menaquinone-6 and geranylgeranyl-pyrophosphate

To date, there is no functional characterization of EmGGPPS (from Elizabethkingia meningoseptica sp.F2) as enzymes catalyzing GGPP. In this research, maltose-binding protein (MBP), disulfide bond A (DbsA), disulfide bond C (DbsC), and two other small protein tags, GB1 (Protein G B1 domain) and ZZ (Protein A IgG ZZ repeat domain), were used as fusion partners to construct an EmGGPPS fusion expression system. The results indicated that the expression of MBP-EmGGPPS was higher than that of the other four fusion proteins in E. coli BL21 (DE3). Additionally, using EmGGPPS as a catalyst for the production of GGPP was verified using a color complementation assay in Escherichia coli. In parallel with it, the enzyme activity experiment in vitro showed that the EmGGPPS protein could produce GGPP, GPP and FPP. Finally, we successfully demonstrated MK-4 production in engineered E. coli by overexpression of EmGGPPS.

Topics: Amino Acid Sequence; Cloning, Molecular; Disulfides; Escherichia coli; Farnesyltranstransferase; Flavobacteriaceae; Gene Expression; Maltose-Binding Proteins; Plasmids; Polyisoprenyl Phosphates; Recombinant Fusion Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Vitamin K 2

Schnyder's crystalline corneal dystrophy (SCCD) is a rare autosomal dominant genetic disorder that is characterized by progressive corneal opacity, owing to aberrant accumulation of cholesterol and phospholipids in the cornea. A number of SCCD affected families have been reported in the world since 1924, when it was first described. In 2007, the molecular basis of SCCD was demonstrated to be associated with a tumor suppressor, UbiA prenyltransferase domain‑containing 1 (UBIAD1), which was isolated from the bladder mucosa and demonstrated to be involved in vitamin K

Topics: Animals; Cholesterol; Cornea; Corneal Dystrophies, Hereditary; Dimethylallyltranstransferase; Endoplasmic Reticulum; Genes, Tumor Suppressor; Genetic Predisposition to Disease; Humans; Mutation; Polyisoprenyl Phosphates; Vitamin K 2

UBIAD1 (UbiA prenyltransferase domain-containing protein-1) utilizes geranylgeranyl pyrophosphate (GGpp) to synthesize vitamin K

Topics: Cells, Cultured; Cholesterol; Dimethylallyltranstransferase; Endoplasmic Reticulum; Endoplasmic Reticulum-Associated Degradation; Fibroblasts; Golgi Apparatus; Humans; Hydroxymethylglutaryl CoA Reductases; Membrane Proteins; Mevalonic Acid; Polyisoprenyl Phosphates; Sterols; Terpenes; Vitamin K 2

UbiA prenyltransferase domain-containing protein-1 (UBIAD1) utilizes geranylgeranyl pyrophosphate (GGpp) to synthesize the vitamin K2 subtype menaquinone-4. Previously, we found that sterols trigger binding of UBIAD1 to endoplasmic reticulum (ER)-localized HMG-CoA reductase, the rate-limiting enzyme in synthesis of cholesterol and nonsterol isoprenoids, including GGpp. This binding inhibits sterol-accelerated degradation of reductase, which contributes to feedback regulation of the enzyme. The addition to cells of geranylgeraniol (GGOH), which can become converted to GGpp, triggers release of UBIAD1 from reductase, allowing for its maximal degradation and permitting ER-to-Golgi transport of UBIAD1. Here, we further characterize geranylgeranyl-regulated transport of UBIAD1. Results of this characterization support a model in which UBIAD1 continuously cycles between the ER and medial-trans Golgi of isoprenoid-replete cells. Upon sensing a decline of GGpp in ER membranes, UBIAD1 becomes trapped in the organelle where it inhibits reductase degradation. Mutant forms of UBIAD1 associated with Schnyder corneal dystrophy (SCD), a human eye disease characterized by corneal accumulation of cholesterol, are sequestered in the ER and block reductase degradation. Collectively, these findings disclose a novel sensing mechanism that allows for stringent metabolic control of intracellular trafficking of UBIAD1, which directly modulates reductase degradation and becomes disrupted in SCD.

Topics: Cell Membrane; Corneal Dystrophies, Hereditary; Dimethylallyltranstransferase; Endoplasmic Reticulum; Golgi Apparatus; Humans; Hydroxymethylglutaryl CoA Reductases; Lipid Metabolism; Polyisoprenyl Phosphates; Protein Transport; Proteolysis; Terpenes; Vitamin K; Vitamin K 2