Karolina Skorupinska-Tudek

Contribution of the mevalonate and methylerythritol phosphate pathways to the biosynthesis of dolichols in plants.

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.

Role of polyisoprenoids in tobacco resistance against biotic stresses

Infection with avirulent pathogens, tobacco mosaic virus (TMV) or Pseudomonas syringae pv. tabaci induced accumulation of polyisoprenoid alcohols, solanesol and a family of polyprenols [from polyprenol composed of 14 isoprene units (Pren-14) to -18, with Pren-16 dominating] in the leaves of resistant tobacco plants Nicotiana tabacum cv. Samsun NN. Upon TMV infection, solanesol content was increased seven- and eight-fold in the inoculated and upper leaves, respectively, while polyprenol content was increased 2.5- and 2-fold in the inoculated and upper leaves, respectively, on the seventh day post-infection. Accumulation of polyisoprenoid alcohols was also stimulated by exogenously applied hydrogen peroxide but not by exogenous salicylic acid (SA). On the contrary, neither inoculation of the leaves of susceptible tobacco plants nor wounding of tobacco leaves caused an increase in polyisoprenoid content. Taken together, these results indicate that polyisoprenoid alcohols might be involved in plant resistance against pathogens. A putative role of accumulated polyisoprenoids in plant response to pathogen attack is discussed. Similarly, the content of plastoquinone (PQ) was increased two-fold in TMV-inoculated and upper leaves of resistant plants. Accumulation of PQ was also stimulated by hydrogen peroxide, bacteria (P. syringae) and SA. The role of PQ in antioxidant defense in cellular membranous compartments is discussed in the context of the enzymatic antioxidant machinery activated in tobacco leaves subjected to viral infection. Elevated activity of several antioxidant enzymes (ascorbate peroxidase, guaiacol peroxidase, glutathione reductase and superoxide dismutase, especially the CuZn superoxide dismutase isoform) and high, but transient elevation of catalase was found in inoculated leaves of resistant tobacco plants but not in susceptible plants.

Divergent pattern of Polyisoprenoid alcohols in the tissues of coluria geoides: A new electrospray lonization ms approach

Polyisoprenoid alcohols of the plant Coluria geoides were isolated and analyzed by HPLC with UV detection to determine the nature of the polyprenol and dolichol mixture in the organs studied. In roots, a family of dolichols (Dol-15 to Dol-23, with Dol-16 dominating, where Dol-n is dolichol composed of n isoprene units) was accompanied by traces of polyprenols of similar chain lengths, whereas in hairy roots grown in vitro, identical patterns with a slightly broader chain-length range were found. Conversely, in leaves and seeds polyprenols were the dominant form, and their pattern was shifted toward longer chains (maximal content of Pren-19, where Pren-n is polyprenol composed of n isoprene units). Interestingly, the pattern of dolichols in seeds and leaves (in which Dol-17 dominated) was similar to that found in roots.Structures of the dolichols and polyprenols isolated were confirmed by the application of a new HPLC/electrospray ionization-MS method, which also offers a much higher sensitivity in detection of these compounds compared to a UV detector. The highest sensitivity was obtained when the [M+Na]+ ions of polyprenols and dolichols were recorded in the selected ion monitoring mode and a small amount of sodium acetate solution was added post-column to enhance the formation of these ions in an electrospray ion source.

Polyisoprenoid alcohols—Recent results of structural studies

Polyisoprenoid alcohols (polyprenols and dolichols) are linear polymers of from several up to more than 100 isoprene units identified in almost all living organisms. Studies of their chemical structures have resulted in the discovery of new variants such as the recently described alloprenols with reversed configuration of the double bond in the alpha-isoprene unit. In parallel, structural elucidation of metabolically labeled plant dolichols has indicated that both the mevalonate and methylerythritol phosphate pathways are involved in the biosynthesis of dolichols in roots, leading to the construction of a spatial model of their biosynthesis. According to this model, in root cells, synthesis of the dolichol molecule is initiated in the plastids, and the resulting intermediates, oligoprenyl diphosphates, are exported to the cytoplasm and are elongated up to the desired chain length. The metabolic consequences of this putative model are discussed in the context of the enzymatic machinery involved.

Biosynthesis of A Water-Soluble Lipid I Analogue and A Convenient Assay for Translocase I

Translocase I (MraY/MurX) is an essential enzyme in growth of the vast majority of bacteria that catalyzes the transformation from UDP-MurNAc-pentapeptide (Parks nucleotide) to prenyl-MurNAc-pentapeptide (lipid I), the first membrane-anchored peptidoglycan precursor. MurX has received considerable attention in the development of new tuberculosis (TB) drugs due to the fact that the MurX inhibitors kill exponentially growing Mycobacterium tuberculosis (Mtb) much faster than clinically used TB drugs. Lipid I isolated from Mtb contains the C50-prenyl unit that shows very poor water solubility; thus, this chemical characteristic of lipid I renders MurX enzyme assays impractical for screening and lacks reproducibility of the enzyme assays. We have established a scalable chemical synthesis of Parks nucleotide-N(ε)-dansylthiourea 2 that can be used as a MurX enzymatic substrate to form lipid I analogues. In our investigation of the minimum structure requirement of the prenyl phosphate in the MraY/MurX-catalyzed lipid I analogue synthesis with 2, we found that neryl phosphate (C10 phosphate) can be recognized by MraY/MurX to generate the water-soluble lipid I analogue in quantitative yield under the optimized conditions. Here, we report a rapid and robust analytical method for quantifying MraY/MurX inhibitory activity of library molecules.

Atmospheric pressure photoionization mass spectrometry as a valuable method for the identification of polyisoprenoid alcohols.

RATIONALE The aim of this study was to determine whether Atmospheric Pressure Photoionization (APPI) was better suited for the mass spectrometric (MS) analysis of polyisoprenoid alcohols than the commonly used Electrospray Ionization (ESI) method. The APPI method should make possible the use of non-polar solvents without any of the additives required by ESI, together with improved detection limits. METHODS The liquid chromatography (LC)/APPI-MS and LC/ESI-MS spectra of polyisoprenoid alcohol standards were acquired in both positive and negative ion mode, in methanol and hexane, using a triple quadrupole/linear ion trap tandem mass spectrometer equipped with both an ESI and an APPI ion source. RESULTS In the positive ion mode peaks corresponding to [M + H - H(2)O](+) and [M + H](+) ions were observed in the APPI-MS spectra of polyprenols and dolichols, respectively. In the negative ion mode peaks corresponding to [M + O(2)](-•) and [M + Cl](-) ions were observed for both classes of polyisoprenoid alcohols. The detection limit of polyisoprenoid alcohols was established at the level of 10 pg. CONCLUSIONS APPI turned out to be a method of choice for the identification and quantitation of polyisoprenoid alcohols by MS using both polar and non-polar solvents. APPI also enabled greater differentiation of polyprenols and dolichols occurring together in natural samples and gave much better TIC chromatograms without the need for the post-column salt addition required by ESI.

Isoprenoid Alcohols are Susceptible to Oxidation with Singlet Oxygen and Hydroxyl Radicals

Isoprenoids, as common constituents of all living cells, are exposed to oxidative agents—reactive oxygen species, for example, singlet oxygen or hydroxyl radicals. Despite this fact, products of oxidation of polyisoprenoids have never been characterized. In this study, chemical oxidation of isoprenoid alcohols (Prenol-2 and -10) was performed using singlet oxygen (generated in the presence of hydrogen peroxide/molybdate or upon photochemical reaction in the presence of porphyrin), oxygen (formed upon hydrogen peroxide dismutation) or hydroxyl radical (generated by the hydrogen peroxide/sonication, UV/titanium dioxide or UV/hydrogen peroxide) systems. The structure of the obtained products, hydroxy-, peroxy- and heterocyclic derivatives, was studied with the aid of mass spectrometry (MS) and nuclear magnetic resonance (NMR) methods. Furthermore, mass spectrometry with electrospray ionization appeared to be a useful analytical tool to detect the products of oxidation of isoprenoids (ESI–MS analysis), as well as to establish their structure on the basis of the fragmentation spectra of selected ions (ESI–MS/MS analysis). Taken together, susceptibility of polyisoprenoid alcohols to various oxidizing agents was shown for the first time.

Medium-Chain Polyprenols Influence Chloroplast Membrane Dynamics in Solanum lycopersicum

The widespread occurrence of polyprenols throughout the plant kingdom is well documented, yet their functional role is poorly understood. These lipophilic compounds are known to be assembled from isoprenoid precursors by a class of enzymes designated as cis-prenyltransferases (CPTs), which are encoded by small CPT gene families in plants. In this study, we report that RNA interference (RNAi)-mediated knockdown of one member of the tomato CPT family (SlCPT5) reduced polyprenols in leaves by about 70%. Assays with recombinant SlCPT5 produced in Escherichia coli determined that the enzyme synthesizes polyprenols of approximately 50-55 carbons (Pren-10, Pren-11) in length and accommodates a variety of trans-prenyldiphosphate precursors as substrates. Introduction of SlCPT5 into the polyprenol-deficient yeast Δrer2 mutant resulted in the accumulation of Pren-11 in yeast cells, restored proper protein N-glycosylation and rescued the temperature-sensitive growth phenotype that is associated with its polyprenol deficiency. Subcellular fractionation studies together with in vivo localization of SlCPT5 fluorescent protein fusions demonstrated that SlCPT5 resides in the chloroplast stroma and that its enzymatic products accumulate in both thylakoid and envelope membranes. Transmission electron microscopy images of polyprenol-deficient leaves revealed alterations in chloroplast ultrastructure, and anisotropy measurements revealed a more disordered state of their envelope membranes. In polyprenol-deficient leaves, CO2 assimilation was hindered and their thylakoid membranes exhibited lower phase transition temperatures and calorimetric enthalpies, which coincided with a decreased photosynthetic electron transport rate. Taken together, these results uncover a role for polyprenols in governing chloroplast membrane dynamics.