Article Figures & Data
Figures
- 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.
- 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.
- 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.
- 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.
- 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
- Table 1.
Comparisons of enzymatic properties among CL190, E. coli, and pepper DXP synthases
DXP synthase source Kmfor: Vmax (U/mg of protein) Optimum temp (°C) Optimum pHa Activation energy (kJ/mol) Divalent cations Molecular mass (kDa) by: Multimeric form Pyruvate (μM) d-Glyceraldehyde 3-phosphate (μM) d-Glyceraldehyde (mM) SDS-PAGE Gel filtration chromatography CL190 65 120 35 370 42–44 9.0 99 Mg2+, Mn2+ 70 130 Dimer E. coli 96 240 38 300 42–44 7.5–8.0 63 Mg2+, Mn2+ 69 120 Dimer Pepperb 500 750
