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 Polyisoprenoid alcohols together with sterols and quinone side chains constitute three main branches of terpene products originating from farnesyl diphosphate (FPP) 4 (1). These linear five-carbon unit polymers are divided into two groups, i.e. polyprenols and dolichols, according to the hydrogenation status of the ␣-terminal isoprene unit (dolichol structure is shown in Fig. 1). In cells, polyprenols and dolichols are always found as mixtures of prenologues, and data collected so far show polyprenols to be typical for bacteria and plants, whereas dolichols are generally attributed to animals and yeast (2). Nevertheless, it should be remembered that dolichols are the predominant form in some plant organs like roots (3). Data on the occurrence and functions of polyisoprenoids are summarized in recently published reviews (4, 5). The formation of the polyisoprenoid chain, starting from the -end of the molecule (Fig. 1), proceeds in a biphasic manner with farnesyl-diphosphate synthase responsible for the synthesis of the all-trans-FPP (three isoprene units of -t 2 structure, t stands for trans-isoprene unit), and its further elongation by cis-prenyltransferase. The latter enzyme, cloned from several prokaryotic and eukaryotic organisms (see Refs. 6, 7 and references therein), including Arabidopsis thaliana (8,9) and Hevea brasiliensis (10), utilizes isopentenyl diphosphate (IPP) for elongation of FPP up to the desired chain length, thus producing a family of polyprenyl diphosphates (n isoprene units of -t 2 -c n-3 structure, c stands for cis-isoprene unit), which are subsequently converted to polyprenols or dolichols according to the "tissue-specific requirements" by a still unknown mechanism.In plant cells two pathways are known to produce IPP utilized by numerous enzymes to finally 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). Both pathways are compartmentalized as follows: the MVA in the cytoplasm to provide sterols, the many sesquiterpenes, and the prenyl chains of ubiquinones, and the MEP one in the plastids Tables 1 and 2

Complete 1H and 13C signal assignment of prenol‐10 with 3D NMR spectroscopy

“…On the other hand, quantitative analysis of the intensity of 13 C NMR signals is essential for more detailed studies on the biosynthesis of polyisoprenoid alcohols in plants. Recently presented spatial model of the biosynthesis of dolichol in plant cell [22] requires further verification, which is possible by application of the method described in this report. In conclusion, we have shown that the application of 3D TOCSY-HSQC and 3D COSY-HMBC experiments resulted in the full spectral assignment of proton and carbon signals of natural abundance prenol-10.…”

“…[17] More accurate assignments have yielded many applications including analysis of compounds with a very large number of isoprene units [18 -21] and accurate tracing of biological pathways in dolichols. [22] Worth noting are 3D models of dolichol-19 and dolichyl-19 phosphate, which were based on 2D 1 H-NMR nuclear Overhauser enhancement spectroscopy (NOESY) results and refined by energy minimization with respect to all atoms using the assisted modelling with energy refinements (AMBER) force field, [23] even though the nuclear Overhauser effect (NOE) constraints used in structural calculations were from broad CH 3 and CH 2 signals.…”

Short-chain polyisoprenoids in the yeast Saccharomyces cerevisiae — New companions of the old guys

“…The MVA pathway is present in the majority of organisms, including yeasts [14], animals [15] and some gram-positive bacteria [16], whereas most gram-negative bacteria (e.g., Escherichia coli and Bacillus subtilis) [17] and green algae use only the MEP pathway [18]. In plants both pathways, the MVA and MEP are used to synthesize dolichols [19]. dominating [20 -22], while upon exponential growth only the former family is detectable.…”

Sugar availability modulates polyisoprenoid and phytosterol profiles in Arabidopsis thaliana hairy root culture

“…dehydrodolichols) [2]. Dolichols have been detected in mammalian and yeast cells and recently in plant roots [4,5]. An interesting feature of the polyisoprenoid alcohols of a given organism is their occurrence as a mixture commonly named 'family'.…”

“…While almost all the enzymatic steps of polyisoprenoid and phytosterol biosynthetic pathways have already been well described [3,5,19,20,21,22], the general mechanisms coordinating the isoprenoid metabolism with the cellular metabolic network are still a subject of extensive studies. One of the well recognized signaling molecules is glucose and hexokinase-dependent and hexokinase-independent signaling pathways are known to participate in sugar sensing [23].…”