Biosynthesis of terpenes: Studies on 1-hydroxy-2-methyl-2-( E )-butenyl 4-diphosphate reductase

Abstract

Earlier in vivo studies showed the involvement of IspH protein in the conversion of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate into isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). We have demonstrated now that cell extract of an Escherichia coli strain engineered for hyperexpression of the ispH (lytB) gene catalyzes the in vitro conversion of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate into IPP and DMAPP. The reaction requires NADH, FAD, divalent cations (preferably Co 2؉ ), and probably one or more as-yet-unidentified proteins. The low intrinsic catalytic activities of wild-type E. coli cell extract and isolated chromoplasts of red pepper (Capsicum annuum) are enhanced by the addition of purified recombinant IspH protein.

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

“…In the experiments with the tritiated substrate, the inseparability of the radioactivity peaks corresponding to the tritiated forms of 7 and 8 did not allow an assessment of their relative rate of formation; however, a value of 6:1 for this ratio was cleanly evidenced by the relative intensities of the corresponding 13 C-NMR signals in the spectrum of the mixture generated from [U-13 C 5 ]-labeled 6. This value, which is in close agreement with earlier findings of in vivo and in vitro experiments (19,20), must represent the outcome of a kinetic control, as demonstrated by the fact that it shifted to the expected equilibrium value of 3:7 (29) on treatment of the solution with the recombinant isopentenyl diphosphate isomerase from E. coli. Not surprisingly, a mixture containing IspG protein, IspH protein, 14 C-labeled 5, and photoreduced deazaflavin afforded predominantly a radioactive peak with a retention volume of 7͞8.…”

“…The ratio of 7 to 8 in Fig. 4A is about 6:1, in close similarity to the ratio observed in our earlier in vivo and in vitro studies (19,20). After incubation with recombinant isopentenyl diphosphate isomerase (Idi protein) this sample displays NMR signals whose intensities had been shifted from the original values to the equilibrium value of 3:7 (28) (Fig.…”

“…We have now expressed in a recombinant E. coli strain a fusion protein encompassing IspG and a maltose binding domain and demonstrated that, whereas the purified protein is inactive, activity can be restored by the addition of a crude cell extract from an ispG-deficient mutant of E. coli. This requirement of auxiliary proteins parallels the one previously detected for the IspH protein, which catalyzes the subsequent transformation of 6 into a mixture of IPP (7) and DMAPP (8) (20). The plausible assumption that such auxiliary proteins catalyze the shuttling of redox equivalents has been corroborated in this work by showing that IspH protein can be activated by the joint action of flavodoxin and flavodoxin reductase in the presence of NADPH.…”

“…Earlier, we had shown that recombinant IspH protein can be activated by unidentified proteins present in the cell extract of wild-type E. coli cells (20). The UV͞visible spectrum is displayed in Fig.…”

“…Subsequently, the in vitro formation of 6 from 5 by crude cell extracts of E. coli overexpressing ispG was confirmed by radiochemical methods (18). Compound 6 was shown by in vivo as well in vitro experiments to serve as the biosynthetic precursor of IPP (7) and DMAPP (8), which were obtained in a ratio of 6:1 by the catalytic action of the IspH protein (19,20).…”