Modeling of Dolichol Mass Spectra Isotopic Envelopes as a Tool to Monitor Isoprenoid Biosynthesis

Abstract

The cooperation of the mevalonate (MVA) and methylerythritol phosphate (MEP) pathways, operating in parallel in plants to generate isoprenoid precursors, has been studied extensively. Elucidation of the isoprenoid metabolic pathways is indispensable for the rational design of plant and microbial systems for the production of industrially valuable terpenoids. Here, we describe a new method, based on numerical modeling of mass spectra of metabolically labeled dolichols (Dols), designed to quantitatively follow the cooperation of MVA and MEP reprogrammed upon osmotic stress (sorbitol treatment) in Arabidopsis (Arabidopsis thaliana). The contribution of the MEP pathway increased significantly (reaching 100%) exclusively for the dominating Dols, while for long-chain Dols, the relative input of the MEP and MVA pathways remained unchanged, suggesting divergent sites of synthesis for dominating and long-chain Dols. The analysis of numerically modeled Dol mass spectra is a novel method to follow modulation of the concomitant activity of isoprenoid-generating pathways in plant cells; additionally, it suggests an exchange of isoprenoid intermediates between plastids and peroxisomes.Isopentenyl diphosphate (IPP), the building block of isoprenoids, the most numerous class of secondary metabolites, is derived in plants from two pathways operating in parallel: the cytoplasmic mevalonate (MVA) and the plastidic methylerythritol phosphate (MEP) pathways (Rohmer, 1999;Pulido et al., 2012).Analysis of over 130 isoprenoids has shown that, in angiosperms under standard growth conditions, carotenoids and chlorophyll phytyl chains on the one hand and phytosterols on the other are made nearly exclusively via a single pathway (MEP and MVA, respectively), while numerous other isoprenoids, including dolichols (Dols; Skorupinska-Tudek et al., 2008), are of mixed origin (Hemmerlin et al., 2012).The cooperation of the two pathways (often called cross talk) is considered essential for plant adaptive responses to biotic and abiotic stresses. Consequently, their relative contributions to the formation of IPP are modulated, and the expression of particular genes of the MVA and MEP pathways is frequently correlated with stress (pathogen attack, elicitation, wounding, etc.;Hemmerlin et al., 2012) or plant development (Opitz et al., 2014). Interestingly, the effect of stress on the regulation of the MEP-MVA balance has so far been analyzed only qualitatively.Several methodologies have been employed to unravel the relative contributions of the MVA and MEP pathways to isoprenoids, such as metabolic labeling with isotopically labeled precursors (stable isotopes or radioisotopes were used), followed by structural analysis of the product of interest or blockage of the pathway (chemical, with pathway-specific inhibitors, or genetic), followed by quantification of the isoprenoid intermediates and/or end products. The advantages

Metabolomics profiling reveals new aspects of dolichol biosynthesis inPlasmodium falciparum

“…3a). The specific labeling pattern of isoprene units resulting from glucose metabolism via glycolysis and incorporation through the mevalonate and MEP pathway are well established 17,18 . Because both the mevalonate and dolichol pathway are inactive in human RBCs 4 , only MEP pathway-specific labeling patterns are observed in P. falciparum infected RBCs 4,8,9 . Highly synchronous infected RBCs in ring stage at 7% parasitemia were first incubated in glucose-free media for 2 h and then supplemented for 24 h with [1-13 C]glucose (4 g/L) which is known to be metabolized into [1,[5][6][7][8][9][10][11][12][13] C]IPP ( Fig.…”

“…S1). The m/z shift toward higher molecular mass mirrors the stochastic distribution of the probability of incorporating differentially labeled 13 C-IPP into 13 C-polyprenols, and therefore 13 C-dolichols, which is determined by the remaining pool of native metabolic precursors in the cell that contain a 13 C natural abundance of ~1% and the number of 13 C-atoms incorporated into IPP based on the isotopic dilution due to metabolism 17,45 . As shown in Fig.…”

“…Currently, we are attempting to generate an inducible knockout of PfCPT to further verify its predicted essentiality 37 . Nevertheless, further investigations are needed to verify that polyprenols can be phosphorylated and glycosylated as dolichols in the malaria parasite.As a result of several breakthroughs in the field of cis-polyisoprenoids 17,19,20 , new biological functions of these lipids are starting to be unveiled besides their known role in protein glycosylation. Further studies are still needed to answer why co-occurrence of polyprenols and dolichols is present in the malaria parasite as well as why cis-polyisoprenoid species change during the parasite's life cycle which may reveal new biological functions of these unique lipids.…”

“…3a). The specific labeling pattern of isoprene units resulting from glucose metabolism via glycolysis and incorporation through the mevalonate and MEP pathway are well established 17,18 . Because both the mevalonate and dolichol pathway are inactive in human RBCs 4 , only MEP pathway-specific labeling patterns are observed in P. falciparum infected RBCs 4,8,9 .…”

“…These lipids are present in all membrane systems 15,16 but their biological functions besides protein glycosylation in the ER remains largely unknown. Recently, cis-polyisoprenoids gained special attention due to several breakthroughs in the field including improvements in the analytical techniques for their analysis 17,18 , partial identification of their enzymatic machinery 19 , and the discovery of new biological functions beyond glycosylation 20,21 . Among some of these biological functions are the regulation of the membrane fluidity 21 , stimulation of spore wall formation in yeast 20 , and scavenging free radicals in cell membranes [22][23][24] .…”