Rohdich, F. and Zepeck, F. and Adam, P. and Hecht, S. and Kaiser, J. and Laupitz, R. and Gräwert, T. and Amslinger, S. and Eisenreich, W. and Bacher, A. and Arigoni, D. (2003) The deoxyxylulose phosphate pathway of isoprenoid biosynthesis: Studies on the mechanisms of the reactions catalyzed by IspG and IspH protein. Proc. Natl. Acad. Sci. USA 100 (4), pp. 1586-1591.
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Earlier in vivo studies have shown that the sequential action of the IspG and IspH proteins is essential for the reductive transformation of 2C-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 2C-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 2C-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.
|Institutions:||Chemistry and Pharmacy > Institut für Organische Chemie > Arbeitskreis Dr. Sabine Amslinger|
|Subjects:||500 Science > 540 Chemistry & allied sciences|
|Refereed:||Yes, this version has been refereed|
|Created at the University of Regensburg:||No|
|Owner:||Dr. Sabine Amslinger|
|Deposited On:||24 Nov 2009 15:30|
|Last Modified:||12 Nov 2012 14:21|
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