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ENZYMES AND PROTEINS

Cloning and Characterization of 1-Deoxy-d-Xylulose 5-Phosphate Synthase fromStreptomyces sp. Strain CL190, Which Uses both the Mevalonate and Nonmevalonate Pathways for Isopentenyl Diphosphate Biosynthesis

Tomohisa Kuzuyama, Motoki Takagi, Shunji Takahashi, Haruo Seto
Tomohisa Kuzuyama
Institute of Molecular and Cellular Biosciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan
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Motoki Takagi
Institute of Molecular and Cellular Biosciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan
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Shunji Takahashi
Institute of Molecular and Cellular Biosciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan
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Haruo Seto
Institute of Molecular and Cellular Biosciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan
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DOI: 10.1128/JB.182.4.891-897.2000
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    Fig. 1.

    IPP biosynthesis via the mevalonate pathway (A) and via the nonmevalonate pathway (B). HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; d-GAP, d-glyceraldehyde 3-phosphate. The pathway leading to IPP from 2-C-methyl-d-erythritol 4-phosphate is undefined.

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    Fig. 2.

    Nucleotide sequence of the 2.9-kbSphI-SphI DNA fragment including thedxs gene from Streptomyces sp. strain CL190 and deduced amino acid sequence. The dxs gene consists of 1,896 bp starting with initiation codon GTG at position 926 and ending with termination codon TGA at position 2819. A putative Shine-Dalgarno sequence, GAAGG, was found 15 bp upstream of the initiation codon. An incomplete ORF product flanking the N terminus of the dxsgene product showed 24% identity in the 120-amino-acid region of overlap with the Helicobacter pylori putative protein (accession no., P56414 ) involved in the biosynthesis of the molybdopterin precursor from guanosine.

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    Fig. 3.

    Multiple alignment of the amino acid sequences of theStreptomyces DXP synthase and other DXP synthase homologs. Identical amino acids among the seven proteins are marked by asterisks. Dashes indicate gaps introduced for the optimization of the alignment. The amino acid sequences used for design of the PCR primers are underlined. CL190, Streptomyces sp. strain CL190; ECOLI,E. coli; HAEIN, H. influenzae; BACSU, B. subtilis; RHOCA, R. capsulatus; SYNY3,Synechocystis sp. strain PCC6803; ARATH, A. thaliana. For accession numbers, see Materials and Methods.

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    Fig. 4.

    Electrophoresis of the purified CL190 and E. coli DXP synthase overexpressed in E. coli. Purified DXP synthases of CL190 and E. coli obtained by using a Ni-nitrolotriacetic acid agarose column were analyzed by SDS–8 to 25% PAGE. Lanes: 1, molecular mass standard; 2, SDS-treated CL190 enzyme (0.2 μg); 3, SDS-treated E. coli enzyme (0.1 μg). Proteins were stained with Coomassie brilliant blue R-250.

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    Fig. 5.

    Temperature dependence of the CL190 DXP synthase activity and the Arrhenius plot (insert). The DXP synthase activity ofStreptomyces sp. strain CL190 was measured in the complete assay mixture as described in Materials and Methods except for the reaction temperature. One hundred percent activity corresponds to 0.42 U. All data are average values for duplicate determinations. The insert shows the Arrhenius plot used to estimate the activation energy of the enzyme.

Tables

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  • Table 1.

    Comparisons of enzymatic properties among CL190, E. coli, and pepper DXP synthases

    DXP synthase sourceKmfor:Vmax (U/mg of protein)Optimum temp (°C)Optimum pHaActivation energy (kJ/mol)Divalent cationsMolecular mass (kDa) by:Multimeric form
    Pyruvate (μM)d-Glyceraldehyde 3-phosphate (μM)d-Glyceraldehyde (mM)SDS-PAGEGel filtration chromatography
    CL190651203537042–449.099Mg2+, Mn2+70130Dimer
    E. coli962403830042–447.5–8.063Mg2+, Mn2+69120Dimer
    Pepperb500750
    • ↵a Assay solutions consisted of 100 mM Tris-HCl at pH 7.0 to 9.5.

    • ↵b From Bouvier et al. (1).

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Cloning and Characterization of 1-Deoxy-d-Xylulose 5-Phosphate Synthase fromStreptomyces sp. Strain CL190, Which Uses both the Mevalonate and Nonmevalonate Pathways for Isopentenyl Diphosphate Biosynthesis
Tomohisa Kuzuyama, Motoki Takagi, Shunji Takahashi, Haruo Seto
Journal of Bacteriology Feb 2000, 182 (4) 891-897; DOI: 10.1128/JB.182.4.891-897.2000

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Cloning and Characterization of 1-Deoxy-d-Xylulose 5-Phosphate Synthase fromStreptomyces sp. Strain CL190, Which Uses both the Mevalonate and Nonmevalonate Pathways for Isopentenyl Diphosphate Biosynthesis
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Cloning and Characterization of 1-Deoxy-d-Xylulose 5-Phosphate Synthase fromStreptomyces sp. Strain CL190, Which Uses both the Mevalonate and Nonmevalonate Pathways for Isopentenyl Diphosphate Biosynthesis
Tomohisa Kuzuyama, Motoki Takagi, Shunji Takahashi, Haruo Seto
Journal of Bacteriology Feb 2000, 182 (4) 891-897; DOI: 10.1128/JB.182.4.891-897.2000
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KEYWORDS

Hemiterpenes
Mevalonic Acid
Organophosphorus Compounds
Streptomyces
Transferases

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