Expression, Purification and Characterization of GDP-D-Mannose 4,6-Dehydratase from Escherichia Coli
| Autor: | Davide Zanardi, Umberto Benatti, Michela Tonetti, Laura Sturla, Angela Bisso, Antonio De Flora |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 1997 |
| Předmět: |
Thymidine diphosphate
Recombinant Fusion Proteins Molecular Sequence Data Biophysics Mannose lac operon Gene Expression Random hexamer medicine.disease_cause Biochemistry l-Fucose metabolism chemistry.chemical_compound Structural Biology (Escherichia coli) Genetics medicine Escherichia coli Enzyme Inhibitors Molecular Biology Chromatography High Pressure Liquid Hydro-Lyases Glutathione Transferase chemistry.chemical_classification GDP-l-fucose biosynthesis Cell Biology NAD Fusion protein Molecular biology Enzyme chemistry NADP+ Dehydratase Chromatography Gel GDP-d-mannose dehydratase NADP |
| Popis: | GDP-D-mannose dehydratase (GMD) catalyzes the first step of the pathway that converts GDP-D-mannose to GDP- L-fucose in bacteria, plants and mammals. Recently, the gene coding for GMD has been identified and sequenced in E. coli. Based on this sequence, we have expressed and purified GMD in E. coli as a glutathione transferase (GST) fusion protein. The fused GST-GMD protein and the thrombin-cleaved GMD were then characterized. The catalytically active form of both enzyme species seems to be a hexamer of 410 and 250 kDa, respectively. The GST-GMD fusion protein has a Km of 0.22 ± 0.04 mM and a specific activity of 2.3 ±0.2 /ffliol/h/mg. Ca2+ and Mg2+ activate GMD, while GDP-L-j3-fucose, the end-product of the pathway, inhibits it specifically. The GST-GMD fusion protein contains one mole of tightly bound NADP+ per mole of hexamer. Apparently, this NADP+ is involved in the catalytic mechanism of GMD. © 1997 Federation of European Biochemical Societies. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|