Contribution of the Mevalonate and Methylerythritol Phosphate Pathways to the Biosynthesis of Dolichols in Plants

Karolina Skorupinska-Tudek,Jaroslaw Poznanski,Jacek Wojcik,Tomasz Bienkowski,Izabela Szostkiewicz,Monika Zelman-Femiak,Agnieszka Bajda,Tadeusz Chojnacki,Olga Olszowska,Jacob Grunler,Odile Meyer,Michel Rohmer,Witold Danikiewicz,Ewa Swiezewska

doi:10.1074/jbc.m706069200

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.

Highlights

In plant cells two pathways are known to produce isopentenyl diphosphate (IPP) utilized by numerous enzymes to give more than 50,000 different isoprenoid structures, the mevalonate pathway (MVA) and the mevalonate-independent methylerythritol phosphate pathway (MEP)
Root cultures were grown for no longer than 3 weeks to keep glucose concentration in the medium sufficiently high to ensure that it was the main carbon and energy source. It is well established in the literature that after labeling with [1-13C]glucose, carbon atoms derived from C-3 of IPP and DMAPP (Fig. 2 and supplemental Fig. 1) should not be labeled either via the MVA or the MEP pathway
C-5 is labeled via either pathway, C-1 will become labeled only when synthesized via the MEP one, whereas C-2 and -4 only via MVA

Summary

Introduction

In plant cells two pathways are known to produce IPP utilized by numerous enzymes to give more than 50,000 different isoprenoid structures, the mevalonate pathway (MVA) and the mevalonate-independent methylerythritol phosphate pathway (MEP) (for reviews, see Refs. 11–13). The ␻-termiphosphate reductoisomerase in the MEP pathway Both nal isoprene unit and several subsequent ones are synthesized inhibitors have recently been used to perturb biosynthetic flux with an involvement of both the MEP and MVA pathways, in in hairy roots [16, 17]. The involvement of both pathways lead- contrast to the very last ␣-terminal and a few preceding units, ing to the formation of a studied isoprenoid compound is most where contribution of the MEP pathway is negligible.

Results

Discussion

Conclusion