A family of transketolases that directs isoprenoid biosynthesis via a mevalonate-independent pathway

Abstract

Isopentenyl diphosphate, the common precursor of all isoprenoids, has been widely assumed to be synthesized by the acetate/mevalonate pathway in all organisms. However, based on in vivo feeding experiments, isopentenyl diphosphate formation in several eubacteria, a green alga, and plant chloroplasts has been demonstrated very recently to originate via a mevalonate-independent route from pyruvate and glyceraldehyde 3-phosphate as precursors. Here we describe the cloning from peppermint (Mentha x piperita) and heterologous expression in Escherichia coli of 1-deoxy-D-xylulose-5-phosphate synthase, the enzyme that catalyzes the first reaction of this pyruvate/glyceraldehyde 3-phosphate pathway. This synthase gene contains an ORF of 2,172 base pairs. When the proposed plastid targeting sequence is excluded, the deduced amino acid sequence indicates the peppermint synthase to be about 650 residues in length, corresponding to a native size of roughly 71 kDa. The enzyme appears to represent a novel class of highly conserved transketolases and likely plays a key role in the biosynthesis of plastid-derived isoprenoids essential for growth, development, and defense in plants.

Figure 1

Proposed mechanism of DXPS. The addition of hydroxyethyl TPP, formed by decarboxylation of pyruvate, to C1 of GAP and subsequent loss of TPP yields 1-deoxy-d-xylulose 5-phosphate, which ultimately gives rise to IPP. The circled P denotes the phosphate moiety.

Figure 2

GC–MS analysis of the biosynthetic product formed by the recombinant peppermint enzyme. (A) Mass spectrum of the biosynthetic product after dephosphorylation and trimethylsilylation (R_t = 6.71 min). (B) Mass spectrum of the silylated derivative of authentic 1-deoxy-d-xylulose (R_t = 6.70 min).

Figure 3

Time course of relative steady-state DXPS mRNA levels (•) and rate of monoterpene biosynthesis as measured by ¹⁴CO₂ incorporation (○) during leaf development in peppermint. Total RNA was isolated from oil gland secretory cells of leaves of different developmental stages in days (d) from emergence. A ³²P-labeled probe derived from DXPS clone pDS29 detected a transcript of about 3 kbp. Ethidium bromide-stained bands of 18S and 28S rRNA are shown as the internal controls. Leaves are fully expanded by 2 weeks, and 2.5-day leaves are the smallest from which oil gland secretory cells can be isolated (16).

Figure 4

Deduced amino acid sequence comparison (upper panel) of DXPS from peppermint (M.p.), CLA1 from A. thaliana (A.t.), ORF 2814 from R. capsulata (R.c.), ORF f620 from E. coli (E.c.), and a protein of unknown function from Synechocystis sp. strain PC6803 (S.sp.). Identical residues are shaded in black, residues of high similarity are indicated by gray shading, and residues of lower similarity are displayed by a pale shade. Asterisks indicate the position of the putative TPP-binding motif. Sequence comparison and clustering relationship analysis (lower panel) were carried out by using GCG version 9.0 of the University of Wisconsin Genetics Computer Group Package (1997). The following transketolase sequences are included: DXPS (M. × piperita, accession number AF019383); CLA1 (A. thaliana, U27099); ORF 2814 (R. capsulata, P26242); ORF f620 (E. coli, U82664); a protein of unknown function (Synechocystis sp. PC6803, D90903); transketolase 1 (human, A45050; yeast, P23254; and E. coli, P27302); transketolase 2 (human, P51854; yeast, P33315; E. coli, P33570); and a plastidial transketolase from potato (Z50099).