependent γ-carboxylation system of enhanced post-translational γ-carboxylation of of VKORC1 in the transfected BHK cells and cells. with the showed enhanced VKORC1 protein in VKOR transfected and the double transfected cells. of are also shown for the of calumenin, which we have shown is an of the γ-carboxylation system in the ER (8Wajih N. Sane D.C. Hutson S.M. Wallin R. J. Biol. Chem. 2004; 279: 25276-25283Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), was in the cells tested of on the in BHK of vitamin K-dependent proteins are with a that is the that the to the γ-carboxylase for post-translational γ-carboxylation of the proteins (1Furie B. Furie C. Cell. 1988; 53: 505-518Abstract Full Text PDF PubMed Scopus (991) Google Scholar). In the the γ-carboxylase, which results in enhanced γ-carboxylation of proteins and J.W. J. A. H. B. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, J.E. J.W. J. Biol. Chem. Full Text PDF PubMed Google Scholar, C. Furie B. Furie J. Biol. Chem. 1989; Full Text PDF PubMed Google Scholar). have an on the recombinant γ-carboxylation system in BHK we the factor to the from the stably transfected cell lines. The of the factor was made upon the of binding to γ-carboxylase Jin D.Y. Lin P.J. Stafford D.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). for of the γ-carboxylation system we used our system, which requires VKOR to the γ-carboxylase with Vit.KH2 cofactor. shown in of the factor the system in cells In γ-carboxylase transfected cells factor the system be that the activity of the system in γ-carboxylase transfected cells was not significantly from the activity measured in that of γ-carboxylase not in enhanced γ-carboxylation of On the other hand, factor the system in VKORC1 transfected cells In the double transfected the system was factor of the system in the double transfected the system in the double transfected cells significantly activity the system in the VKORC1 transfected cells. VKORC1 with an CXXC of VKORC1 from a CXXC redox center in all proteins (16Goodstadt L. Ponting C.P. Trends Biochem. Sci. 2004; 29: 289-292Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). In the the CXXC center is at (16Goodstadt L. Ponting C.P. Trends Biochem. Sci. 2004; 29: 289-292Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). shown in the center is to be located in the transmembrane We two mutant cDNA constructs of VKORC1 in which of the two Cys residues in the CXXC center were by Ser residues, mutant VKOR activities measured in BHK cells stably transfected with the mutant and cDNA constructs, respectively, and the VKORC1 cDNA VKOR activity measured in cells transfected with the was enhanced the activity measured in cells The activity measured in cells transfected with the mutant was from the activity measured in cells. The activity measured in cells transfected with the mutant was the activity measured in cells. with the peptide of the protein and the mutant proteins not The results the (16Goodstadt L. Ponting C.P. Trends Biochem. Sci. 2004; 29: 289-292Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) that the 132CXXC135 center in VKORC1 functions as the redox center in VKOR. This is the to demonstrate engineering of stably transfected cells a recombinant vitamin K-dependent γ-carboxylation system. These BHK cells are shown to have an enhanced capacity to the γ-carboxylase peptide substrate FLEEL to contain γ-carboxyglutamic acid residues γ-carboxylation of by liver preparations has been shown to enhanced γ-carboxylation of of vitamin K-dependent proteins present in the J.W. J. A. H. B. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, J.E. J.W. J. Biol. Chem. Full Text PDF PubMed Google Scholar, C. Furie B. Furie J. Biol. Chem. 1989; Full Text PDF PubMed Google Scholar). the enhanced capacity of our engineered cells to FLEEL is to also the of cells to increased of Gla containing finding be of for of recombinant vitamin K-dependent proteins used as in of with of is with of factor J.D. 2004; PubMed Scopus Google Scholar). requires of factor recombinant factor produced by hamster cells is used as a C.M. B. Blood. 2001; 98: PubMed Scopus Google Scholar). with of recombinant vitamin K-dependent coagulation factors has been of proteins produced by and from transfected cells Furie Furie B. J. Biol. Chem. Full Text PDF PubMed Google Scholar). cloning of the γ-carboxylase (10Wu S.M. Cheung W.F. Frazier D. Stafford D.W. Science. 1991; 254: 1634-1636Crossref PubMed Scopus (171) Google Scholar), cDNA was into hamster cells stably transfected with a factor cDNA with the of of factor by the cells A. A. Furie B. Furie Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus (65) Google Scholar). was not A. A. Furie B. Furie Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus (65) Google Scholar). This finding is in with our results where we show that the γ-carboxylation system in BHK cells stably transfected with a γ-carboxylase cDNA exhibits γ-carboxylation activity that is from the activity measured in BHK cells. results demonstrate that VKOR not is the rate-limiting step in the system. This is with earlier data by our on the γ-carboxylation system in liver R. Sane D.C. Hutson S.M. Res. Full Text Full Text PDF Scopus Google Scholar) and also with results by B.A. Biochemistry. 2002; PubMed Scopus Google Scholar). the successful cloning of the VKORC1 subunit of the putative VKOR enzyme complex, have new experimental approaches to understand how the vitamin K-dependent γ-carboxylation system works at the molecular level in the ER membrane. earlier that are involved in the catalytic mechanism of Vit.K>O reduction by VKOR and that warfarin to to a redox center in VKOR. The for the mechanism of of VKOR by warfarin from work by Fasco et al. M.J. Biochemistry. PubMed Scopus (91) Google Scholar). experimental which also were by R. J. Biochem. PubMed Scopus Google Scholar), to the that warfarin to an redox center and reduction of the center by an The of the has not been with the VKORC1 subunit of VKOR for the earlier proposed redox center M.J. Biochemistry. 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PubMed Scopus (91) Google Scholar). not the two binding are located on the VKORC1 protein are present on proteins a VKOR enzyme complex to be The for the existence of a VKOR enzyme complex is by carried out by our (12Cain D. Hutson S.M. Wallin R. J. Biol. Chem. 1998; 273: 4982-4989Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) and by et al. D.C. J. 2001; 53: PubMed Scopus Google Scholar) where increased VKOR activity be measured by protein from of ER proteins and by the finding that can be produced from Vit.K>O by an ER enzyme that is to warfarin I. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). of the in the ER C. H.F. Science. PubMed Scopus Google Scholar, J. Biol. Chem. 2004; Full Text Full Text PDF Scopus (137) Google Scholar), VKOR and γ-carboxylase in the ER membrane as a γ-carboxylation system (7Wallin R. Hutson S.M. Trends Mol. Med. 2004; 10: 299-302Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). This is to the reduced Vit.KH2 cofactor produced by VKOR and used by γ-carboxylase (7Wallin R. Hutson S.M. Trends Mol. Med. 2004; 10: 299-302Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). We that the two enzymes be in the ER membrane the two enzymes can be by (12Cain D. Hutson S.M. Wallin R. J. Biol. Chem. 1998; 273: 4982-4989Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). the existence of a vitamin K-dependent γ-carboxylation system of where ER membrane also an (12Cain D. Hutson S.M. Wallin R. J. Biol. Chem. 1998; 273: 4982-4989Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). In this work we have engineered a recombinant vitamin K-dependent γ-carboxylation system in BHK cells that be used to the molecular mechanism by which this system carries out γ-carboxylation of vitamin K-dependent