Identification of a thiamin-dependent synthase in Escherichia coli required for the formation of the 1-deoxy-D-xylulose 5-phosphate precursor to isoprenoids, thiamin, and pyridoxol

Abstract

In Escherichia coli, 1-deoxy-D-xylulose (or its 5-phosphate, DXP) is the biosynthetic precursor to isopentenyl diphosphate [Broers, S. T. J. (1994) Dissertation (Eidgenössische Technische Hochschule, Zürich)], thiamin, and pyridoxol [Himmeldirk, K., Kennedy, I. A., Hill, R. E., Sayer, B. G. & Spenser, I. D. (1996) Chem. Commun. 1187-1188]. Here we show that an open reading frame at 9 min on the chromosomal map of E. coli encodes an enzyme (deoxyxylulose-5-phosphate synthase, DXP synthase) that catalyzes a thiamin diphosphate-dependent acyloin condensation reaction between C atoms 2 and 3 of pyruvate and glyceraldehyde 3-phosphate to yield DXP. We have cloned and overexpressed the gene (dxs), and the enzyme was purified 17-fold to a specific activity of 0.85 unit/mg of protein. The reaction catalyzed by DXP synthase yielded exclusively DXP, which was characterized by 1H and 31P NMR spectroscopy. Although DXP synthase of E. coli shows sequence similarity to both transketolases and the E1 subunit of pyruvate dehydrogenase, it is a member of a distinct protein family, and putative DXP synthase sequences appear to be widespread in bacteria and plant chloroplasts.

Figure 1

1-Deoxy-d-xylulose 5-phosphate as a common precursor of thiamin, pyridoxol, and isoprenoids.

Figure 2

Alignment of DXP synthase sequence from E. coli, CLA1 protein of A. thaliana, and transketolase A of E. coli. ECODXS, E. coli DXP synthase (ref. , this paper); CLA1AT, A. thaliana CLA1 protein, underlined is the N-terminal putative chloroplast transit peptide (26); ECOTKT1, transketolase A of E. coli (23). The shaded sequences denote the putative ThDP-binding site. The boldface characters denote amino acid residues that are highly conserved in all putative DXP synthase sequences, and asterisks below the sequence denote residues that are also identical in transketolase A from E. coli. # denotes amino acid residues that form the substrate channel of transketolase (27).

Figure 3

SDS/PAGE of different steps in DXP synthase purification. Lanes 1 and 7, Combithek size markers (Boehringer Mannheim). Lane 2, Cell-free extract of strain JM109/pBM20dxs without IPTG; lane 3, same plus IPTG; lane 4, after ammonium sulfate precipitation (40% saturation); lane 5, after anion-exchange chromatography on Q Sepharose HP; lane 6, after anion-exchange chromatography on DEAE-650S tentacle column. DXS denotes the protein band containing DXP synthase.

Figure 4

UV (A₁₈₅) and ¹⁴C elution profiles of DXP synthase assay mixtures after HPLC on an Aminex HPX-87H column (Bio-Rad). The column eluate was directed through a UV (arbitrary units) and a radioactivity monitor connected in series. (A) UV and ¹⁴C elution profiles of the assay mixture with cell-free extract of E. coli JM109 pUCBM20dxs after 2 h of incubation at 30°C. Phosphorylated (Cx-P) and ¹⁴C-labeled reaction products (¹⁴Cx-P) appeared in the void volume. (B) Elution profiles of the assay mixture shown in A after treatment with alkaline phosphatase, showing a novel UV and ¹⁴C peak (DOX, ¹⁴C-DOX). Peaks at retention times of 9.3 min (UV) and 10.0 min (¹⁴C) represent pyruvate; other peaks stem from components of the assay mixture or other reaction products.

Figure 5

Unrooted tree of DXP synthases (DXS family), transketolases, E1 proteins, and related proteins. A multiple alignment of sequences was constructed by a computer program (PepPepSearch) with an algorithm of Smith and Waterman (28). The alignment was obtained by using a deletion scoring function (29) and a Dayhoff matrix (30). The tree was generated by using an algorithm of Gonnet (31). Swiss-Prot/GenBank accession numbers of sequences are given for further identification. Characters a–g indicate DXS family sequences (a = E. coli U82664; b = Haemophilus influenzae P45205; c = A. thaliana U27099; d = Rhodobacter capsulatus P26242; e = Synechocystis sp. D90903; f = Bacillus subtilis P54523; g = Mycobacterium leprae P46708), h and i are sequences of unknown function consisting of 316 amino acid residues (Methanococcus jannaschii G64384) and 345 residues (Rhizobium sp. P55573). The transketolase family consists of j and k representing two human transketolases (P29401 and P51854), l = E. coli transketolase 1, P27302; m = H. influenzae P43757; n = Saccharomyces cerevisiae P23254; o = B. subtilis P45694; p = Alcaligenes eutrophus P21725; q = Mycoplasma genitalium P47312; r = Solanum tuberosum S58083, and s is dihydroxyacetone synthase (DAS, formaldehyde transketolase) from Hansenula polymorpha P06834. The E₁ p family consists of E1 protein sequences from PHDCs (t = A. thaliana P52901; u = human M24848; v = S. cerevisiae P32473; w = B. subtilis P21881; x = Mycoplasma capricolum MCU62057; y = Thiobacillus ferrooxidans TFU81808) and E1 proteins of oxoisovalerate dehydrogenase complexes (z = B. subtilis P37941; A = Pseudomonas putida P09061.