Jean-François Hoeffler

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 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.

A cytosolic Arabidopsis D-xylulose kinase catalyzes the phosphorylation of 1-deoxy-D-xylulose into a precursor of the plastidial isoprenoid pathway.

Plants are able to integrate exogenous 1-deoxy-d-xylulose (DX) into the 2C-methyl-d-erythritol 4-phosphate pathway, implicated in the biosynthesis of plastidial isoprenoids. Thus, the carbohydrate needs to be phosphorylated into 1-deoxy-d-xylulose 5-phosphate and translocated into plastids, or vice versa. An enzyme capable of phosphorylating DX was partially purified from a cell-free Arabidopsis (Arabidopsis thaliana) protein extract. It was identified by mass spectrometry as a cytosolic protein bearing d-xylulose kinase (XK) signatures, already suggesting that DX is phosphorylated within the cytosol prior to translocation into the plastids. The corresponding cDNA was isolated and enzymatic properties of a recombinant protein were determined. In Arabidopsis, xylulose kinases are encoded by a small gene family, in which only two genes are putatively annotated. The additional gene is coding for a protein targeted to plastids, as was proved by colocalization experiments using green fluorescent protein fusion constructs. Functional complementation assays in an Escherichia coli strain deleted in xk revealed that the cytosolic enzyme could exclusively phosphorylate xylulose in vivo, not the enzyme that is targeted to plastids. xk activities could not be detected in chloroplast protein extracts or in proteins isolated from its ancestral relative Synechocystis sp. PCC 6803. The gene encoding the plastidic protein annotated as “xylulose kinase” might in fact yield an enzyme having different phosphorylation specificities. The biochemical characterization and complementation experiments with DX of specific Arabidopsis knockout mutants seedlings treated with oxo-clomazone, an inhibitor of 1-deoxy-d-xylulose 5-phosphate synthase, further confirmed that the cytosolic protein is responsible for the phosphorylation of DX in planta.

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.

Chemical Synthesis of Enantiopure 2-C-Methyl-d-Erythritol 4-Phosphate, the Key Intermediate in the Mevalonate-Independent Pathway for Isoprenoid Biosynthesis

Abstract Optically pure 2- C -methyl- d -erythritol 4-phosphate, a key intermediate in the mevalonate-independent route for isoprenoid biosynthesis, is conveniently synthesized from 1,2- O -isopropylidene-α- d -xylofuranose, including the possibility of radiolabeling of this substrate.

A review of tobacco BY-2 cells as an excellent system to study the synthesis and function of sterols and other isoprenoids.

In plants, two pathways are utilized for the synthesis of isopentenyl diphosphate (IPP), the universal precursor for isoprenoid biosynthesis. In this paper we review findings and observations made primarily with tobacco BY-2 cells (TBY-2), which have proven to be an excellent system in which to study the two biosynthetic pathways. A major advantage of these cells as an experimental system is their ability to readily take up specific inhibitors and stably- and/or radiolabeled precursors. This permits the functional elucidation of the role of isoprenoid end products and intermediates. Because TBY-2 cells undergo rapid cell division and can be synchronized within the cell cycle, they constitute a highly suitable test system for determination of those isoprenoids and intermediates that act as cell cycle inhibitors, thus giving an indication of which branches of the isoprenoid pathway are essential. Through chemical complementation, and use of precursors, intracellular compartmentation can be elucidated, as well as the extent to which the plastidial and cytoslic pathways contribute to the syntheses of specific groups of isoprenoids (e.g., sterols) via exchange of intermediates across membranes. These topics are discussed in the context of the pertinent literature.

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.

Synthesis of tritium labelled 2-C-methyl-d-erythritol, a useful substrate for the elucidation of the methylerythritol phosphate pathway for isoprenoid biosynthesis

Abstract Free 2-C-methyl- d -erythritol is utilised for isoprenoid biosynthesis by Escherichia coli mutants lacking the two first enzymes of the methylerythritol phosphate pathway, the deoxyxylulose phosphate synthase and isomero-reductase. For feeding experiments, this tetrol was synthesised with an overall 43% yield from readily available 1,2-O-isopropylidene-α- d -xylofuranose, including the possibility of tritium labelling.

Synthesis of [3,5,5,5-2H4]-2-C-methyl-d-erythritol, a substrate designed for the elucidation of the mevalonate independent route for isoprenoid biosynthesis

Abstract 2- C -Methyl- d -erythritol 4-phosphate is a key intermediate in the mevalonate independent pathway for isoprenoid biosynthesis. In order to investigate the fate of the protons in this metabolic route, [3,5,5,5- 2 H 4 ]-2- C -methyl- d -erythritol was synthesized. The relevant steps allowing deuterium introduction were a palladium(II)-catalyzed hydrostannation and a coupling reaction between a vinyl iodide and a methylcyanocuprate.