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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

A Cytosolic Arabidopsis D-Xylulose Kinase Catalyzes the Phosphorylation of 1-Deoxy-D-Xylulose into a Precursor of the Plastidial Isoprenoid Pathway¹

Centre National de la Recherche Scientifique, UPR 2357, associated to the Université Louis Pasteur, Institut de Biologie Moléculaire des Plantes, 67083 Strasbourg cedex, France (A.H., M.H., T.J.B.); Université Louis Pasteur/Centre National de la Recherche Scientifique, Institut de Chimie de Strasbourg, LC3-UMR 7177, 67070 Strasbourg cedex, France (D.T., M.R.); AlsaChim, Institut de Science et d'Ingénierie Supramoléculaires, 67000 Strasbourg, France (J.-F.H.); and Université Louis Pasteur/Centre National de la Recherche Scientifique, Laboratoire de Spectrométrie de Masse Bio-Organique, Institut Pluridisciplinaire Hubert Curien, LC4-UMR 7178, Ecole Européenne de Chimie, Polymères et Matériaux, F–67087 Strasbourg cedex, France (K.P., A.v.D.)

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.

² These authors contributed equally to the paper.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Andréa Hemmerlin (andrea.hemmerlin{at}ibmp-ulp.u-strasbg.fr).

www.plantphysiol.org/cgi/doi/10.1104/pp.106.086652

^* Corresponding author; e-mail andrea.hemmerlin{at}ibmp-ulp.u-strasbg.fr; fax 33–390–24–18–84.

Received July 12, 2006; accepted August 9, 2006; published August 18, 2006.

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