The mevalonate pathway for the biosynthesis of the universal terpenoid precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), is known in considerable detail. Only recently, the existence of a second mevalonate-independent pathway for the biosynthesis of IPP and DMAPP was detected in plants and certain eubacteria. Experiments with 13C and/or 2H-labelled precursors were crucial in the elucidation of this novel route. The pathway is essential in plants, many eubacteria and apicomplexan parasites, but not in archaea and animals. The genes, enzymes and intermediates of this pathway were rapidly unravelled over the past few years. Detailed knowledge about the mechanisms of this novel route may benefit the development of novel antibiotics, antimalarials and herbicides.

Recent studies have uncovered the existence of an alternative, non-mevalonate pathway for the formation of isopentenyl pyrophosphate and dimethylallyl pyrophosphate, the two building blocks of terpene biosynthesis.

The incorporation of [1-13 C]-and [2,3,4,5-13 C 4 ]1-deoxy-D-xylulose into ␤-carotene, lutein, phytol, and sitosterol in a cell culture of Catharanthus roseus was analyzed by NMR spectroscopy. The labeling patterns of the isoprene precursors, isopentenyl pyrophosphate and dimethylallyl pyrophosphate, were obtained from the terpenes by a retrobiosynthetic approach. 13 C Enrichment and 13 C 13 C coupling patterns showed conclusively that 1-deoxy-D-xylulose and not mevalonate is the predominant isoprenoid precursor of phytol, ␤-carotene, and lutein. Label from 1-deoxyxylulose was also diverted to phytosterols to a minor extent (6% relative to carotene and phytol formation). The data demonstrate that the formation of isopentenyl pyrophosphate from pentulose occurs strictly by an intramolecular rearrangement process.

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.

Isopentenyl diphosphate and dimethylallyl diphosphate serve as the universal precursors for the biosynthesis of terpenes. Although their biosynthesis by means of mevalonate has been studied in detail, a second biosynthetic pathway for their formation by means of 1-deoxy-D-xylulose 5-phosphate has been discovered only recently in plants and certain eubacteria. Earlier in vivo experiments with recombinant Escherichia coli strains showed that exogenous 1-deoxy-Dxylulose can be converted into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate by the consecutive action of enzymes specified by the xylB and ispCDEFG genes. This article describes the transformation of exogenous [U-13 C5]1-deoxy-D-xylulose into a 5:1 mixture of [U-13 C5]isopentenyl diphosphate and [U-13 C5]dimethylallyl diphosphate by an E. coli strain engineered for the expression of the ispH (lytB) gene in addition to recombinant xylB and ispCDEFG genes.T erpenes are one of the largest groups of natural products comprising numerous medically relevant compounds (e.g., vitamins, hormones, and antitumor agents such as Taxol) (1). Bloch, Lynen, Cornforth, and their coworkers showed that the universal terpenoid precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), are biosynthesized by means of the mevalonate pathway in yeasts and animals (for review see refs. 2-5). These studies served as the basis for the development of metabolic inhibitors that are widely used for the treatment of hypercholesterolemia.For a period of several decades, these milestone achievements completely eclipsed the existence of a second terpenoid pathway for the biosynthesis of IPP and DMAPP. Recently, however, knowledge on that pathway has been unfolding rapidly on the basis of seminal discoveries by the research groups of . Briefly, the mevalonate independent pathway starts from 1-deoxy-D-xylulose 5-phosphate, which is assembled from pyruvate and D-glyceraldehyde 3-phosphate (13, 14) and was already known to serve as a biosynthetic precursor of vitamins B 1 (thiamine) and B 6 (pyridoxal) (Fig. 1) (15-17). 1-Deoxy-D-xylulose 5-phosphate is converted into 2C-methyl-D-erythritol 4-phosphate, the first committed intermediate of the nonmevalonate pathway, by isomerization followed by a two-electron reduction step catalyzed by 2C-methyl-D-erythritol 4-phosphate synthase specified by the ispC gene (formerly designated yaeM and then dxr) (18) (Fig. 1).In the next step, 2C-methyl-D-erythritol 4-phosphate is converted into the 2C-methyl-D-erythritol 2,4-cyclodiphosphate by the sequential action of three enzymes specified by the ispD, ispE, and ispF genes (19-24). The last known step of the sequence, the reductive transformation of the cyclic diphosphate into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate has been identified recently in work with a recombinant Escherichia coli strain engineered for hyperexpression of the ispG gene (previously designated gcpE) (25). Comparative genomics suggest that the protein specified by the lytB gene is involved in the conversion of ...

The biogenetic isoprene rule in its application to the triterpenes is discussed from a stereochemical standpoint. On the basis of a well defined system of arbitrary assumptions a scheme has been developed leading from squalene to the formulae of the basic representatives of all known cyclic triterpene groups ‐ i.e. euphol, tirucallol, lupeol, taraxasterol, germanicol, β‐amyrin, taraxerol, friedelin, α‐amyrin, lanosterol ‐ in their full structural and configurational detail. This result is considered to support the squalene hypothesis of the biogenesis of cyclic triterpenes.

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.

Recombinant Escherichia coli cells engineered for the expression of the xylB gene in conjunction with genes of the nonmevalonate pathway were supplied with 13 C-labeled 1-deoxy-D-xylulose. Cell extracts were analyzed directly by NMR spectroscopy. 13 C-labeled 2C-methyl-D-erythritol 2,4-cyclodiphosphate was detected at high levels in cells expressing xylB, ispC, ispD, ispE, and ispF. The additional expression of the gcpE gene afforded 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate as an intermediate of the nonmevalonate pathway. Hypothetical mechanisms involving conserved cysteine residues are proposed for the enzymatic conversion of 2C-methyl-D-erythritol 2,4-cyclodiphosphate into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate catalyzed by the GcpE protein.isoprenoid biosynthesis ͉ 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate