GcpE Is Involved in the 2- C -Methyl- d -Erythritol 4-Phosphate Pathway of Isoprenoid Biosynthesis in Escherichia coli

Abstract

In a variety of organisms, including plants and several eubacteria, isoprenoids are synthesized by the mevalonate-independent 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Although different enzymes of this pathway have been described, the terminal biosynthetic steps of the MEP pathway have not been fully elucidated. In this work, we demonstrate that the gcpE gene of Escherichia coli is involved in this pathway. E. coli cells were genetically engineered to utilize exogenously provided mevalonate for isoprenoid biosynthesis by the mevalonate pathway. These cells were then deleted for the essential gcpE gene and were viable only if the medium was supplemented with mevalonate or the cells were complemented with an episomal copy of gcpE.In all organisms studied so far, isoprenoids derive from the common isoprene units, isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP). In mammals and in fungi, IPP and DMAPP are formed exclusively by the mevalonate pathway (11). In contrast, many eubacteria (including Escherichia coli), algae, and the plastids of higher plants synthesize IPP and DMAPP by the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway (9, 34). The MEP pathway was also identified in a plastid-like organelle of malaria parasites (15). Since the MEP pathway is absent in humans, it has been validated as a drug target for the treatment of both bacterial and parasitic infections (15,29).The pathway initiates with the formation of 1-deoxy-D-xylulose 5-phosphate (DOXP) by condensation of pyruvate and D-glyceraldehyde 3-phosphate catalyzed by the DOXP synthase (Dxs) (1,6,20,22,24,25,35,38). DOXP is then converted by the DOXP reductoisomerase (Dxr) into MEP (Fig.

Regulation of Mycobacterium tuberculosis cell envelope composition and virulence by intramembrane proteolysis

“…Figure 1b shows the genomic location of Rv2869c between dxr and gcpE, two genes in the non-mevalonate pathway of isoprenoid biosynthesis 19,20. Southern blotting (Figure 1c) confirmed successful replacement of Rv2869c with a Hygromycin resistance cassette in both BCG and M. tuberculosis, demonstrating that, in contrast to YaeL in E. coli, Rv2869c is not an essential gene for M. tuberculosis viability.…”

Studies on the nonmevalonate pathway to terpenes: The role of the GcpE (IspG) protein

“…From this fact and from the established metabolic function of dxs, ispC, ispD, ispE, and ispF for the conversion of pyruvate and D-glyceraldehyde 3-phosphate into 2C-methyl-D-erythritol 2,4-cyclodiphosphate, it was deduced that gcpE and lytB specify enzymes for the conversion of 2C-methyl-D-erythritol 2,4-cyclodiphosphate into isopentenyl diphosphate and dimethylallyl diphosphate (20). Subsequently, the involvement of these genes in the nonmevalonate pathway has been confirmed by gene-targeting studies (21)(22)(23), but their metabolic roles are still unknown.…”

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].…”