Incorporation of 2-C-Methyl-d-erythritol, a Putative Isoprenoid Precursor in the Mevalonate-Independent Pathway, into Ubiquinone and Menaquinone of Escherichia coli

Duvold, Tore; Calí, Patrizia; Bravo, Jean-Michel; Rohmer, Michel

doi:10.1016/s0040-4039(97)01392-0

Cited by 100 publications

(77 citation statements)

References 18 publications

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“…Oligonucleotide primers and linkers were synthesized by the Protein/DNA Core Facility of the Utah Regional Cancer Center. ME was synthesized by the procedure of Duvold et al (8). DXP and 1-deoxy-D-xylulose (DXS) were synthesized by the procedure of Blagg and Poulter (3).…”

Section: Materials Sodium [2-mentioning

confidence: 99%

“…A 25-l portion was loaded onto a silica column as described above for DXS. The radioactivity in [2][3][4][5][6][7][8][9][10][11][12][13][14] C]DXS was measured as described above. Initial velocities were determined from the slope of a plot of product versus time.…”

Section: Vol 183 2001 Two R Capsulatus Dxp Synthase Genes Dxsa Anmentioning

confidence: 99%

“…rescued at 44°C by addition of ME, an advanced intermediate in the pathway, to the medium (8). To establish that ORF 2895 encodes a functional DXPase, FH11 cells were transformed with pFMH39.…”

Section: R Capsulatus Orf 2816 Carries Dxsa the Gene For Dxpase-amentioning

confidence: 99%

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1-Deoxy- d -Xylulose 5-Phosphate Synthase, the Gene Product of Open Reading Frame (ORF) 2816 and ORF 2895 in Rhodobacter capsulatus

et al. 2001

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Section: Materials Sodium [2-mentioning

confidence: 99%

Section: Vol 183 2001 Two R Capsulatus Dxp Synthase Genes Dxsa Anmentioning

confidence: 99%

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1-Deoxy- d -Xylulose 5-Phosphate Synthase, the Gene Product of Open Reading Frame (ORF) 2816 and ORF 2895 in Rhodobacter capsulatus

et al. 2001

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“…1). ME could only be used for incorporation into bacteria (Duvold et al, 1997;Kuzuyama et al, 1999;Fontana et al, 2001) and was found toxic to plant cells (Hemmerlin et al, 2003a), though the free tetrol or the corresponding lactone was shown present in many plants ( Dittrich and Angyal, 1988). While there is only weak evidence that DX has a functional role in vivo, it was already substantially used as a stable or radioactive isotope-labeled precursor for studies of the biosynthetic pathway in plants (Schwender et al, 1997;Sagner et al, 1998;Arigoni et al, 1999;Hoeffler et al, 2002;Luan and Wü st, 2002), as well as in bacteria (Hill et al, 1989;Broers, 1994;Giner et al, 1998;Rosa-Putra et al, 1998b;Spiteller et al, 2002).…”

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

et al. 2006

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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

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“…strain CL190 (7). In the second step, the intramolecular rearrangement of DXP was assumed to give a hypothetical rearrangement product, 2-C-methylerythrose 4-phosphate, which was then converted to 2-C-methyl-D-erythritol 4-phosphate (MEP) by an unspecified reduction process (8,9). We have succeeded in the first cloning and overexpression of the gene (dxr, formerly yaeM) encoding the DXP reductoisomerase from E. coli and have shown that the recombinant enzyme catalyzed the formation of MEP from DXP in a single step in the presence of both NADPH and a divalent cation, such as Mn 2ϩ , Mg 2ϩ , or Co 2ϩ (10,11).…”

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confidence: 99%

Characterization of 1-Deoxy-d-xylulose 5-Phosphate Reductoisomerase, an Enzyme Involved in Isopentenyl Diphosphate Biosynthesis, and Identification of Its Catalytic Amino Acid Residues

Kuzuyama¹,

Takahashi²,

Takagi

et al. 2000

Journal of Biological Chemistry

106

104

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1-Deoxy-D-xylulose 5-phosphate (DXP) reductoisomerase, which simultaneously catalyzes the intramolecular rearrangement and reduction of DXP to form 2-C-methyl-D-erythritol 4-phosphate, constitutes a key enzyme of an alternative mevalonate-independent pathway for isopentenyl diphosphate biosynthesis. The dxr gene encoding this enzyme from Escherichia coli was overexpressed as a histidine-tagged protein and characterized in detail. DNA sequencing analysis of the dxr genes from 10 E. coli dxr-deficient mutants revealed base substitution mutations at four points: two nonsense mutations and two amino acid substitutions (Gly 14 to Asp 14 and Glu 231 to Lys 231 ). Diethyl pyrocarbonate treatment inactivated DXP reductoisomerase, and subsequent hydroxylamine treatment restored the activity of the diethyl pyrocarbonate-treated enzyme. To characterize these defects, we overexpressed the mutant enzymes G14D, E231K, H153Q, H209Q, and H257Q. All of these mutant enzymes except for G14D were obtained as soluble proteins. Although the purified enzyme E231K had wildtype K m values for DXP and NADPH, the mutant enzyme had less than a 0.24% wild-type k cat value. K m values of H153Q, H209Q, and H257Q for DXP increased to 3.5-, 7.6-, and 19-fold the wild-type value, respectively. These results indicate that Glu 231 of E. coli DXP reductoisomerase plays an important role(s) in the conversion of DXP to 2-C-methyl-D-erythritol 4-phosphate, and that His 153

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Incorporation of 2-C-Methyl-d-erythritol, a Putative Isoprenoid Precursor in the Mevalonate-Independent Pathway, into Ubiquinone and Menaquinone of Escherichia coli

Cited by 100 publications

References 18 publications

1-Deoxy- d -Xylulose 5-Phosphate Synthase, the Gene Product of Open Reading Frame (ORF) 2816 and ORF 2895 in Rhodobacter capsulatus

1-Deoxy- d -Xylulose 5-Phosphate Synthase, the Gene Product of Open Reading Frame (ORF) 2816 and ORF 2895 in Rhodobacter capsulatus

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

Characterization of 1-Deoxy-d-xylulose 5-Phosphate Reductoisomerase, an Enzyme Involved in Isopentenyl Diphosphate Biosynthesis, and Identification of Its Catalytic Amino Acid Residues

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