Terpenoid biosynthesis from 1-deoxy-D-xylulose in higher plants by intramolecular skeletal rearrangement

Abstract

The incorporation of [1-13C]- and [2,3,4,5-13C4]1-deoxy-D-xylulose into beta-carotene, lutein, phytol, and sitosterol in a cell culture of Catharanthus roseus was analyzed by NMR spectroscopy. The labeling patterns of the isoprene precursors, isopentenyl pyrophosphate and dimethylallyl pyrophosphate, were obtained from the terpenes by a retrobiosynthetic approach. 13C Enrichment and 13C13C coupling patterns showed conclusively that 1-deoxy-D-xylulose and not mevalonate is the predominant isoprenoid precursor of phytol, beta-carotene, and lutein. Label from 1-deoxyxylulose was also diverted to phytosterols to a minor extent (6% relative to carotene and phytol formation). The data demonstrate that the formation of isopentenyl pyrophosphate from pentulose occurs strictly by an intramolecular rearrangement process.

Figure 1

Structure (A) and ¹³C labeling pattern (B) of trans-phytol obtained by saponification of chlorophyll. Solid circles represent carbon atoms that acquired label from [1-¹³C]deoxyxylulose. Bold lines connect carbon atoms that were transferred from the same molecule of [2,3,4,5-¹³C₄]deoxyxylulose. Arrow indicates the carbon atom documented by the NMR pattern in Fig. 3A.

Figure 2

Structure of β-carotene (A) and lutein (B). The same labeling pattern (C) was obtained for both compounds. Arrow indicates the carbon atom documented by the NMR patterns in Fig. 3 B and C. For additional details see the legend to Fig. 1.

Figure 3

NMR patterns of metabolites derived from [2,3,4,5-¹³C₄]deoxyxylulose. (A) C-2 of phytol. (B) C-14 of β-carotene. (C) C-14 of lutein. (D) C-5 of sitosterol. Signals of natural ¹³C abundance isotopomers biosynthesized from proffered glucose are marked by asterisks. ¹³C coupling patterns are indicated. Coupled carbon atoms are shown in italics. Arrows indicate the predicted signal position of isotopomers with fewer than four contiguous ¹³C atoms per biosynthetic isoprenoid intermediate.

Figure 4

Section of INADEQUATE spectrum of β-carotene biosynthesized from [2,3,4,5-¹³C₄]deoxyxylulose. (Inset) The relevant part of the β-carotene structure. Contiguous ¹³C atoms are connected by bold lines.

Figure 5

Structure (A) and ¹³C labeling pattern (B) of sitosterol. For details see Fig. 1. Arrow indicates the position documented by the NMR spectrum in Fig. 3C.

Figure 6

The skeletal rearrangement conducive to the observed labeling patterns of IPP as reconstructed from the terpenoid compounds studied. ¹³C labels from [1-¹³C]deoxyxylulose are shown as solid circles. Labeling from [2,3,4,5-¹³C₄]deoxyxylulose is shown by bold lines. Arrow indicates the carbon atom documented by the NMR patterns in Fig. 4. R = H or PO₃H₂.

Figure 7

Hypothetical partitioning of the mevalonate pathway and the alternative pathway in C. roseus. R = H or PO₃H₂.