title: Conserved loop cysteines of vitamin K epoxide reductase complex subunit 1-like 1 (VKORC1L1) are involved in its active site regeneration.
abstract: Vitamin K epoxide reductase complex subunit 1 (VKORC1) reduces vitamin K epoxide in the vitamin K cycle for post-translational modification of proteins that are involved in a variety of biological functions. However, the physiological function of VKORC1-like 1 (VKORC1L1), a paralogous enzyme sharing about 50% protein identity with VKORC1, is unknown. Here we determined the structural and functional differences of these two enzymes using fluorescence protease protection (FPP) assay and an in vivo cell-based activity assay. We show that in vivo VKORC1L1 reduces vitamin K epoxide to support vitamin K-dependent carboxylation as efficiently as does VKORC1. However, FPP assays show that unlike VKORC1, VKORC1L1 is a four-transmembrane domain protein with both its termini located in the cytoplasm. Moreover, the conserved loop cysteines, which are not required for VKORC1 activity, are essential for VKORC1L1's active site regeneration. Results from domain exchanges between VKORC1L1 and VKORC1 suggest that it is VKORC1L1's overall structure that uniquely allows for active site regeneration by the conserved loop cysteines. Intermediate disulfide trapping results confirmed an intra-molecular electron transfer pathway for VKORC1L1's active site reduction. Our results allow us to propose a concerted action of the four conserved cysteines of VKORC1L1 for active site regeneration; the second loop cysteine, Cys-58, attacks the active site disulfide, forming an intermediate disulfide with Cys-139; the first loop cysteine, Cys-50, attacks the intermediate disulfide resulting in active site reduction. The different membrane topologies and reaction mechanisms between VKORC1L1 and VKORC1 suggest that these two proteins might have different physiological functions.
authors.affiliation: From the Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599.
authors.forename: Jian-Ke
authors.initials: JK
authors.lastname: Tie
authors.forename: Da-Yun
authors.initials: DY
authors.lastname: Jin
authors.forename: Darrel W
authors.initials: DW
authors.lastname: Stafford
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002462:Cell Membrane / Q000378:metabolism; D017124:Conserved Sequence; D004220:Disulfides / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D013329:Structure-Activity Relationship; D014812:Vitamin K / Q000031:analogs & derivatives / Q000378:metabolism; D064417:Vitamin K Epoxide Reductases / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D004220:Disulfides; D014812:Vitamin K; C578523:VKORC1 protein, human; C531327:VKORC1L1 protein, human; D064417:Vitamin K Epoxide Reductases
keywords: Disulfide; Electron Transfer; Endoplasmic Reticulum (ER); Enzymes; Membrane Proteins; Post-translational Modification; Protein Carboxylation; Vitamin K