Plant sterol-C24-methyl transferases: different profiles of tobacco transformed with SMT1 or SMT2.

Higher plant cells contain a mixture of 24-desmethyl, 24-methyl(ene), and 24-ethyl(idene) sterols in given proportions according to species but also to cell type. As a first step to investigate the function of such sterol compositions in the physiology of a plant, we have illustrated in the present work the coexistence of two distinct (S)-adenosyl-L-methionine sterol-C24-methyltransferases (SMT) in transgenic Nicotiana tabacum L. Indeed, modulation of the expression of the tobacco gene SMT1-1, which encodes a cycloartenol-C24-methyltransferase, results in variations of the proportion of cycloartenol and a concomitant effect on the proportion of 24-ethyl sterols. Overexpression in tobacco of the Arabidopsis thaliana (L.) Heynh. gene SMT2-1 which encodes a 24-methylene lophenol-C24(1)-methyltransferase, results in a dramatic modification of the ratio of 24-methyl cholesterol to sitosterol associated with a reduced growth, a topic discussed in the present work.

Expression in yeast of an acyl-CoA:diacylglycerol acyltransferase cDNA from Caenorhabditis elegans.

We have identified a cDNA from the nematode worm Caenorhabditis elegans that encodes an acyl-CoA:diacylglycerol acyltransferase (DGAT). Its expression in Saccharomyces cerevisiae resulted in an increase both in triacylglycerol content and in microsomal oleyl-CoA:diacylglycerol acyltransferase activity. Such effects were similar to those of characterized plant DGAT genes. This is the first DGAT gene isolated from an invertebrate. The phylogenetic relationships between DGATs and animal and yeast acyl-CoA:sterol acyltransferases are illustrated.

Expression in yeast and tobacco of plant cDNAs encoding acyl CoA:diacylglycerol acyltransferase.

During the course of a search for cDNAs encoding plant sterol acyltransferases, an expressed sequence tag clone presenting substantial identity with yeast and animal acyl CoA:cholesterol acyltransferases was used to screen cDNA libraries from Arabidopsis and tobacco. This resulted in the isolation of two full-length cDNAs encoding proteins of 520 and 532 amino acids, respectively. Attempts to complement the yeast double-mutant are1 are2 defective in acyl CoA:cholesterol acyltransferase were unsuccessful, showing that neither gene encodes acyl CoA:cholesterol acyltransferase. Their deduced amino acid sequences were then shown to have 40 and 38% identity, respectively, with a murine acyl CoA:diacylglycerol acyltransferase and their expression in are1 are2 or wild-type yeast resulted in a strong increase in the incorporation of oleyl CoA into triacylglycerols. Incorporation was 2-3 times higher in microsomes from yeast transformed with these plant cDNAs than in yeast transformed with the void vector, clearly showing that these cDNAs encode acyl CoA:diacylglycerol acyltransferases. Moreover, during the preparation of microsomes from the Arabidopsis DGAT-transformed yeast, a floating layer was observed on top of the 100 000 g supernatant. This fraction was enriched in triacylglycerols and exhibited strong acyl CoA:diacylglycerol acyltransferase activity, whereas almost no activity was detected in the corresponding clear fraction from the control yeast. Thanks to the use of this active fraction and dihexanoylglycerol as a substrate, the de novo synthesis of 1,2-dihexanoyl 3-oleyl glycerol by AtDGAT could be demonstrated. Transformation of tobacco with AtDGAT was also performed. Analysis of 19 primary transformants allowed detection, in several individuals, of a marked increase (up to seven times) of triacylglycerol content which correlated with the AtDGAT mRNA expression. Furthermore, light-microscopy observations of leaf epidermis cells, stained with a lipid-specific dye, showed the presence of lipid droplets in the cells of triacylglycerol-overproducer plants, thus illustrating the potential application of acyl CoA:diacylglycerol acyltransferase-transformed plants.

Overexpression of an Arabidopsis cDNA encoding a sterol-C24(1)-methyltransferase in tobacco modifies the ratio of 24-methyl cholesterol to sitosterol and is associated with growth reduction.

Higher plants synthesize 24-methyl sterols and 24-ethyl sterols in defined proportions. As a first step in investigating the physiological function of this balance, an Arabidopsis cDNA encoding an S-adenosyl-L-methionine 24-methylene lophenol-C24(1)-methyltransferase, the typical plant enzyme responsible for the production of 24-ethyl sterols, was expressed in tobacco (Nicotiana tabacum L.) under the control of a constitutive promoter. Transgenic plants displayed a novel 24-alkyl-Delta5-sterol profile: the ratio of 24-methyl cholesterol to sitosterol, which is close to 1 in the wild type, decreased dramatically to values ranging from 0.01 to 0.31. In succeeding generations of transgenic tobacco, a high S-adenosyl-L-methionine 24-methylene lophenol-C24(1)-methyltransferase enzyme activity and, consequently, a low ratio of 24-methyl cholesterol to sitosterol, was associated with reduced growth compared with the wild type. However, this new morphological phenotype appeared only below the threshold ratio of 24-methyl cholesterol to sitosterol of approximately 0.1. Because the size of cells was unchanged in small, transgenic plants, we hypothesize that a radical decrease of 24-methyl cholesterol and/or a concomitant increase of sitosterol would be responsible for a change in cell division through as-yet unknown mechanisms.

Two families of sterol methyltransferases are involved in the first and the second methylation steps of plant sterol biosynthesis.

Two methyl transfers are involved in the biosynthesis of 24-methyl and 24-ethyl sterols, which play major roles in plant growth and development. The first methyl transfer applies to cycloartenol, the second to 24-methylene lophenol. About ten cDNA clones encoding S-adenosyl-L-methionine (AdoMet) sterol methyltransferases (SMTs) have been isolated so far from various plants. According to their deduced amino acid sequences, they were classified in two families, smtl and smt2; in addition, smt2 cDNAs were shown to encode a 24-methylene lophenol C24 methyltransferase [Bouvier-Navé, P., Husselstein, T., Desprez, T. & Benveniste, P. (1997) Eur. J. Biochem. 246, 518-529]. We now report the comparison of two cDNAs isolated from Nicotiana tabacum, Ntsmt1-1 which belongs to the first SMT cDNA family and Ntsmt2-1 which belongs to the second. Both cDNAs were expressed in the yeast null mutant erg6, deficient in SMT. Whereas erg6 is devoid of 24-alkyl sterols, erg6 Ntsmt1-1 contained a majority of 24-methylene sterols and erg6 Ntsmt2-1, a majority of 24-ethylidene sterols, indicating distinct functions for the expression products of these cDNAs. In the presence of AdoMet, delipidated microsomes from erg6 Ntsm1-1 efficiently converted cycloartenol into 24-methylene cycloartanol, but did not produce any 24-ethylidene lophenol upon incubation with 24-methylene lophenol. This demonstrates that cDNA Ntsmt1-1 (and most probably the other plant SMT cDNAs of the first family) encode(s) a cycloartenol C24 methyltransferase. In contrast, delipidated microsomes of erg6 Ntsmt2-1 were shown to methylate preferentially 24-methylene lophenol, as expected from an SMT encoded by an smt2 cDNA. In summary, among various cDNAs isolated from N. tabacum, one (Ntsmt1-1) belongs to the first family of plant SMT cDNAs according to its deduced amino acid sequence and was shown to encode a cycloartenol C24 methyltransferase, whereas another (Ntsmt2-1) belongs to the second family and was shown to encode a 24-methylene lophenol C24 methyltransferase. Meanwhile, two cDNAs were isolated from Oriza sativa and shown to belong to smtl and to smt2 families, respectively. These data disclose the coexistence, in a given plant species, of two distinct SMTs, each catalyzing one step of methylation in the sterol biosynthesis pathway.

Identification of cDNAs encoding sterol methyl-transferases involved in the second methylation step of plant sterol biosynthesis.

Two methyl transfers are involved in the course of plant sterol biosynthesis and responsible for the formation of 24-alkyl sterols (mainly 24-ethyl sterols) which play major roles in plant growth and development. The first methyl transfer applies to cycloartenol, the second one to 24-methylene lophenol. Five cDNA clones encoding two Arabidopsis thaliana, two Nicotiana tabacum and one Ricinus communis S-adenosyl-L-methionine (AdoMet) sterol methyltransferases (SMT) were isolated. The deduced amino acid sequences of A. thaliana and N. tabacum SMT are about 80% identical in all possible combinations. In contrast they are about 40% identical with the deduced amino acid sequence of R. communis SMT and the published Glycine max sequence. Both A. thaliana and one N. tabacum SMT cDNAs were expressed in a yeast null mutant erg6, deficient in AdoMet zymosterol C24-methyltransferase and containing C24-non-alkylated sterols. In all cases, several 24-ethylidene sterols were synthesized. A thorough study of the sterolic composition of erg6 expressing the A. thaliana cDNA 411 (erg6-4118-pYeDP60) showed 24-methylene and 24-ethylidene derivatives of 4-desmethyl, 4alpha-methyl and 4,4-dimethyl sterols as well as 24-methyl and 24-ethyl derivatives of 4-desmethyl sterols. The structure of 5alpha-stigmasta-8, Z-24(24(1))-dien-3beta-ol, the major sterol of transformed yeasts, was demonstrated by 400 MHz 1H NMR. Microsomes from erg6-4118-pYeDP60 were shown to possess AdoMet-dependent sterol-C-methyltransferase activity. Delipidated preparations of these microsomes converted cycloartenol into 24-methylene cycloartanol and 24-methylene lophenol into 24-ethylidene lophenol, thus allowing the first identification of a plant sterol-C-methyltransferase cDNA. The catalytic efficiency of the expressed SMT was 17-times higher with 24-methylene lophenol than with cycloartenol. This result provides evidence that the A. thaliana cDNA 411 (and most probably the 3 plant SMT cDNAs presenting 80% identity with it) encodes a 24-methylene lophenol-C-24(1) methyltransferase catalyzing the second methylation step of plant sterol biosynthesis.

UDP-glucose sterol beta-D-glucosyltransferase, a plasma membrane-bound enzyme of plants: enzymatic properties and lipid dependence.

UDP-glucose sterol beta-D-glucosyltransferase (UDPG-SGTase) catalyzes the glucosylation of plant sterols. This enzyme has been shown to be membrane-bound, most of its activity being associated with plasma membrane in etiolated maize coleoptiles. After solubilization with detergents, total delipidation and purification, kinetic studies performed with a purified enzyme preparation in the presence of detergent and soybean phosphatidylcholine (PC) strongly suggest an ordered bi-bi mechanism for the glucosylation of sterols. A reduced sulfhydryl group and an arginyl residue were shown to be essential for activity. Lipid dependence studies have been performed on the delipidated enzyme in two systems: a micellar one composed of a mixture of enzyme, detergent and phospholipids and another one where the enzymatic activity was reconstituted in unilamellar lipid vesicles. In both systems it was shown that the UDPG-SGTase activity was stimulated to a large extent by negatively charged phospholipids. Enzymatic assays were performed with membrane fractions originating from plants whose sterol content was profoundly modified following treatment with a sterol biosynthesis inhibitor. Results showed that the sterol glucosylating activity was strongly inhibited in these fractions in accordance with sterol substrate specificity studies. All these results show that the UDPG-SGTase is exquisitely sensitive to its lipid environment. Physiological implications of these data are discussed in the light of the putative role of sterols in the plant cell.

Phospholipid-Dependence of Plant UDP-Glucose Sterol beta-d-Glucosyl Transferase : IV. Reconstitution into Small Unilamellar Vesicles.

The phospholipid dependence of the UDP-glucose sterol glucosyl transferase (UDPG-SGTase) from maize coleoptiles was previously demonstrated using the partially purified and highly delipidated enzyme, in the presence of the detergent Triton X-100 (P Ullmann, P Bouvier-Navé, P Benveniste [1987] Plant Physiol 85: 51-55). We now report the reconstitution of the enzyme activity into unilamellar lipid vesicles. This was achieved by adding phospholipids, sterols and beta-octylglucoside to the solubilized enzyme and passing the mixture through Sephadex G-50. The treatment led to almost complete removal of the detergents. The incorporation of UDPG-SGTase in the lipid vesicles was demonstrated by (a) coelution of the enzyme activity with the labeled lipid vesicles (average diameter: 260A) on a Sephacryl S-1000 column and (b) flotation experiments on metrizamide density gradients. Release of dithiobis-(2-nitro-benzoic acid) (DTNB) from DTNB-preloaded vesicles was very slow, indicating good membrane integrity of the vesicles. Treatment of the intact vesicles with the nonpermeant reagent p-chloro-mercuribenzene sulfonate led to more than 95% inactivation of the total enzyme activity, i.e. the activity measured in the presence of Triton X-100 at permeabilizing concentration. This suggests an outward orientation for the active site of the enzyme. Finally, the enzyme was incorporated into vesicles of various phospholipid compositions and the kinetic parameters of the reactions were determined. Our results clearly show that the reconstituted UDPG-SGTase activity is stimulated to a large extent by negatively charged phospholipids.

Cyclopropyl sterol and phospholipid composition of membrane fractions from maize roots treated with fenpropimorph.

Maize (Zea mays L.) caryopses were grown in the presence of fenpropimorph, a systemic fungicide, for 7 days in the dark. Membrane fractions enriched, respectively, in endoplasmic reticulum, plasma membrane, and mitochondria were isolated from control and treated maize roots and analyzed for their free sterol, phospholipid, and fatty acid composition. In treated plants, the intracellular distribution of free sterols was dramatically modified both qualitatively and quantitatively. The normally occurring Delta(5)-sterols disappeared almost completely and were replaced by 9beta, 19-cyclopropyl sterols, mainly cycloeucalenol and 24-methyl pollinastanol. These new compounds were found to accumulate in all the membrane fractions in such a way that the endoplasmic reticulum-rich fraction became the richest one in free sterols instead of the plasma membrane. In contrast, the fenpropimorph treatment of maize roots was shown not to affect either the relative proportions or the amounts of the individual phospholipids, but an increase in the unsaturation index of phospholipid-fatty acyl chains of the endoplasmic reticulum-rich fraction was observed. The present data suggest that, in higher plant membranes, cyclopropyl sterols could play a structural role similar to that of the bulk of Delta(5)-sterols.

Regulation by Phospholipids and Kinetic Studies of Plant Membrane-Bound UDP-Glucose Sterol beta-d-Glucosyl Transferase.

Solubilization and partial purification of the microsomal UDP-glucose sterol glucosyl transferase activity from maize coleoptiles by chromatography on DEAE-cellulose resulted in a highly delipidated (>95%) and inactive enzymic preparation. Addition of sterols revealed part of the activity and subsequent addition of phospholipids further increased the activity. Negatively charged phospholipids were shown to be by far the best activators. The purification step also produced the elimination of two interfering microsomal enzymic activities: UDPase and steryl glucoside acyl transferase. The removal of these two enzymic activities was a prerequisite for kinetic studies including product-inhibition studies, since the substrates of these two latter enzymes are the products of UDPG-SGTase activity. The results of the kinetic studies strongly suggest an ordered bi-bi mechanism for the glucosylation of sterols. Finally the effect of different phospholipids on the kinetic parameters of the reaction was studied. Both phosphatidylcholine and phosphatidylglycerol significantly decrease K(m-sterol) (and not K(m-UDPglucose)) and increase the reaction V(max). The decrease of K(m-sterol) is similar with both phospholipids whereas the increase of V(max) is much greater with phosphatidylglycerol than with phosphatidylcholine.

The squalene-2,3-epoxide cyclase as a model for the development of new drugs.

The 2,3-oxido squalene (SO) cyclases represent a group of enzymes which convert SO into polycyclic triterpenoids such as lanosterol, cycloartenol, cucurbitadienol and beta-amyrin. Taking into account the postulated model of the enzymatic cyclization of SO, we have investigated the possibility of designing compounds that would be selective and potent inhibitors of SO cyclases. Due to the fundamental role of sterols in animal, higher plant and fungal tissues, these inhibitors might behave as very selective (ipocholesterolemic, antifungal or phytotoxic) drugs. Our first approach was the synthesis and biological evaluation of 2-aza-2,3-dihydrosqualene and its derivatives which, being protonated at physiological pH, would present some similarities to the C-2 carbon ion generated by the opening of the oxirane ring of SO. Microsomes from different sources (germinated pea cotyledons, maize seedlings, rat liver and yeasts) were utilized to determine the inhibition values (I50: concentration of inhibitor producing 50% inhibition at a given substrate concentration). From the results obtained so far we conclude that 2-aza-2-dihydrosqualene and its derivatives strongly inhibited the cyclases, the site of the enzyme responsible for binding to the inhibitor is quite sensitive to the steric hindrance, and the degree of the inhibitory activity is greater in higher plants than in rat liver or fungi.

Design of high energy intermediate analogues to study sterol biosynthesis in higher plants.

Several enzymes of plant sterol biosynthesis involve during their catalysis postulated or demonstrated carbocationic high energy intermediates (HEI). The aim of this study was to interfere with plant sterol biosynthesis by means of rationally designed species able to mimic these carbocationic HEI. It has been demonstrated previously that the design of transition state (TS) or HEI analogues could lead to powerful and specific inhibitors of enzymes. We applied this approach to the following target enzymes: 2,3-epoxy-2,3-dihydroqualene cyclase, AdoMet-cycloartenol-C-24-methyltransferase (AdoMet CMT), cycloeucalenol-obtusifoliol isomerase (COI) and Δ(8)-Δ(7)-sterol isomerase. Very potent inhibitors have been obtained in the four cases. As an example, analogues of cycloartenol substituted at C-25 by a charged heteroatom (N, As, S) have been synthesized and shown to be able to mimic the C-25 carbocationic HEI involved in the reaction catalyzed by the AdoMet CMT. These compounds were shown to be very potent and specific inhibitors of this enzyme both in vitro (Ki=2.10(-8) M, Ki/Km=10(-3)) and in vivo. The potent inhibitors described are powerful tools to control in vivo the sterol profile of plant cells and therefore to study the structural and functional roles of sterols in cell membranes. Moreover, these compounds constitute leader molecules of a new class of rationally designed inhibitors which could be of value in plant protection.

In vitro inhibition of animal and higher plants 2,3-oxidosqualene-sterol cyclases by 2-aza-2,3-dihydrosqualene and derivatives, and by other ammonium-containing molecules.

2-Aza-2,3-dihydrosqualene and related molecules, a series of new compounds designed as analogues of the transient carbocationic high energy intermediate, occurring in the oxirane ring opening during the cyclization of 2,3-oxidosqualene, were tested in vitro as inhibitors of the microsomal 2,3-oxidosqualene cyclase of animals (rat liver) and of higher plants (maize, pea). These molecules proved to be good and specific inhibitors for the cyclases of both phyla. The inhibition is due to positively charged species and is sensitive to the steric hindrance around the nitrogen-atom. 4,4,10 beta-Trimethyl-trans-decal-3 beta-ol and 4,10 beta-dimethyl-trans-decal-3 beta-ol, which have previously been described (J.A. Nelson et al., J. Am. chem. Soc. 100, 4900 (1978] as inhibitors of the 2,3-oxidosqualene cyclase of chinese hamster ovary cells, were found to be non-competitive inhibitors of the rat liver microsomal enzyme and presented no activity towards the higher plants cyclases. Aza derivatives of these decalines (A. Rahier et al., Phytochemistry, in press), which were aimed to mimic the C-8 carbocationic intermediate occurring during later steps of the 2,3-oxidosqualene cyclization did not inhibit the cyclases. This result underlines the theoretical limitations of the high energy analogues concept in designing enzyme inhibitors. Amongst other molecules tested, 2,3-epiminosqualene was found to be a reversible, non-competitive inhibitor of the cyclases; similarly U18666A was a very potent inhibitor of the microsomal cyclases. In contrast AMO 1618, a known anticholesterolemic agent reported previously to act at the level of the 2,3-oxidosqualene cyclization step, was not found per se to act on the cyclases.