The deoxyxylulose phosphate pathway of isoprenoid biosynthesis: studies on the mechanisms of the reactions catalyzed by IspG and IspH protein

Abstract

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 x min(-1) x 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 micromol x min(-1) x mg(-1). IspH protein could also be activated by a mixture of flavodoxin, flavodoxin reductase, and NADPH at a rate of 3 nmol x min(-1) x mg(-1). The striking similarities of IspG and IspH protein are discussed, and plausible mechanistic schemes are proposed for the two reactions.

Figure 1

The deoxyxylulose phosphate pathway of isoprenoid biosynthesis.

Figure 2

Enzymatic conversion of ¹⁴C-labeled 5 into 6 and 7/8. Radiochemical assays of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase activity were performed as described in Experimental Procedures. (A–D) Method A. (E and F) Method B. The assay mixtures contained 3 mg of crude cell extract of E. coli cells SK6600 (A); 3 mg of crude cell extract of E. coli cells SK6600ispG∷neo^R (B); 180 μg of recombinant MalE/IspG fusion protein (C); 3 mg of crude cell extract of E. coli cells SK6600ispG∷neo^R and 180 μg of recombinant MalE/IspG fusion protein (D); 180 μg of recombinant MalE/IspG fusion protein and 0.5 mM photoreduced deazaflavin (E); 180 μg of recombinant MalE/IspG fusion protein, 48 μg of recombinant MalE/IspH fusion protein, and 0.5 mM photoreduced deazaflavin (F).

Figure 3

¹³C NMR signals of 6 obtained from [1,3,4-¹³C₃]5 (A) and [2,2′-¹³C₂]5 (B). Assay mixtures containing 50 mM Tris⋅HCl (pH 8.0), 0.5 mM deazaflavin, 100 μM ¹³C-labeled 5, and recombinant MalE/IspG fusion protein were irradiated as described in Experimental Procedures. *, A signal from an impurity.

Figure 4

¹³C NMR signals of 7 (green) and 8 (red) generated by irradiation from [U-¹³C₅]6 in the presence of MalE/IspH fusion protein and deazaflavin (A) and after incubation of the latter sample with Idi protein (B). The ¹³C coupling patterns are indicated. *, A signal of an impurity.

Figure 5

Proposed mechanism for the conversion of 5 into 6 catalyzed by IspG protein.

Figure 6

Proposed mechanism for the conversion of 6 into 7/8 catalyzed by IspH protein.

Figure 7

Postulated conformation of 6 at the active site of the IspH protein. The indicated reference plane corresponds to the nodal plane of the olefinic bond.