ABSTRACT
The membrane topology of vitamin K epoxide reductase (VKOR) is controversial with data supporting both a three transmembrane and a four transmembrane model. The positioning of the transmembrane domains and the loops between these domains is critical if we are to understand the mechanism of vitamin K oxidation and its recycling by members of the thioredoxin family of proteins and the mechanism of action of warfarin, an inhibitor of VKOR. Here we show that both mammalian VKOR isoforms adopt the same topology, with the large loop between transmembrane one and two facing the lumen of the endoplasmic reticulum (ER). We used a redox sensitive green fluorescent protein (GFP) fused to the N- or C-terminus to show that these regions face the cytosol, and introduction of glycosylation sites along with mixed disulfide formation with thioredoxin-like transmembrane protein (TMX) to demonstrate ER localization of the major loop. The topology is identical with the bacterial homologue from Synechococcussp., for which the structure and mechanism of recycling has been characterized. Our results provide a resolution to the membrane topology controversy and support previous results suggesting a role for members of the ER protein disulfide isomerase (PDI) family in recycling VKOR.
Subject(s)
Bacterial Proteins/chemistry , Cell Membrane/chemistry , Synechococcus/chemistry , Vitamin K Epoxide Reductases/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cell Line , Cell Membrane/genetics , Cell Membrane/metabolism , Humans , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Synechococcus/genetics , Synechococcus/metabolism , Vitamin K Epoxide Reductases/genetics , Vitamin K Epoxide Reductases/metabolismABSTRACT
Alcohols are converted into to their corresponding carbonyl compounds using catalytic amounts of 1,4-hydroquinone with a copper nanoparticle electron transfer mediator with oxygen as the terminal oxidant in acetone as solvent under visible light irradiation. These conditions employing biorenewable hydroquinone as reagent were developed from initial experiments using stoichiometric amounts of 1,4-benzoquinone as oxidant. A range of benzylic and aliphatic primary and secondary alcohols are oxidized, affording the corresponding aldehydes or ketones in moderate to excellent yields. The methodology is also applicable to the oxidative degradation of lignin model compounds that undergo C-C bond cleavage to give simple aromatic compounds.
