Cloning and characterization of a gene from Escherichia coli encoding a transketolase-like enzyme that catalyzes the synthesis of D-1-deoxyxylulose 5-phosphate, a common precursor for isoprenoid, thiamin, and pyridoxol biosynthesis

Abstract

For many years it was accepted that isopentenyl diphosphate, the common precursor of all isoprenoids, was synthesized through the well known acetate/mevalonate pathway. However, recent studies have shown that some bacteria, including Escherichia coli, use a mevalonate-independent pathway for the synthesis of isopentenyl diphosphate. The occurrence of this alternative pathway has also been reported in green algae and higher plants. The first reaction of this pathway consists of the condensation of (hydroxyethyl)thiamin derived from pyruvate with the C1 aldehyde group of D-glyceraldehyde 3-phosphate to yield D-1-deoxyxylulose 5-phosphate. In E. coli, D-1-deoxyxylulose 5-phosphate is also a precursor for the biosynthesis of thiamin and pyridoxol. Here we report the molecular cloning and characterization of a gene from E. coli, designated dxs, that encodes D-1-deoxyxylulose-5-phosphate synthase. The dxs gene was identified as part of an operon that also contains ispA, the gene that encodes farnesyl-diphosphate synthase. D-1-Deoxyxylulose-5-phosphate synthase belongs to a family of transketolase-like proteins that are highly conserved in evolution.

Figures

Figure 1

Restriction map of the genomic region containing the ispA gene from E. coli. The genomic region cloned in λ19F6 (21) is shown on top. The fragments subcloned into plasmids pLR1, pLR2, and pLR3 are also indicated. The position of the ispA gene is represented by a solid box. Restriction sites are as follows: B, BglII; H, HindIII; N, NdeI; P, PstI; Sm, SmaI; Sp, SphI; X, XhoI.

Figure 2

Analysis of a transcription unit from E. coli containing ORF1, ispA, ORF2 (dxs), and ORF3. (A) The previously reported genes ispA and pgpA and the short ORF cotranscribed with ispA (ORF1) are represented by open boxes. ORF2 and ORF3 are represented by shaded boxes. ATG and TAA indicate translation start and termination codons, respectively. The length (in bp) of the coding and intergenic regions is indicated in the line below. (B) A transcript containing ispA, ORF2, and ORF3 was detected by reverse transcription–PCR (RT-PCR) analysis with primers P1, P2, P3, and P4. Primers P2 and P4 were used for the synthesis of first strand cDNA by using reverse transcriptase. The PCR products obtained with the sets of primers P1/P2 and P3/P4 are shown below the mRNA. The enzymes used for the restriction enzyme mapping of the amplification products are also indicated.

Figure 3

Amino acid sequence alignment of d-1-deoxyxylulose-5-phosphate synthase from different organisms. Dots indicate the absence of particular amino acid residues. Numbers indicate the position of amino acid residues in the sequences. White-on-black letters denote amino acid residues common to at least six polypeptides. A consensus sequence denoting identical and conserved amino acid residues in all polypeptides is shown below the alignment. Conserved residues are indicated with the symbol +. Amino acid residues putatively involved in the binding of thiamin diphosphate are indicated with the symbol #. The conserved motif containing the histidine residue putatively involved in proton transfer during catalysis is indicated with ∗. Ecol, E. coli (GenBank AF035440); Hinf, H. influenzae (Swiss-Prot P45205); Rcap, R. capsulatus (Swiss-Prot P26242); Syne, Synechocystis sp. (PCC6803) (GenBank D90903); Bsub, B. subtilis (Swiss-Prot P54523); Hpyl, H. pylori (GenBank AE000552); Mtub, M. tuberculosis (GenBank Z96072); Atha, A. thaliana (GenBank U27099); Mpip, Mentha × piperita [GenBank AF019383; Lange et al. (40)].

Figure 4

Overexpression of the dxs gene product in E. coli. (A) E. coli XL1-Blue cells harboring plasmid pTAC-ORF2 or pTACTAC were induced with isopropyl β-d-thiogalactoside, and samples were withdrawn at the indicated times. Proteins were analyzed by SDS/PAGE and Coomassie blue staining. The position of molecular mass markers is indicated on the left.

Figure 5

Thin layer chromatograms of the reaction products obtained from pyruvate and d-glyceraldehyde or dl-glyceraldehyde 3-phosphate. Cell-free extracts from induced E. coli XL1-Blue cells harboring plasmid pTAC-ORF2 (25 μg of protein) were incubated for 2 h under the conditions described in Materials and Methods with the following substrates: pyruvate and d-glyceraldehyde (lanes 2 and 5) and pyruvate and dl-glyceraldehyde 3-phosphate (lanes 3 and 4). The reactions were stopped, and reaction mixture aliquots (10 μl) were treated with 1.25 units of calf intestine alkaline phosphatase for 1 h (lanes 4 and 5). Lane 1 corresponds to a standard of chemically synthesized d-1-deoxyxylulose. Reaction products were detected by staining with p-anisaldehyde/sulfuric acid. The positions of d-1-deoxyxylulose (1-DX) and d-1-deoxyxylulose 5-phosphate (1-DX-5-P) are indicated.