Plant acyl-CoA:lysophosphatidylcholine acyltransferases (LPCATs) have different specificities in their forward and reverse reactions.

Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) enzymes have central roles in acyl editing of phosphatidylcholine (PC). Plant LPCAT genes were expressed in yeast and characterized biochemically in microsomal preparations of the cells. Specificities for different acyl-CoAs were similar for seven LPCATs from five different species, including species accumulating hydroxylated acyl groups in their seed oil, with a preference for C18-unsaturated acyl-CoA and low activity with palmitoyl-CoA and ricinoleoyl (12-hydroxyoctadec-9-enoyl)-CoA. We showed that Arabidopsis LPCAT1 and LPCAT2 enzymes catalyzed the acylation and de-acylation of both sn positions of PC, with a preference for the sn-2 position. When acyl specificities of the Arabidopsis LPCATs were measured in the reverse reaction, sn-2-bound oleoyl, linoleoyl, and linolenoyl groups from PC were transferred to acyl-CoA to a similar extent. However, a ricinoleoyl group at the sn-2-position of PC was removed 4-6-fold faster than an oleoyl group in the reverse reaction, despite poor utilization in the forward reaction. The data presented, taken together with earlier published reports on in vivo lipid metabolism, support the hypothesis that plant LPCAT enzymes play an important role in regulating the acyl-CoA composition in plant cells by transferring polyunsaturated and hydroxy fatty acids produced on PC directly to the acyl-CoA pool for further metabolism or catabolism.

Cholesterol glucosylation promotes immune evasion by Helicobacter pylori.

Helicobacter pylori infection causes gastric pathology such as ulcer and carcinoma. Because H. pylori is auxotrophic for cholesterol, we have explored the assimilation of cholesterol by H. pylori in infection. Here we show that H. pylori follows a cholesterol gradient and extracts the lipid from plasma membranes of epithelial cells for subsequent glucosylation. Excessive cholesterol promotes phagocytosis of H. pylori by antigen-presenting cells, such as macrophages and dendritic cells, and enhances antigen-specific T cell responses. A cholesterol-rich diet during bacterial challenge leads to T cell-dependent reduction of the H. pylori burden in the stomach. Intrinsic alpha-glucosylation of cholesterol abrogates phagocytosis of H. pylori and subsequent T cell activation. We identify the gene hp0421 as encoding the enzyme cholesterol-alpha-glucosyltransferase responsible for cholesterol glucosylation. Generation of knockout mutants lacking hp0421 corroborates the importance of cholesteryl glucosides for escaping phagocytosis, T cell activation and bacterial clearance in vivo. Thus, we propose a mechanism regulating the host-pathogen interaction whereby glucosylation of a lipid tips the scales towards immune evasion or response.

Two pathways of sphingolipid biosynthesis are separated in the yeast Pichia pastoris.

Although the yeast Saccharomyces cerevisiae has only one sphingolipid class with a head group based on phosphoinositol, the yeast Pichia pastoris as well as many other fungi have a second class, glucosylceramide, which has a glucose head group. These two sphingolipid classes are in addition distinguished by a characteristic structure of their ceramide backbones. Here, we investigate the mechanisms controlling substrate entry into the glucosylceramide branch of the pathway. By a combination of enzymatic in vitro studies and lipid analysis of genetically engineered yeast strains, we show that the ceramide synthase Bar1p occupies a key branching point in sphingolipid biosynthesis in P. pastoris. By preferring dihydroxy sphingoid bases and C(16)/C(18) acyl-coenzyme A as substrates, Bar1p produces a structurally well defined group of ceramide species, which is the exclusive precursor for glucosylceramide biosynthesis. Correlating with the absence of glucosylceramide in this yeast, a gene encoding Bar1p is missing in S. cerevisiae. We could not successfully investigate the second ceramide synthase in P. pastoris that is orthologous to S. cerevisiae Lag1p/Lac1p. By analyzing the ceramide and glucosylceramide species in a collection of P. pastoris knock-out strains in which individual genes encoding enzymes involved in glucosylceramide biosynthesis were systematically deleted, we show that the ceramide species produced by Bar1p have to be modified by two additional enzymes, sphingolipid Δ4-desaturase and fatty acid α-hydroxylase, before the final addition of the glucose head group by the glucosylceramide synthase. Together, this set of four enzymes specifically defines the pathway leading to glucosylceramide biosynthesis.

Conformational studies on the Delta8(E,Z)-sphingolipid desaturase from Helianthus annuus with chiral fluoropalmitic acids as mechanistic probes.

The Delta(8)-sphingolipid desaturase from sunflower (Helianthus annuus) converts phytosphinganine into a mixture of Delta(8)-(E)- and -(Z)-phytosphingenines by removal of two syn-hydrogen atoms from anti-, and gauche-conformations of the substrate. With chiral (R)-6-, (S)-6-, (R)-7-, and (S)-7-fluoropalmitic acids the importance of conformations for the formation of (E)- and (Z)-isomers was investigated by using growing yeast cells expressing the desaturase from H. annuus. The fluoropalmitic acids were readily incorporated into a series of fluorinated phytosphinganines. The desaturation products of the major C(18)-fluorophytosphinganine demonstrate that different conformations of the relevant aliphatic segment of the sphingolipids can be exposed to the active center of the enzyme resulting in (E)- or (Z)-fluoroalkenes. The presence of a fluorine atom at the position of the initial hydrogen removal C8-H(R) led to a complete suppression of the desaturation reaction, while replacement of C8-H(S) with fluorine generated a mixture of mainly (Z)- and trace amounts of (E)-fluoroolefine. Fluorine at C9 of the phytosphinganine precursors did not interfere with the initial C-H activation step and produced (E)- and (Z)-fluoroalkenes in the same ratio as observed for the nonfluorinated precursors. Hydroxylated byproducts of the desaturation process were not observed. These results strongly support the importance of conformations of the transition states during desaturation as the relevant criterion for the relative ratio of (E)- and (Z)-alkenes.

Stepwise engineering to produce high yields of very long-chain polyunsaturated fatty acids in plants.

Very long chain polyunsaturated fatty acids (VLCPUFAs) such as arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are valuable commodities that provide important human health benefits. We report the transgenic production of significant amounts of AA and EPA in Brassica juncea seeds via a stepwise metabolic engineering strategy. Using a series of transformations with increasing numbers of transgenes, we demonstrate the incremental production of VLCPUFAs, achieving AA levels of up to 25% and EPA levels of up to 15% of total seed fatty acids. Both fatty acids were almost exclusively found in triacylglycerols, with AA located preferentially at sn-2 and sn-3 positions and EPA distributed almost equally at all three positions. Moreover, we reconstituted the DHA biosynthetic pathway in plant seeds, demonstrating the practical feasibility of large-scale production of this important omega-3 fatty acid in oilseed crops.

Plasma membrane lipid alterations induced by cold acclimation and abscisic acid treatment of winter wheat seedlings differing in frost resistance.

Cold acclimation of plants affects many aspects of metabolism. Changes in plasma membrane lipids have always been considered to be important for development of frost resistance and survival at subzero temperatures. We studied different cultivars of winter wheat (Triticum aestivum L.) that differed in frost resistance induced either by cold acclimation or treatment with the plant hormone abscisic acid (ABA). Plasma membranes were isolated from non-acclimated and cold- as well as from ABA-acclimated plants, and were subjected to detailed lipid analysis. Cold acclimation affected virtually all plasma membrane lipid components and their constituents, resulting in both increases and decreases, which varied between the three groups of plants investigated. Including the cold-induced variations observed in the few plant species studied in detail previously, cerebrosides were the only components reduced by cold acclimation in all plants. In wheat, more uniform and consistent patterns were obtained when considering colligative parameters such as total free sterols, phospholipids or glycolipids, either as the proportion of total lipids or based on plasma membrane protein. The parameter which changed most significantly in parallel to the increase of inducible frost resistance in the three groups of plants was the ratio of free sterols/glycolipids, which increased. ABA treatment resulted in qualitatively similar effects in only one cultivar, but in general these changes were less pronounced. Compared to changes in transcription rates of several cold-induced genes and in the concentration of various compatible solutes reported for other plants, the observed changes in plasma membrane lipids are minor ones. This may indicate that acclimation-induced changes can be accomplished by posttranscriptional regulation of enzymatic activities, which is in agreement with the failure to detect significant changes in transcription of the corresponding genes during cold induction.

Relief for fish stocks: oceanic fatty acids in transgenic oilseeds.

Three recent reports (Baoxiu Qi et al., Amine Abbadi et al. and Anthony J. Kinney et al.) describe the production of very long-chain polyunsaturated fatty acids in transgenic plants. This might lead to a sustainable source of these valuable fatty acids for use in human food and animal feed. At present they are mainly available via consumption of fish, which is a limited and endangered resource.

A possible role of sphingolipids in the aluminium resistance of yeast and maize.

The plasma membrane is most likely the major target for sensing of aluminium (Al), leading to inhibition of plant root-growth. As a result of high external Al, alterations in plasma membrane composition may be expected in order to maintain its properties. As sphingolipids are characteristic components of this membrane, their involvement in membrane adjustment to increased Al concentrations was investigated. Heterologous expression of a stereounselective long-chain base (LCB) (8E/Z)-desaturase from Arabidopsis thaliana, Brassica napus and Helianthus annuus in Saccharomyces cerevisiae improved the Al resistance of the transgenic yeast cells. This encouraged us to investigate whether Al affects the LCB composition, and whether genetic engineering of the LCB profile modifies the Al resistance of the Al-sensitive plant species maize (Zea mays, L.). Constitutive expression of the LCB (8E/Z)-desaturase from Arabidopsis thaliana in maize roots led to an 8- to 10-fold increase in (8E)-4-hydroxysphing-8-enine in total roots. Less marked but similar changes were observed in 3 mm root apices. Al treatment of the Al-sensitive maize cv Lixis resulted in a significant increase in the proportion of (8Z)-LCB and in the content of total LCBs in root tips, which was not observed in the Al-resistant cv ATP-Y. When root tips of transgenic plants were exposed to Al, only minor changes of both (8Z)- and (8E)-unsaturated LCBs as well as of the total LCB were observed. Al treatment of the wild type parental line H99 decreased the (8Z)-unsaturated LCBs and the total LCB content. Based on Al-induced callose production, a marker for Al sensitivity, the parental line H99 was as Al-resistant as cv ATP-Y, whereas the transgenic line became as sensitive as cv Lixis. Taken together, these data suggest that, in particular, the loss of the ability to down-regulate the proportion of (8Z)-unsaturated LCBs may be related to increased Al sensitivity.

In vivo characterization of the first acyl-CoA Delta6-desaturase from a member of the plant kingdom, the microalga Ostreococcus tauri.

Genomic DNA of Ostreococcus tauri, a fully sequenced marine unicellular alga from the phytoplankton, was used to amplify a gene coding for a typical front-end desaturase involved in polyunsaturated fatty acid biosynthesis. Heterologous expression in Saccharomyces cerevisiae revealed very high desaturation activity with Delta6-regioselectivity. Short-time kinetic experiments showed that the desaturase product was detected in the acyl-CoA pool 5 min after addition of the exogenous substrate to the yeast medium and long before its appearance in the total fatty acids. When this desaturase was co-expressed with the acyl-CoA Delta6-elongase from Physcomitrella patens and the lipid-linked Delta5-desaturase from Phaeodactylum tricornutum, high proportions of arachidonic or eicosapentaenoic acid were obtained, because nearly all of the Delta6-desaturated products were elongated. Furthermore, the product/educt ratios calculated in each glycerolipid for the Delta6-desaturase or for the acyl-CoA Delta6-elongase were in about the same range, whereas this ratio showed a very uneven profile in the case of the lipid-linked Delta5-desaturase. Finally, a sequence-based comparison of all the functionally characterized Delta6-desaturases showed that this enzyme was not related to any previously described sequence. Altogether, our data suggest that this desaturase from O. tauri is an acyl-CoA Delta6-desaturase, the first one cloned from a photosynthetically active organism.

Plant sphingolipids: structural diversity, biosynthesis, first genes and functions.

In mammals and Saccharomyces cerevisiae, sphingolipids have been a subject of intensive research triggered by the interest in their structural diversity and in mammalian pathophysiology as well as in the availability of yeast mutants and suppressor strains. More recently, sphingolipids have attracted additional interest, because they are emerging as an important class of messenger molecules linked to many different cellular functions. In plants, sphingolipids show structural features differing from those found in animals and fungi, and much less is known about their biosynthesis and function. This review focuses on the sphingolipid modifications found in plants and on recent advances in the functional characterization of genes gaining new insight into plant sphingolipid biosynthesis. Recent studies indicate that plant sphingolipids may be also involved in signal transduction, membrane stability, host-pathogen interactions and stress responses.

Are glucocerebrosides the predominant sphingolipids in plant plasma membranes?

Long-chain sphingobases have been analyzed in various fractions prepared from different organs (leaf, root, storage tissue) from five dicotyledoneous plants (Arabidopsis thaliana, Brassica oleracea, Nicotiana tabacum, Pisum sativum, Spinacia oleracea). The resulting sphingobase profiles from cerebrosides and plasma membranes (PMs) show large qualitative and quantitative differences. Assuming that cerebrosides from all cellular membranes have similar sphingobase profiles, these data suggest that cerebrosides, considered to be characteristic glycolipids of plant PMs and specified by large proportions of sphingobases with an 8Z-double bond motif, do not represent the major sphingolipids of PMs. The fraction of unidentified complex sphingolipids, containing mainly 8E-phytosphingenine, exceeds the cerebroside proportion in PMs by several factors and may be as abundant as diacylglycerol-based phospholipids. These results are discussed with respect to the distribution of various lipids between the bilayer halves of plant PM.

Formation of glucosylceramide and sterol glucoside by a UDP-glucose-dependent glucosylceramide synthase from cotton expressed in Pichia pastoris.

In plants, glucosylceramide (GlcCer) biosynthesis is poorly understood. Previous investigations suggested that sterol glucoside (SG) acts as the actual glucose donor for the plant GlcCer synthase (GCS). We addressed this question by generating a Pichia pastoris double mutant devoid of GlcCer and SG. This mutant was used for heterologous expression of the plant GCS. The activity of the GCS resulted in the accumulation of GlcCer and, surprisingly, a small proportion of SG. The synthesis of GlcCer in the transformed double mutant shows that the GCS is SG-independent, while the detection of SG suggests that in addition to the sterol glucosyltransferase, also the GCS may contribute in planta to SG biosynthesis.

Metal-mediated reactions modeled after nature.

The Collaborative Research Center (CRC) 436 'Metal-Mediated Reactions Modeled after Nature' was founded for the express purpose of analyzing the catalytic principles of metallo-enzymes in order to construct efficient catalysts on a chemical basis. The structure of the active center and neighboring chemical environment in enzymes serves as a focal point for developing reactivity models for the chemical redesign of catalysts. Instead of simply copying enzyme construction, we strive to achieve new chemical intuition based on the results of long-lasting natural evolution. We hope for success, since nature uses a limited set of building blocks, whereas we can apply the full repertoire of chemistry. Key substrates in this approach are small molecules, such as CO2, O2 NO3- and N2. Nature complexes these substrates, activates them and performs chemical transformations--all within the active center of a metalloenzyme. In this article, we report on some aspects and first results of the Collaborative Research Center (CRC) 436, such as nitrate reductase, sphingolipid desaturase, carbonic anhydrase, leucine aminopeptidase and dopamine beta-monooxygenase.

Glycolipid headgroup replacement: a new approach for the analysis of specific functions of glycolipids in vivo.

Glycolipids with one or two sugar residues attached to different lipid backbones are found in biomembranes of bacteria, fungi, plants and animals in the form of steryl glycosides, glycosylceramides and diacylglycerol glycosides. They contain different sugar residues, mainly glucose and galactose, in either alpha- or beta-configuration. Many of the isolated compounds have been studied in great detail with regard to their biophysical behavior in artificial membrane systems. With the availability of cloned genes, the methods of reverse genetics were used to study glycolipid functions in living cells. The deletion of a lipid glycosyltransferase gene leads to the loss of the corresponding glycolipid in the transformed pro- and eukaryotic organisms. Often, these glycosyltransferase deletion mutants showed many differences to the wild-type organisms and thus demonstrated the biological importance of the glycolipid. When extensive deletion-induced glycolipid losses were not complemented by higher proportions of other membrane lipids, the mutants could display severe phenotypes due to a serious dysfunction or even collapse of an entire membrane system. On the other hand, by this approach the specific contribution of characteristic head group details cannot be recognized and separated from more general glycolipid functions. Many of these difficulties can be circumvented by a glycolipid headgroup replacement approach. This new approach requires the exchange of a lipid glycosyltransferase in an organism by a heterologous glycosyltransferase having a different headgroup specificity, e.g. the substitution of a galactosyltransferase by a glucosyltransferase. The resulting transgenic organism produces a novel glycolipid which differs from that of the native organism not in proportion, but only in structural details of its headgroup. Therefore, such rescued mutants are comparable to suppressor mutants and show less severe phenotypes than the intermediate deletion mutants. A comparison between the wild type, the simple deletion mutant and the mutant rescued by glycolipid replacement will not only disclose general functions of glycolipids, but also additional roles of headgroup details.

Rapid characterization of the fatty acyl composition of complex lipids by collision-induced dissociation time-of-flight mass spectrometry.

Profiling of leaf extracts from mutants of Arabidopsis with defects in lipid desaturation demonstrates the utility of collision-induced dissociation time-of-flight mass spectrometry (CID-TOF MS) for screening biological samples for fatty acid compositional alterations. CID-TOF MS uses the collision cell of a quadrupole time-of-flight mass spectrometer to simultaneously fragment all of the ions produced by an ionization source. Electrospray ionization CID-TOF MS in the negative mode can be used to analyze fatty acyl anions derived from complex lipids as well as free fatty acids. Although acyl anion yield is shown to be a function of the lipid class and the position on the glycerol backbone, acyl compositional profiles can be determined, and the TOF detector provides resolution of nominally isobaric acyl species in the profiles. Good precision is obtained when data are acquired for approximately 1 min per sample.

Channeling of eukaryotic diacylglycerol into the biosynthesis of plastidial phosphatidylglycerol.

Plastidial glycolipids contain diacylglycerol (DAG) moieties, which are either synthesized in the plastids (prokaryotic lipids) or originate in the extraplastidial compartment (eukaryotic lipids) necessitating their transfer into plastids. In contrast, the only phospholipid in plastids, phosphatidylglycerol (PG), contains exclusively prokaryotic DAG backbones. PG contributes in several ways to the functions of chloroplasts, but it is not known to what extent its prokaryotic nature is required to fulfill these tasks. As a first step toward answering this question, we produced transgenic tobacco plants that contain eukaryotic PG in thylakoids. This was achieved by targeting a bacterial DAG kinase into chloroplasts in which the heterologous enzyme was also incorporated into the envelope fraction. From lipid analysis we conclude that the DAG kinase phosphorylated eukaryotic DAG forming phosphatidic acid, which was converted into PG. This resulted in PG with 2-3 times more eukaryotic than prokaryotic DAG backbones. In the newly formed PG the unique Delta3-trans-double bond, normally confined to 3-trans-hexadecenoic acid, was also found in sn-2-bound cis-unsaturated C18 fatty acids. In addition, a lipidomics technique allowed the characterization of phosphatidic acid, which is assumed to be derived from eukaryotic DAG precursors in the chloroplasts of the transgenic plants. The differences in lipid composition had only minor effects on measured functions of the photosynthetic apparatus, whereas the most obvious phenotype was a significant reduction in growth.

Identification of fungal sphingolipid C9-methyltransferases by phylogenetic profiling.

Fungal glucosylceramides play an important role in plant-pathogen interactions enabling plants to recognize the fungal attack and initiate specific defense responses. A prime structural feature distinguishing fungal glucosylceramides from those of plants and animals is a methyl group at the C9-position of the sphingoid base, the biosynthesis of which has never been investigated. Using information on the presence or absence of C9-methylated glucosylceramides in different fungal species, we developed a bioinformatics strategy to identify the gene responsible for the biosynthesis of this C9-methyl group. This phylogenetic profiling allowed the selection of a single candidate out of 24-71 methyltransferase sequences present in each of the fungal species with C9-methylated glucosylceramides. A Pichia pastoris knock-out strain lacking the candidate sphingolipid C9-methyltransferase was generated, and indeed, this strain contained only non-methylated glucosylceramides. In a complementary approach, a Saccharomyces cerevisiae strain was engineered to produce glucosylceramides suitable as a substrate for C9-methylation. C9-methylated sphingolipids were detected in this strain expressing the candidate from P. pastoris, demonstrating its function as a sphingolipid C9-methyltransferase. The enzyme belongs to the superfamily of S-adenosylmethionine-(SAM)-dependent methyltransferases and shows highest sequence similarity to plant and bacterial cyclopropane fatty acid synthases. An in vitro assay showed that sphingolipid C9-methylation is membrane-bound and requires SAM and Delta4,8-desaturated ceramide as substrates.

Functional differences between galactolipids and glucolipids revealed in photosynthesis of higher plants.

Galactolipids represent the most abundant lipid class in thylakoid membranes, where oxygenic photosynthesis is performed. The identification of galactolipids at specific sites within photosynthetic complexes by x-ray crystallography implies specific roles for galactolipids during photosynthetic electron transport. The preference for galactose and not for the more abundant sugar glucose in thylakoid lipids and their specific roles in photosynthesis are not understood. Introduction of a bacterial glucosyltransferase from Chloroflexus aurantiacus into the galactolipid-deficient dgd1 mutant of Arabidopsis thaliana resulted in the accumulation of a glucose-containing lipid in the thylakoids. At the same time, the growth defect of the dgd1 mutant was complemented. However, the degree of trimerization of light-harvesting complex II and the photosynthetic quantum yield of transformed dgd1 plants were only partially restored. These results indicate that specific interactions of the galactolipid head group with photosynthetic protein complexes might explain the preference for galactose in thylakoid lipids of higher plants. Therefore, galactose in thylakoid lipids can be exchanged with glucose without severe effects on growth, but the presence of galactose is crucial to maintain maximal photosynthetic efficiency.