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.

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 cytosolic 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.

Cross-talk between the cytosolic mevalonate and the plastidial methylerythritol phosphate pathways in tobacco bright yellow-2 cells.

In plants, two pathways are utilized for the synthesis of isopentenyl diphosphate, the universal precursor for isoprenoid biosynthesis. The key enzyme of the cytoplasmic mevalonic acid (MVA) pathway is 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR). Treatment of Tobacco Bright Yellow-2 (TBY-2) cells by the HMGR-specific inhibitor mevinolin led to growth reduction and induction of apparent HMGR activity, in parallel to an increase in protein representing two HMGR isozymes. Maximum induction was observed at 24 h. 1-Deoxy-d-xylulose (DX), the dephosphorylated first precursor of the plastidial 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, complemented growth inhibition by mevinolin in the low millimolar concentration range. Furthermore, DX partially re-established feedback repression of mevinolin-induced HMGR activity. Incorporation studies with [1,1,1,4-2H4]DX showed that sterols, normally derived from MVA, in the presence of mevinolin are synthesized via the MEP pathway. Fosmidomycin, an inhibitor of 1-deoxy-d-xylulose-5-phosphate reductoisomerase, the second enzyme of the MEP pathway, was utilized to study the reverse complementation. Growth inhibition by fosmidomycin of TBY-2 cells could be partially overcome by MVA. Chemical complementation was further substantiated by incorporation of [2-13C]MVA into plastoquinone, representative of plastidial isoprenoids. Best rates of incorporation of exogenous stably labeled precursors were observed in the presence of both inhibitors, thereby avoiding internal isotope dilution.

Isoprenoid biosynthesis via the methylerythritol phosphate pathway. Mechanistic investigations of the 1-deoxy-D-xylulose 5-phosphate reductoisomerase.

The 1-deoxyxylulose 5-phosphate reductoisomerase (DXR, EC 1.1.1.267) catalyzes the conversion of 1-deoxy-d-xylulose 5-phosphate (DXP) into 2-C-methyl-d-erythritol 4-phosphate (MEP). This transformation is a two-step process involving a rearrangement of DXP into the putative intermediate 2-C-methyl-d-erythrose 4-phosphate followed by a NADPH-dependent reduction of the latter aldehyde. By using [1-(13)C]DXP as a substrate, the rearrangement of DXP into [5-(13)C]2-C-methyl-d-erythrose 4-phosphate was shown to be NADPH dependent, although it does not involve areduction step. The putative aldehyde intermediate, obtained by chemical synthesis, was converted into MEP by the DXR in the presence of NADPH and into DXP in the presence of NADP(+), indicating the reversibility of the reaction catalyzed by the DXR. This reversibility was confirmed by the conversion of MEP into DXP in the presence of NADP(+). The equilibrium was, however, largely displaced in favour of the formation of MEP. The reduction step required the presence of a divalent cation such as Mg(2+) or Mn(2+).

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.