Dimethylallyl pyrophosphate is not the committed precursor of isopentenyl pyrophosphate during terpenoid biosynthesis from 1-deoxyxylulose in higher plants

Abstract

Cell cultures of Catharanthus roseus were supplied with [2-13C, 3-2H]-deoxyxylulose or [2-13C,4-2H]1-deoxyxylulose. Lutein and chlorophylls were isolated from the cell mass, and hydrolysis of the chlorophyll mixtures afforded phytol. Isotope labeling patterns of phytol and lutein were determined by 2H NMR and 1H,2H-decoupled 13C NMR. From the data it must be concluded that the deuterium atom in position 3 of deoxyxylulose was incorporated into both isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate with a rate of 75% (with respect to the internal 13C label). The detected stereochemical signature implies that the label is located preferentially in the (E)-hydrogen atom of IPP. This preferential labeling, in turn, rules out dimethylallyl pyrophosphate as the compulsory precursor of IPP. In the experiment with [2-13C, 4-2H]1-deoxyxylulose, the 13C label was efficiently transferred to the terpenoids whereas the 2H label was completely washed out, most probably after IPP formation as a consequence of the isomerization and elongation process. In addition, the data cast light on the stereochemical course of the dehydrogenation and cyclization steps involved in the biosynthesis of lutein.

Figure 1

Biosynthesis of the terpenoid precursors IPP and DMAPP via the deoxyxylulose pathway (for review see refs. and 27).

Figure 2

Structures and atom numbering of phytol (7) and lutein (8).

Figure 3

¹³C NMR signals of phytol. (A) From the experiment with [2-¹³C,3-²H]deoxyxylulose; signals from a two-dimensional CH- correlated spectroscopy experiment are displayed (Upper). (B) From the experiment with [2-¹³C,4-²H]deoxyxylulose. (C) From a sample with natural ¹³C abundance.

Figure 4

13C NMR signals of lutein. (A) From the experiment with [2-¹³C,3-²H]deoxyxylulose; signals from a two-dimensional correlated spectroscopy via long-range coupling (COLOC) experiment are displayed (Upper). (B) From the experiment with [2-¹³C,4-²H]deoxyxylulose.

Figure 5

Part of the ¹H NMR spectrum (A) and ²H NMR spectrum (B) of phytol from the experiment with [2-¹³C,3-²H]deoxyxylulose. ²H NMR spectrum (C) of phytol from the experiment with [2-¹³C,4-²H]deoxyxylulose.

Figure 6

Labeling pattern of phytol (7) from the experiment with [2-¹³C,3-²H]deoxyxylulose. The labeling patterns of geranylgeranyl pyrophosphate (9), DMAPP (6), and IPP (5) are reconstructed retrobiosynthetically. ■ indicates ¹³C enrichment from ¹³C-2 of deoxyxylulose. H_a and H_b indicate positions of ²H enrichment from ²H-3 of the deoxyxylulose precursor. The numbers in italics indicate atomic ratios with respect to the value for the 16 position arbitrarily set at 1.

Figure 7

Parts of the ¹H NMR (A) and ²H NMR (B) of lutein from the experiment with [2-¹³C,3-²H]deoxyxylulose.

Figure 8

Labeling pattern of lutein (8) from the experiment with [2-¹³C,3-²H]deoxyxylulose. The labeling pattern of phytoene (10) was reconstructed from the labeling pattern of phytol (see also Fig. 6). The labeling pattern of lycopene (11) was adjusted to accommodate the observed labeling pattern of lutein. For other details see Fig. 6 and text.

Figure 9

Biosynthetic origin of hydrogen atoms in geranylgeranyl pyrophosphate (9) derived from IPP (5) and DMAPP (6) assembled via the deoxyxylulose pathway. ∗ and ● indicate positions of deuterium label. For details see Fig. 6 and text.

Figure 10

Stereochemical course of ring formation processes during biosynthesis of lutein.