Beschreibung:
<p>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<tex-math>$1\>nmol\!\cdot\!min^{-1}\!\cdot\!mg^{-1}$</tex-math>. 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<tex-math>$0.4\>\mu mol\!\cdot\!min^{-1}\!\cdot\!mg^{-1}$</tex-math>. IspH protein could also be activated by a mixture of flavodoxin, flavodoxin reductase, and NADPH at a rate of<tex-math>$3\>nmol\!\cdot\!min^{-1}\!\cdot\!mg^{-1}$</tex-math>. The striking similarities of IspG and IspH protein are discussed, and plausible mechanistic schemes are proposed for the two reactions.</p>