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-deazaisoalloxazine, 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 nmol⅐min ؊1 ⅐mg ؊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 mol⅐min ؊1 ⅐mg ؊1 . IspH protein could also be activated by a mixture of flavodoxin, flavodoxin reductase, and NADPH at a rate of 3 nmol⅐min ؊1 ⅐mg ؊1 . The striking similarities of IspG and IspH protein are discussed, and plausible mechanistic schemes are proposed for the two reactions.

2-C-Methyl-D-erythritol 4-phosphate synthase (IspC) is the first enzyme committed to isoprenoid biosynthesis in the methylerythritol phosphate pathway, which represents an alternative route to the classical mevalonate pathway. As it is present in many pathogens and plants, but not in man, this pathway has attracted considerable interest as a target for novel antibiotics and herbicides. Fosmidomycin represents a specific high-affinity inhibitor of IspC. Very recently, its anti-malaria activity in man has been demonstrated in clinical trials. Here, we present the crystal structure of Escherichia coli IspC in complex with manganese and fosmidomycin at 2.5 Å resolution. The (N-formyl-N-hydroxy)amino group provides two oxygen ligands to manganese that is present in a distorted octahedral coordination, whereas the phosphonate group is anchored in a specific pocket by numerous hydrogen bonds. Both sites are connected by a spacer of three methylene groups. The substrate molecule, 1-D-deoxyxylulose 5-phosphate, can be superimposed onto fosmidomycin, explaining the stereochemical course of the reaction.

The ispH gene of Escherichia coli specifies an enzyme catalyzing the conversion of 1-hydroxy-2-methyl-2-(E)-butenyl diphosphate into a mixture of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in the nonmevalonate isoprenoid biosynthesis pathway. The implementation of a gene cassette directing the overexpression of the isc operon involved in the assembly of iron-sulfur clusters into an Escherichia coli strain engineered for ispH gene expression increased the catalytic activity of IspH protein anaerobically purified from this strain by a factor of at least 200. For maximum catalytic activity, flavodoxin and flavodoxin reductase were required in molar concentrations of 40 and 12 microM, respectively. EPR experiments as well as optical absorbance indicate the presence of a [3Fe-4S](+) cluster in IspH protein. Among 4 cysteines in total, the 36 kDa protein carries 3 absolutely conserved cysteine residues at the amino acid positions 12, 96, and 197. Replacement of any of the conserved cysteine residues reduced the catalytic activity by a factor of more than 70 000.

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