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J. Biol. Chem., Vol. 283, Issue 30, 21024-21035, July 25, 2008

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

Karolina Skorupinska-Tudek¹, Jaroslaw Poznanski¹, Jacek Wojcik, Tomasz Bienkowski, Izabela Szostkiewicz, Monika Zelman-Femiak, Agnieszka Bajda, Tadeusz Chojnacki, Olga Olszowska^¶, Jacob Grunler^||, Odile Meyer^**, Michel Rohmer^**, Witold Danikiewicz², and Ewa Swiezewska³

From the Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland, Institute of Organic Chemistry, Polish Academy of Sciences, 01-224 Warsaw, Poland, the ^¶Medical University of Warsaw, 02-090 Warsaw, Poland, ^||Rolf Luft Research Centre for Diabetes and Endocrinology, Karolinska Institutet, 17176 Stockholm, Sweden, and ^**Université Louis Pasteur/CNRS, Institut de Chimie, 6700 Strasbourg Cedex, France

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 ¹³C-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 [5-²H]mevalonate or [5,5-²H₂]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.

Received for publication, July 24, 2007 , and in revised form, April 17, 2008.

^* This work was supported by the State Committee for Scientific Research Grant PBZ-KBN-110/P04/19 and by European Community Grant LSHB-CT-2004-005151. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1–5, Tables 1 and 2, and additional text.

¹ Both authors contributed equally to this work.

² To whom correspondence may be addressed: Kasprzaka 44/52, 01-224 Warsaw, Poland. Fax: 48-22-6326681; E-mail: witold{at}icho.edu.pl. ³ To whom correspondence may be addressed: Pawinskiego 5a, 02-106 Warsaw, Poland. Fax: 48-22-5922190; E-mail: ewas{at}ibb.waw.pl.

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