Catherine Grosdemange-Billiard

Genetic evidence of branching in the isoprenoid pathway for the production of isopentenyl diphosphate and dimethylallyl diphosphate in Escherichia coli.

An alternative mevalonate‐independent pathway for isoprenoid biosynthesis has been recently discovered in eubacteria (including Escherichia coli) and plant plastids, although it is not fully elucidated yet. In this work, E. coli cells were engineered to utilize exogenously provided mevalonate and used to demonstrate by a genetic approach that branching of the endogenous pathway results in separate synthesis of the isoprenoid building units isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). In addition, the IPP isomerase encoded by the idi gene was shown to be functional in vivo and to represent the only possibility for interconverting IPP and DMAPP in this bacterium.

Cutting edge: human gamma delta T cells are activated by intermediates of the 2-C-methyl-D-erythritol 4-phosphate pathway of isoprenoid biosynthesis

Activation of Vγ9/Vδ2 T cells by small nonprotein Ags is frequently observed after infection with various viruses, bacteria, and eukaryotic parasites. We suggested earlier that compounds synthesized by the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway of isopentenyl pyrophosphate synthesis are responsible for the Vγ9/Vδ2 T cell reactivity of many pathogens. Using genetically engineered Escherichia coli knockout strains, we now demonstrate that the ability of E. coli extracts to stimulate γδ T cell proliferation is abrogated when genes coding for essential enzymes of the MEP pathway, dxr or gcpE, are disrupted or deleted from the bacterial genome.

Isoprenoid biosynthesis as a target for antibacterial and antiparasitic drugs: phosphonohydroxamic acids as inhibitors of deoxyxylulose phosphate reducto-isomerase

Isoprenoid biosynthesis via the methylerythritol phosphate pathway is a target against pathogenic bacteria and the malaria parasite Plasmodium falciparum. 4-(Hydroxyamino)-4-oxobutylphosphonic acid and 4-[hydroxy(methyl)amino]-4-oxobutyl phosphonic acid, two novel inhibitors of DXR (1-deoxy-D-xylulose 5-phosphate reducto-isomerase), the second enzyme of the pathway, have been synthesized and compared with fosmidomycin, the best known inhibitor of this enzyme. The latter phosphonohydroxamic acid showed a high inhibitory activity towards DXR, much like fosmidomycin, as well as significant antibacterial activity against Escherichia coli in tests on Petri dishes.

Isoprenoid biosynthesis in higher plants and in Escherichia coli: on the branching in the methylerythritol phosphate pathway and the independent biosynthesis of isopentenyl diphosphate and dimethylallyl diphosphate

In the bacterium Escherichia coli, the mevalonic-acid (MVA)-independent 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway is characterized by two branches leading separately to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). The signature of this branching is the retention of deuterium in DMAPP and the deuterium loss in IPP after incorporation of 1-[4-(2)H]deoxy-d-xylulose ([4-(2)H]DX). Feeding tobacco BY-2 cell-suspension cultures with [4-(2)H]DX resulted in deuterium retention in the isoprene units derived from DMAPP, as well as from IPP in the plastidial isoprenoids, phytoene and plastoquinone, synthesized via the MEP pathway. This labelling pattern represents direct evidence for the presence of the DMAPP branch of the MEP pathway in a higher plant, and shows that IPP can be synthesized from DMAPP in plant plastids, most probably via a plastidial IPP isomerase.

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

Abstract 2- C -Methyl- d -erythritol cyclodiphosphate is converted into ( E )-4-hydroxy-3-methylbut-2-enyl diphosphate by a cell-free system from an Escherichia coli strain overexpressing the gcpE gene. The latter diphosphate, representing probably the last intermediate in the MEP pathway for isoprenoid biosynthesis, was identified by comparison with reference material obtained by chemical synthesis.

Isoprenoid Biosynthesis via the Methylerythritol Phosphate Pathway: Structural Variations around Phosphonate Anchor and Spacer of Fosmidomycin, a Potent Inhibitor of Deoxyxylulose Phosphate Reductoisomerase

Fosmidomycin and its analogue FR-900098 are potent inhibitors of 1-deoxy-d-xylulose 5-phosphate reducto-isomerase (DXR), the second enzyme of the MEP pathway for the biosynthesis of isoprenoids. This paper describes the synthesis of analogues of the two reverse phosphonohydroxamic acids 3 and 4, in which the length of the carbon spacer is modified, the N-methyl group of 3 is replaced by an ethyl group, and the phosphate group is replaced by potential isosteric moieties, i.e., sulfonate or carboxylate functionalities. The potential of the synthesized analogues to inhibit the E. coli DXR was evaluated.

Isoprenoid biosynthesis via the methylerythritol phosphate pathway: accumulation of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate in a gcpE deficient mutant of Escherichia coli

Abstract In the bacterium Escherichia coli , gcpE is an essential gene in the methylerythritol phosphate pathway for isoprenoid biosynthesis. Incubation of [1- 3 H]methylerythritol with an E . coli mutant defective in the gcpE gene resulted in the accumulation of [1- 3 H]methylerythritol 2,4-cyclodiphosphate. This suggests that the GCPE protein is involved in the further conversion of methylerythritol cyclodiphosphate into isoprenoids.

Isoprenoid biosynthesis via the MEP pathway. Synthesis of (3R,4S)-3,4-dihydroxy-5-oxohexylphosphonic acid, an isosteric analogue of 1-deoxy-D-xylulose 5-phosphate, the substrate of the 1-deoxy-D-xylulose 5-phosphate reducto-isomerase

(3,4)-3,4-Dihydroxy-5-oxohexylphosphonic acid, an isosteric analogue of 1-deoxy-D-xylulose 5-phosphate (DXP), was obtained in enantiomerically pure form from (+)-2,3--benzylidene--threitol by a seven-step sequence. This phosphonate did not affect the growth of. It did not inhibit the 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), but was converted by this enzyme into (3,4)-3,4,5-trihydroxy-3-methylpentylphosphonic acid, an isosteric analogue of 2-C-methyl-D-erythritol 4-phosphate. The enzyme was, however, less efficient with the methylene phosphonate analogue than with the natural substrate.

Enzymatic synthesis of 1-deoxysugar-phosphates using E. coli 1-deoxy-d-xylulose 5-phosphate synthase

Abstract The thiamine diphosphate-dependent enzyme 1-deoxy- d -xylulose 5-phosphate synthase from E. coli can use d -erythrose 4-phosphate and d -ribose 5-phosphate as alternative substrates. These reactions were used for the production of 1-deoxy- d -fructose 6-phosphate and 1-deoxy- d -sedoheptulose 7-phosphate and have potential application for the biosynthesis of other 1-deoxysugar phosphates.

Elucidation of the 2-C-methyl-d-erythritol 4-phosphate pathway for isoprenoid biosynthesis: straightforward syntheses of enantiopure 1-deoxy-d-xylulose from pentose derivatives

Optically pure 1-deoxy-d-xylulose, a key metabolite for feeding experiments in the methylerythritol phosphate pathway for isoprenoid biosynthesis, is conveniently synthesised from 1,2-O-isopropylidene-α-d-xylofuranose or from d-arabinose. This renders labelling with hydrogen isotopes possible.