Contribution of the mevalonate and methylerythritol phosphate pathways to the biosynthesis of dolichols in plants

Abstract

Plant isoprenoids are derived from two biosynthetic pathways, the cytoplasmic mevalonate (MVA) and the plastidial methylerythritol phosphate (MEP) pathway. In this study their respective contributions toward formation of dolichols in Coluria geoides hairy root culture were estimated using in vivo labeling with (13)C-labeled glucose as a general precursor. NMR and mass spectrometry showed that both the MVA and MEP pathways were the sources of isopentenyl diphosphate incorporated into polyisoprenoid chains. The involvement of the MEP pathway was found to be substantial at the initiation stage of dolichol chain synthesis, but it was virtually nil at the terminal steps; statistically, 6-8 isoprene units within the dolichol molecule (i.e. 40-50% of the total) were derived from the MEP pathway. These results were further verified by incorporation of [5-(2)H]mevalonate or [5,5-(2)H(2)]deoxyxylulose into dolichols as well as by the observed decreased accumulation of dolichols upon treatment with mevinolin or fosmidomycin, selective inhibitors of either pathway. The presented data indicate that the synthesis of dolichols in C. geoides roots involves a continuous exchange of intermediates between the MVA and MEP pathways. According to our model, oligoprenyl diphosphate chains of a length not exceeding 13 isoprene units are synthesized in plastids from isopentenyl diphosphate derived from both the MEP and MVA pathways, and then are completed in the cytoplasm with several units derived solely from the MVA pathway. This study also illustrates an innovative application of mass spectrometry for qualitative and quantitative evaluation of the contribution of individual metabolic pathways to the biosynthesis of natural products.

FIGURE 1.

Structure of dolichols. t and c indicate the number of internal trans- and cis-isoprene units; α and ω indicate the terminal isoprene units.

FIGURE 2.

MVA and MEP pathway-specific labeling pattern of isoprene units resulting from glucose catabolism via glycolysis (according to Ref, 27) shown separately for [1-¹³C]glucose and [1,6-¹³C₂]glucose. Half-black circle indicates ¹³C abundance in isoprene units, which is 50% of the initial one in [1-¹³C]glucose (see “Results”). Numbering of carbon atoms is indicated.

FIGURE 3.

Profiles of dolichol isotopomer signals obtained from in vivo labeling with glucose. A, overlaid HPLC/ESI-MS spectra of Dol-n (-14 up to -19) obtained from [1-¹³C]glucose. B, spectra acquired after all three feeding experiments: red, [1-¹³C]glucose; blue, [1,6-¹³C₂]glucose, and green, [U-¹³C₆]glucose. In all samples, the group of low mass signals represents residual population of native dolichols remaining from root inoculum.

FIGURE 4.

Comparison of experimental and predicted MS spectra of Dol-16. Overlay of experimentally recorded MS spectrum of Dol-16 obtained from [1-¹³C]glucose feeding (red curve, cf. Fig. 3B) with two model theoretical spectra (hatched bars) calculated for Dol-16 synthesized exclusively by either the MEP or the MVA pathway. Location of the experimental data between the two theoretical distributions indicates mixed biosynthetic origin of dolichol. The signals of native dolichol (Fig. 3B) are overlaid with the theoretical spectrum (open bars) calculated for native Dol-16.

FIGURE 5.

Estimation of average molecular mass of dolichol. Gaussian distribution was fitted to experimentally recorded signals of isotopomers with highest intensity, as presented for representative Dol-16. Overlaid spectra obtained for Dol-16 isolated from roots grown on [1-¹³C]glucose (filled triangles), [1,6-¹³C₂]glucose (filled circles), and [U-¹³C₆]glucose (filled diamonds). Empty triangles and circles represent residual native dolichols from the inoculum of [1-¹³C]glucose and [1,6-¹³C₂]glucose. Solid lines represent Gaussian profiles for Dol-16 obtained after feeding by [1-¹³C]glucose (red lines), [1,6-¹³C₂]glucose (blue lines), [U-¹³C₆]glucose (green lines), and native dolichols (black lines). Arrows indicate the estimated average masses. Each point represents the intensity of a single Dol-16 isotopomer signal in the mass spectrum.

FIGURE 6.

The effect of ¹³C labeling on the increase of dolichol molecular mass. Markers represent average molecular masses of individual dolichols; Dol-14, Dol-15, Dol-16, Dol-17, and Dol-18 relate to the theoretical enrichment in the MVA pathway indicated by vertical dotted lines, 1.49, 2.99, and 4.95 Da for mono-, di-, and uniformly labeled glucose, respectively (see Table 3). Solid lines follow the expected values calculated for masses of dolichols synthesized exclusively via the MVA pathway. The observed mass deficit of 7–15 Da found for mono- and di-labeled glucose experiments indicates substantial participation of the alternative MEP pathway in dolichol synthesis.

FIGURE 7.

Incorporation of deuterium-labeled pathway-specific precursors into dolichols. Overlaid HPLC/ESI-MS spectra of representative Dol-16 of the deuterium-enriched (solid line) and native (dotted line) samples obtained from feeding with [5,5-²H₂]deoxyxylulose (A) and[5-²H]mevalonate (B).

FIGURE 8.

Compartmentalization of dolichol biosynthesis in root cells. Proposed cooperation of MEP and MVA pathways.