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

Abstract

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.In plants, the biosynthesis of the active isoprene units (D 2 -and D 3

The Plastidial 2-C-Methyl-d-Erythritol 4-Phosphate Pathway Provides the Isoprenyl Moiety for Protein Geranylgeranylation in Tobacco BY-2 Cells

“…Addition of Ca 2+ or the calmodulin inhibitor calmidazolium (cf. Merret et al, 2007) As predicted (Hemmerlin et al, 2006), DX restored plasma membrane localization of His 6 -GFP-BD-CVIL in the presence of OC (Figure 9). The ability of DX to bypass the DXS reaction in the presence of OC confirmed the prediction that OC inhibits DXS (see also Sakakibara et al, 2005).…”

“…At confocal resolution, individual cells representative of the various populations were analyzed, and images were acquired and analyzed as described (Hemmerlin et al, 2006). At high resolution, pictures were taken such that the focal plane included the nucleus and nucleolus.…”

The Arabidopsis thaliana Type I Isopentenyl Diphosphate Isomerases Are Targeted to Multiple Subcellular Compartments and Have Overlapping Functions in Isoprenoid Biosynthesis

“…The transport of bona fide MEP pathway intermediates such as 1-deoxy D-xylulose 5-phosphate (DXP) from the cytosol into plastids has been shown to proceed via an inorganic phosphate antiport transporter (Flü gge and Gao, 2005). In fact, a cytosolic xylulose kinase was recently identified in Arabidopsis that converts 1-deoxy D-xylulose to DXP (Hemmerlin et al, 2006), although a role for DXP in the cytosol is unclear at present.…”

Two nearly identical terpene synthases catalyze the formation of nerolidol and linalool in snapdragon flowers

“…Although the MVA pathway is localized in the cytosol, the results obtained in fruits do not exclude the possible transport of IPP formed from labeled MVL to plastids for linalool biosynthesis. The absence of detectable [ 2 H 2 ]DOX labeling of terpenoids in fruit tissue could result from a lack of D-xylulose kinase, which catalyzes the phosphorylation of exogenously supplied DOX (Hemmerlin et al, 2006) in order for it to enter the pathway and be utilized. Unfortunately, D-xylulose kinase activity was not checked in fruit tissue, leaving the actual contributions of the MEP pathway to terpenoid biosynthesis in strawberry fruits yet to be determined.…”