Isoprenoid biosynthesis via the methylerythritol phosphate pathway. (E)-4-Hydroxy-3-methylbut-2-enyl diphosphate: chemical synthesis and formation from methylerythritol cyclodiphosphate by a cell-free system from Escherichia coli

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

“…As far as the IspG protein is concerned, such a requirement rules out the previously considered alternative of a purely ionic mechanism patterned after the mode of action of vitamin K epoxyquinone reductase (16). The postulated analogy with anaerobic ribonucleotide reductase (16,18) is invalidated by the demonstrated absence of S-adenosylmethionine as a radical initiator, and the suggestion of a resemblance with the mechanism of ascarylose biosynthesis (18) must be considered irrelevant, inasmuch as in the latter process the reductive elimination of the critical hydroxy group rests entirely on ionic steps catalyzed by the essential pyridoxamine cofactor and the radical part of the reaction deals exclusively with the subsequent stepwise reduction of the oxidized cofactor (for review, see ref. 37).…”

“…The additional implementation of a recombinant ispG gene resulted in the in vivo formation of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (6) (16), which was also isolated from an ispH-deficient E. coli mutant (17). 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).…”

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

“…In vivo experiments showed that the reductive ring opening of 7 affording 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate 8 requires a protein specified by the ispG (gcpE) gene (18,19). The same compound was shown also to accumulate in an ispH-deficient Escherichia coli strain (20), and its formation from 14 C-labeled 7 was observed in cell extracts from E. coli overexpressing the ispG (gcpE) gene (21). Subsequent in vivo studies with a recombinant E. coli strain showed that the conversion of 8 into 9 and 10 requires a protein specified by the ispH (lytB) gene (22).…”

Possible direct involvement of the active‐site [4Fe–4S] cluster of the GcpE enzyme from Thermus thermophilus in the conversion of MEcPP

“…This procedure, however, can result in the presence of a mix of clusters types. Sequence alignments of the available GcpE sequences [13][14][15][16][17] show that there are only three highly conserved cysteine residues that in principle can coordinate a [4Fe-4S] cluster that contains a unique Fe atom or a [3Fe-4S] cluster. In the case of enzyme from Thermosynechococcus elongatus the reconstitution procedure resulted in an enzyme preparation that showed an absorption spectrum with bands at 305, 395 and 585 nm, which was interpreted as being due to a [4Fe-4S] + cluster [12].…”