The deoxyxylulose phosphate pathway of isoprenoid biosynthesis: Studies on the mechanisms of the reactions catalyzed by IspG and IspH protein
| Autor: | Stefan Hecht, Adelbert Bacher, Felix Rohdich, Petra Adam, Tobias Gräwert, Duilio Arigoni, Wolfgang Eisenreich, Johannes Kaiser, Sabine Amslinger, Ralf Laupitz, Ferdinand Zepeck |
|---|---|
| Rok vydání: | 2003 |
| Předmět: |
Flavodoxin
Blotting Western Protein domain Mutant Catalysis Chromatography Affinity law.invention Xylulose Maltose-binding protein Bacterial Proteins Polyisoprenyl Phosphates law Escherichia coli Bioorganic chemistry Nuclear Magnetic Resonance Biomolecular Multidisciplinary biology Chemistry Escherichia coli Proteins Biological Sciences Fusion protein Transformation (genetics) Biochemistry biology.protein Recombinant DNA Oxidoreductases |
| Zdroj: | Proceedings of the National Academy of Sciences. 100:1586-1591 |
| ISSN: | 1091-6490 0027-8424 |
| DOI: | 10.1073/pnas.0337742100 |
| Popis: | Earlier in vivo studies have shown that the sequential action of the IspG and IspH proteins is essential for the reductive transformation of 2 C -methyl- d -erythritol 2,4-cyclodiphosphate into dimethylallyl diphosphate and isopentenyl diphosphate via 1-hydroxy-2-methyl-2-( E )-butenyl 4-diphosphate. A recombinant fusion protein comprising maltose binding protein and IspG protein domains was purified from a recombinant Escherichia coli strain. The purified protein failed to transform 2 C -methyl- d -erythritol 2,4-cyclodiphosphate into 1-hydroxy-2-methyl-2-( E )-butenyl 4-diphosphate, but catalytic activity could be restored by the addition of crude cell extract from an ispG -deficient E. coli mutant. This indicates that auxiliary proteins are required, probably as shuttles for redox equivalents. On activation by photoreduced 10-methyl-5-deaza-isoalloxazine, the recombinant protein catalyzed the formation of 1-hydroxy-2-methyl-2-( E )-butenyl 4-diphosphate from 2 C -methyl- d -erythritol 2,4-cyclodiphosphate at a rate of 1 nmol⋅min −1 ⋅mg −1 . Similarly, activation by photoreduced 10-methyl-5-deaza-isoalloxazine enabled purified IspH protein to catalyze the conversion of 1-hydroxy-2-methyl-2-( E )-butenyl 4-diphosphate into a 6:1 mixture of isopentenyl diphosphate and dimethylallyl diphosphate at a rate of 0.4 μmol⋅min −1 ⋅mg −1 . IspH protein could also be activated by a mixture of flavodoxin, flavodoxin reductase, and NADPH at a rate of 3 nmol⋅min −1 ⋅mg −1 . The striking similarities of IspG and IspH protein are discussed, and plausible mechanistic schemes are proposed for the two reactions. |
| Databáze: | OpenAIRE |
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