Evolutionarily conserved Δ25(27)-olefin ergosterol biosynthesis pathway in the alga Chlamydomonas reinhardtii
2012; Elsevier BV; Volume: 53; Issue: 8 Linguagem: Inglês
10.1194/jlr.m027482
ISSN1539-7262
AutoresMatthew B. Miller, Brad A. Haubrich, Qian Wang, William J. Snell, W. David Nes,
Tópico(s)Natural product bioactivities and synthesis
ResumoErgosterol is the predominant sterol of fungi and green algae. Although the biosynthetic pathway for sterol synthesis in fungi is well established and is known to use C24-methylation-C24 (28)-reduction (Δ24(28)-olefin pathway) steps, little is known about the sterol pathway in green algae. Previous work has raised the possibility that these algae might use a novel pathway because the green alga Chlamydomonas reinhardtii was shown to possess a mevalonate-independent methylerythritol 4-phosphate not present in fungi. Here, we report that C. reinhardtii synthesizes the protosterol cycloartenol and converts it to ergosterol (C24β-methyl) and 7-dehydroporiferasterol (C24β-ethyl) through a highly conserved sterol C24- methylation-C25-reduction (Δ25(27)-olefin) pathway that is distinct from the well-described acetate-mevalonate pathway to fungal lanosterol and its conversion to ergosterol by the Δ24 (28)-olefin pathway. We isolated and characterized 23 sterols by a combination of GC-MS and proton nuclear magnetic resonance spectroscopy analysis from a set of mutant, wild-type, and 25-thialanosterol-treated cells. The structure and stereochemistry of the final C24-alkyl sterol side chains possessed different combinations of 24β-methyl/ethyl groups and Δ22(23)E and Δ25 (27)-double bond constructions. When incubated with [methyl-2H3]methionine, cells incorporated three (into ergosterol) or five (into 7-dehydroporiferasterol) deuterium atoms into the newly biosynthesized 24β-alkyl sterols, consistent only with a Δ25 (27)-olefin pathway. Thus, our findings demonstrate that two separate isoprenoid-24-alkyl sterol pathways evolved in fungi and green algae, both of which converge to yield a common membrane insert ergosterol. Ergosterol is the predominant sterol of fungi and green algae. Although the biosynthetic pathway for sterol synthesis in fungi is well established and is known to use C24-methylation-C24 (28)-reduction (Δ24(28)-olefin pathway) steps, little is known about the sterol pathway in green algae. Previous work has raised the possibility that these algae might use a novel pathway because the green alga Chlamydomonas reinhardtii was shown to possess a mevalonate-independent methylerythritol 4-phosphate not present in fungi. Here, we report that C. reinhardtii synthesizes the protosterol cycloartenol and converts it to ergosterol (C24β-methyl) and 7-dehydroporiferasterol (C24β-ethyl) through a highly conserved sterol C24- methylation-C25-reduction (Δ25(27)-olefin) pathway that is distinct from the well-described acetate-mevalonate pathway to fungal lanosterol and its conversion to ergosterol by the Δ24 (28)-olefin pathway. We isolated and characterized 23 sterols by a combination of GC-MS and proton nuclear magnetic resonance spectroscopy analysis from a set of mutant, wild-type, and 25-thialanosterol-treated cells. The structure and stereochemistry of the final C24-alkyl sterol side chains possessed different combinations of 24β-methyl/ethyl groups and Δ22(23)E and Δ25 (27)-double bond constructions. When incubated with [methyl-2H3]methionine, cells incorporated three (into ergosterol) or five (into 7-dehydroporiferasterol) deuterium atoms into the newly biosynthesized 24β-alkyl sterols, consistent only with a Δ25 (27)-olefin pathway. Thus, our findings demonstrate that two separate isoprenoid-24-alkyl sterol pathways evolved in fungi and green algae, both of which converge to yield a common membrane insert ergosterol. The defining feature of eukaryote membranes, other than animals that possess the C27 cholesterol, is the presence of C28- to C30-steroidal compounds of varied side-chain constructions characterized by a C24-alkyl group. The addition of C1 to C3 side chains is derived by transmethylation reactions requiring S-adenosyl-L-methionine as the methyl donor and catalyzed by the sterol C24-methyltransferase (24-SMT) family of enzymes (1Nes W.D. Biosynthesis of cholesterol and other sterols.Chem. Rev. 2011; 111: 6423-6451Crossref PubMed Scopus (375) Google Scholar, 2Goodwin, T. W., 1981. Biosynthesis of plants sterols and other triterpenoids. In, Biosynthesis of isoprenoids, vol. I. Porter, J. W., Spurgeon, S. L., editors. Wiley, New York. 443–480.Google Scholar). Using differences in the C24-alkyl group size, stereochemistry, and complexity in further transalkylations at C22, C23, C27, and C28 as taxonomic traits, and linking them to steroidogenesis, we are able to reason the grouping of 24-alkyl sterol- containing organisms into more or less primitive and advanced forms of life (3Nes, W. D, Nes, W. R., . 1980. Lipids in evolution. Plenum Press, New York, 244, pp.Google Scholar). Fossil steranes identified from their diagenetic remains in sedimentary rocks confirm the evolution of 24-alkyl sterol diversity in Eukarya noted in the chemotaxonomy studies and further suggest an ancient origin (< 2.7 billion years ago) of the sterol frame (4Brocks J.J. Logan G.A. Buick R. Summons R.E. Archean molecular fossils and the early rise of eukaryotes.Science. 1999; 285: 1033-1036Crossref PubMed Scopus (942) Google Scholar–6Volkman J.K. Sterols and other triterpenoids: source specificity and evolution of biosynthetic pathways.Org. 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These compounds can be assembled modularly in three stages: In module I, synthesis of the basic C5-unit, isopentenyl diphosphate (Δ3-IPP) from glucose, can originate from two independent and nonhomologous metabolic pathways, namely the acetate-mevalonate or the mevalonate-independent 2-C-methyl-D-erythritol 4-phosphate pathways (Fig. 1) (1Nes W.D. Biosynthesis of cholesterol and other sterols.Chem. Rev. 2011; 111: 6423-6451Crossref PubMed Scopus (375) Google Scholar, 10Disch A. Schwender A.J. Muller C. Lichtenthaler H.K. Rohmer M. Distribution of the mevalonate and glycerlaldehyde phosphate/pyruvate pathways for isopreniod biosynthesis in unicellular alga and the cyanobacterium Synechocystis PCC 6714.Biochem. J. 1988; 333: 381-388Crossref Scopus (172) Google Scholar–12Hunter W.N. The non-mevalonate pathway of isoprenoid precursor biosynthesis.J. Biol. Chem. 2007; 282: 21573-21577Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). In module II, Δ3-IPP is converted to the protosterols, lanosterol (nonphotosynthetic lineage), or cycloartenol (photosynthetic lineage) (2Goodwin, T. W., 1981. Biosynthesis of plants sterols and other triterpenoids. In, Biosynthesis of isoprenoids, vol. I. Porter, J. W., Spurgeon, S. L., editors. Wiley, New York. 443–480.Google Scholar, 3Nes, W. D, Nes, W. R., . 1980. Lipids in evolution. Plenum Press, New York, 244, pp.Google Scholar, 13Nes W.D. Norton R.A. Crumley F.G. Madigan S.J. Katz E.R. Sterol phylogenesis and algal evolution.Proc. Natl. Acad. Sci. USA. 1990; 87: 7565-7569Crossref PubMed Scopus (76) Google Scholar, 14Phillips D.R. Rasbery J.M. Bartell B. Matsuda S.P.T. Biosynthetic diversity in plant triterpene cyclization.Curr. Opin. Plant Biol. 2006; 9: 305-314Crossref PubMed Scopus (300) Google Scholar). In module III, protosterols are converted to Δ5-sterols via a C24-reduction or coupled C24-alkylation-reduction pathway that yields the exquisite 24-alkyl sterol patterns observed throughout nature (1Nes W.D. Biosynthesis of cholesterol and other sterols.Chem. Rev. 2011; 111: 6423-6451Crossref PubMed Scopus (375) Google Scholar, 7Benveniste P. Biosynthesis and accumulation of sterols.Annu. Rev. Plant Biol. 2004; 55: 429-457Crossref PubMed Scopus (302) Google Scholar). A striking finding to emerge from in vivo isotopically labeling, sterol biosynthesis inhibitor-treatments, and cell-free 24-SMT enzyme studies among phylogenetically diverse algae that include the green, brown, and golden brown algae (15Nes W.R. McKean M.R. Biochemistry of steroids and other isopentenoids. University Park Press, Baltimore, MD1977Google Scholar–18Giner J-L. Djerassi C. Biosynthetic studies of marine lipids. 33. Biosynthesis of dinosterol, peridinosterol and gorgosterol: Unusual pattern of bioalkylation in dinoflagellate sterols.J. Org. Chem. 1991; 56: 2357-2363Crossref Scopus (32) Google Scholar) that is not necessarily obvious by current bioinformatic analyses (19Desmond E. Gribaldo S. Phylogenomics of sterol synthesis: Insights into the origin, evolution and diversity of a key eukaryotic feature.Genome Biol. Evol. 2009; 1: 364-381Crossref PubMed Google Scholar) is that ergosterol biosynthesis in the green algae may use a different set of enzymatic reactions in module III from fungi. A crucial difference between ergosterol biosynthesis in these organisms involves the sterol C24-methyl transferase activities and side-chain reductase specificities involved in generating the final ergosterol side chain. Thus, green algae express a 24-SMT that recognizes cycloartenol (16Mangla A.T. Nes W.D. Sterol C-methyl transferase from Prototheca wickerhamii: mechanism, sterol specificity and inhibition.Bioorg. Med. Chem. 2000; 8: 925-936Crossref PubMed Scopus (35) Google Scholar) and a sterol C25(27)-reductase (25-SR) of unknown substrate preference that operate in tandem to introduce stereoselectively the methyl cation at C24 and the hydride ion at C25 from S-adenosyl-L-methionine and NADPH, respectively (Fig. 2). Alternatively, fungi synthesize a 24-SMT that prefers substrates zymosterol or lanosterol, depending on the organism, and a sterol C24(28)-reductase the prefers ergosta-5,7,24(28)-trienol as substrate (1Nes W.D. Biosynthesis of cholesterol and other sterols.Chem. Rev. 2011; 111: 6423-6451Crossref PubMed Scopus (375) Google Scholar). In both sterol C-methylation-reduction pathways, the resulting ergosterol contains the same stereochemistry at C24 and C25 (1Nes W.D. Biosynthesis of cholesterol and other sterols.Chem. Rev. 2011; 111: 6423-6451Crossref PubMed Scopus (375) Google Scholar). Therefore, it was surprising that several investigations on sterol biosynthesis in the green alga Chlamydomonas reinhardtii reported that 24-alkyl(idene) sterol biosynthesis followed the fungal Δ24(28)-olefin pathway (20Brumfield K.M. Moroney J.V. Moore T.S. Simms T.A. Donze D. Functional characterization of the Chlamydomonas reinhardtii ERG3 ortholog, a gene involved in the biosynthesis of ergosterol.PLoS ONE. 2010; 5 (e8659)Crossref PubMed Scopus (22) Google Scholar, 21Salimova E. Boschetti A. Eichenberger W. Lutova L. Sterol mutants of Chlamydomonas reinhardtii: characterisation of three strains deficient in C24(28)-reductase.Plant Physiol. Biochem. 1999; 37: 241-249Crossref Scopus (14) Google Scholar). To deduce generalities for a Δ25(27)-olefin pathway in the synthesis of algal ergosterol and its 24-ethyl homolog, we have examined the ability of C. reinhardtii to synthesize sterol intermediates in the presence and absence of an inhibitor of the sterol C24-methylation reaction and after genetic manipulation to induce intermediates to accumulate in the cell. In a painstaking analysis of the minor and trace compounds of mutant and treated cells, 23 different sterols were detected, many of which contained a Δ25(27) bond consistent with a Δ25(27)-olefin pathway to ergosterol. Moreover, the sterol profiles of these cells failed to show C24(28)-ethylidene derivatives required in the synthesis of 24β-ethyl(idene) sterols synthesized in golden brown or brown algae or which can serve as precursor of land plant sitosterol (22Goad L.J. Lenton J.B. Knapp F.F. Phytosterol side chain biosynthesis.Lipids. 1974; 9: 582-595Crossref PubMed Scopus (87) Google Scholar, 23McKean M.L. Nes W.R. Evidence for separate intermediates in the biosynthesis of 24α- and 24β-alkylsterols in tracheophytes.Phytochemistry. 1977; 16: 683-686Crossref Scopus (34) Google Scholar). Analysis of isotopically labeled ergosterol and 7-dehydroporiferasterol isolated from C. reinhardtii grown in the presence of [methyl-2H3]methionine provided further verification for the Δ25(27)-olefin pathway to Δ5,7-C24β-alkyl sterols, which ultimately become the architectural components of algal cell membranes. C. reinhardtii wild-type strains 21gr (mt+ CC-1690 and 6145c; CC-1691) and ergosterol mutants KD7 and KD21 (24Bard M. Wilson K.J. Thompson R.M. Isolation of sterol mutants in Chlamydomonas reinhardtii: chromatographic analysis.Lipids. 1978; 13: 533-539Crossref PubMed Scopus (11) Google Scholar), obtained from the Chlamydomonas Genetics Center, Duke University (Durham, NC), were grown at 23°C on a 13:11 h light:dark cycle with aeration in medium I or medium II of Sager and Granick (25Sanger R. Granick S. Nutritional control of sexuality in Chlamydomonas reinhardtii.J. Can. Microbiol. 1954; 18: 729-742Google Scholar) as previously described (26Wang Q. Pan J. Snell W.J. Intraflagellar transport particles participate directly in cilium-generated signaling in Chlamydomonas.Cell. 2006; 25: 549-562Abstract Full Text Full Text PDF Scopus (116) Google Scholar). The KD7PY mutant was a product of a cross between 6145c and KD7 and was selected due its its ability to grow on agar plates in medium I containing nystatin (2 mM). For the inhibitor studies, cells (1 × 106/ml) were cultured for 3 days in medium I containing 1 µM 25-thialanosterol iodide salt. Cell number was determined using a hemocytometer. Sterol analysis was performed as described previously (27Kanagasabai R. Zhou W. Liu J. Nguyen T.T. Veeramachaneni P. Nes W.D. Disruption of ergosterol biosynthesis, growth and the morphological transition in Candida albicans by sterol methyltransferase inhibitors containing sulfur at C-25 in the sterol side chain.Lipids. 2004; 39: 737-746Crossref PubMed Scopus (18) Google Scholar). Briefly, algal cells at approximately 1 × 107 cells/ml were harvested by centrifugation and saponified in 10% aqueous methanolic KOH (10% w/v) at reflux for 30 min to give hexane-soluble neutral lipids. The neutral lipids were routinely examined by GC-MS (30 m HP-5 capillary column coupled to a HP 6890 gas chromatograph interfaced to a 5973 mass spectrometer at 70 eV; GC flow rate of He was set at 1.2 ml/min, injector port was 250°C, and the initial temperature was set at 170°C, held for 1 min, and increased at 20°C/min to 280°C) and HPLC equipped with a photodiode array detector used to provide UV spectra relevant to double bond character in the molecule. In several cases, sterols isolated from the nonsaponifiable lipid fraction and purified by HPLC (analytical Phenomenex Luna column, ODS-100A, eluted with methanol at 20°C at 1 ml/min or analytical TOSOHAAS TSK gel column, ODS-120A with acetonitrile/isopropanol [65/35, v/v] at 35°C at 1 ml/min) were examined by proton nuclear magnetic resonance spectroscopy (1HNMR) (spectra measured in deuterochloroform solutions on a Varian Unity Inova 500 MHz spectrometer with the chemical shifts referenced to chloroform resonating at 7.265 ppm and reported as δ in ppm, ppm) to confirm structure and stereochemistry of the side-chain C24-alkyl group. Authentic reference specimens for comparative GC-MS and 1HNMR analyses are taken from our sterol collection reported in references 27Kanagasabai R. Zhou W. Liu J. Nguyen T.T. Veeramachaneni P. Nes W.D. Disruption of ergosterol biosynthesis, growth and the morphological transition in Candida albicans by sterol methyltransferase inhibitors containing sulfur at C-25 in the sterol side chain.Lipids. 2004; 39: 737-746Crossref PubMed Scopus (18) Google Scholar–31Zhou W. Cross G.A.M. Nes W.D. Cholesterol import fails to prevent catalyst-based inhibition of ergosterol synthesis and cell proliferation in Trypanosoma brucei.J. Lipid Res. 2007; 48: 665-673Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar and from literature values (32Goad, L. J., Akihisa, T., editors. 1997. Analysis of sterols. Blackie Academic & Professional, New York.Google Scholar). Sterols are referenced to the retention time of cholesterol in capillary GC at 13.8 min (old column) or 14.5 min (new column) and in HPLC at 16.5 min (Luna column) or 26.8 min (TSK gel) affording the relative retention times to cholesterol in GC as the RRTc or in HPLC as the αc values. L-[methyl-2H3]methionine (98 atom % of 2H) (Sigma, St. Louis) was administered to wild-type C. reinhardtii cultures inoculated with 1 × 107 cells/ml at 1 mg/ml. After 3 days inoculation under light, the cells were harvested by centrifugation, and the total sterols from the cultures were examined by GC-MS. When this work was undertaken, little information was available on the sterol composition of Chlamydomonas. Several recent reports documented that this alga synthesizes two major sterol products, ergosterol and 7-dehydroporiferasterol, and variably a minor compound, ergost-7-enol (20Brumfield K.M. Moroney J.V. Moore T.S. Simms T.A. Donze D. Functional characterization of the Chlamydomonas reinhardtii ERG3 ortholog, a gene involved in the biosynthesis of ergosterol.PLoS ONE. 2010; 5 (e8659)Crossref PubMed Scopus (22) Google Scholar, 21Salimova E. Boschetti A. Eichenberger W. Lutova L. Sterol mutants of Chlamydomonas reinhardtii: characterisation of three strains deficient in C24(28)-reductase.Plant Physiol. Biochem. 1999; 37: 241-249Crossref Scopus (14) Google Scholar, 33Gealt M.A. Adler J.A. Nes W.R. The sterols and fatty acids from purified flagella of Chlamydomonas reinhardtii.Lipids. 1981; 16: 133-135Crossref Scopus (27) Google Scholar–35Schwender J. Gemunden C. Licthentaler H.K. Chlorophyta exclusively use the 1-deoxyxylulose 5-phosphate/2-C-methylerthritol 4-phosphate pathway for the biosynthesis of isoprenoids.Planta. 2001; 212: 416-423Crossref PubMed Scopus (110) Google Scholar). Moreover, [3-3H]squalene-2,3-oxide incubated in a microsomal enzyme preparation converts exclusively to cycloartenol (36Giner J-L. Wunsche L. Andersen R.A. Djerassi C. Dinoflagellates cyclize squalene oxide to lanosterol.Biochem. Syst. Ecol. 1991; 19: 143-145Crossref Scopus (21) Google Scholar). On the other hand, information is lacking about the types and amount of C4 methyl- or C24(28)-ethylidene intermediates involved in the sequence of chemicals to ergosterol or its 24-ethyl homolog from which a reliable sterol biosynthesis pathway could be constructed. In the present study, the total sterol fraction of C. reinhardtii was analyzed by GC-MS, and 11 sterols were detected (Fig. 3), including ergosterol, 7-dehydroporiferasterol, and cycloar tenol, which possessed mass and UV spectra similar to authentic specimens (Fig. 4). The major sterols in the sterol composition, ergosterol and 7-dehydroporiferasterol, were purified by HPLC, and 1HNMR analysis confirmed their structure and C24β-methyl/ethyl group stereochemistry (supplementary Table I). Minor compounds detected in the GC chromatogram were identified according to GC retention times and mass spectra relative to standards corresponding to cycloartenol, 4α,14α-dimethylergosta-8,25(27)-dienol, 4α,14α-dimethylergosta-8,24(28)-dienol (obtusifoliol) ergosta-7,25(27)-dienol, ergosta-8,25(27)-dienol, ergost-7-enol, porifersta-7,25(27)-dienol, porifersta-8,25(27)-dienol, and poriferst-7-enol (Tables 1 and 2). The finding of a set of C4-methyl intermediates, including cycloartenol, in the sterol composition of wild-type cells was significant (Fig. 4C) because it confirmed the "photosynthetic lineage" of sterol biosynthesis in this alga. Moreover, the natural occurrence of Δ25(27)-sterols in cells was consistent with ergosterol formation proceeding from a Δ25(27)-olefin pathway.Fig. 4Mass spectra and UV spectra (inset) for select sterols from control C. reinhardtii cells analyzed by GC-MS shown in Figure 1. A: GC peak 1. B: Peak 7. C: Peak 11.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 1.Chromatographic and spectral properties of sterols from Chlamydomonas reinhardtiiSystematic NameStructureaStructures of sterols are shown in Fig. 7.GC (RRTc)UV (λmax)MW (M )Cycloart-24(25)-enol11.43EA42624β-Methyl cycloart-25(27)-enol21.57EA440Cycloart-24(28)-enol31.59EA4404α, 14α-Dimethyl ergosta-8,25(27)-dienol41.23EA4264α, 14α-Dimethyl ergosta-8,24(28)-dienol51.25EA426Ergosta-8,25(27)-dienol61.15EA3984α-,14α-Dimethyl porifersta-8,25(27)-dienol71.40EA440Ergosta-7,25(27)-dienol81.17EA398Porifersta-8,25(27)-dienol91.29EA412Ergost-7-enol101.23EA400Porifersta-7,25(27)-dienol111.34EA412Ergosta-5,7-dienol121.2282398Poriferst-7-enol131.39EA414Ergosta-5,7,22-trienol141.10282396Poriferata-5,7-dienol151.35282412Porifersta-5,7,22-trienol161.28282410Lanosta-8,24-dienol bLanosterol was not detected in the cells and is given for reference purpose only.171.33EA4264α-,14α-Dimethyl cholesta-8,24-dienol181.15EA41214α-Methyl cholesta-8,24-dienol191.07EA398Cholesta-7,24-dienol201.12EA384Ergosta-5,7,25(27)-trienol211.18282396Ergosta-5,7,22,25(27)-tetraenol221.12282394Porifersta-5,7,25(27)-trienol231.33282410Porifersta-5,7,22,25(27)-tetraenol241.26282408a Structures of sterols are shown in Fig. 7.b Lanosterol was not detected in the cells and is given for reference purpose only. Open table in a new tab TABLE 2.Sterol composition of Chlamydomonas reinhardtii cellsSterolaStructures of sterols are shown in Fig. 7.WTbWT, wild-type cells.KD7cMutant cell lines.KD21cMutant cell lines.25-TLd25TL, 25-thialanosterol salt treated cells.KD7PYcMutant cell lines.10.30.72.51.120.330.34tretr, trace amount of sterol at less than 0.3%; blank refers to no sterol detected in cells.trtr0.95trtrtrtrtr62.77tr82.614.98.121.491.6103.530.5110.56.410.61221.3130.94.61450.836.61570.41637.227.51.5171815.5190.5208.82122.621.52219.412.7231.72.9243526.8a Structures of sterols are shown in Fig. 7.b WT, wild-type cells.c Mutant cell lines.d 25TL, 25-thialanosterol salt treated cells.e tr, trace amount of sterol at less than 0.3%; blank refers to no sterol detected in cells. Open table in a new tab To generate a more robust sterol profile from which a sterol biosynthesis pathway for C. reinhardtii could be established, we next examined the sterol composition of cells engineered to produce modified sterol compositions using mutant strains generated previously by Bard et al. (24Bard M. Wilson K.J. Thompson R.M. Isolation of sterol mutants in Chlamydomonas reinhardtii: chromatographic analysis.Lipids. 1978; 13: 533-539Crossref PubMed Scopus (11) Google Scholar), one mutant created by us from the Bard strains, and mutants after inhibitor treatment of wild-type cells. The sterol composition of KD7 was examined first because this mutant strain was reported to accumulate six unconventional C28 and C29Δ25(27)-sterol products, specifically a C28-7,25(27)-diene, C28-5,7,25(27)-triene, and C28-5,7,22,25(27)-tetraene and the corresponding C29-ethyl homologs (37Dennis A.L. Nes W.D. Sterol methyltransferase. Evidence for successive C-methyl transfer reactions generating Δ24(28)- and Δ25(27)-olefins by a single plant enzyme.Tetrahedron Lett. 2002; 43: 7017-7021Crossref Scopus (13) Google Scholar); two of them, ergosta-7,25(27)-dienol and porifersta-7,25(27)-dienol, were detected in our analysis of the sterol composition of wild-type cells (Table 2), suggesting that the Δ25(27)-sterol pathway might be operational in C. reinhardtii. In our investigation of the sterols from KD7, we detected eight sterols, including the six reported by Bard et al. (24Bard M. Wilson K.J. Thompson R.M. Isolation of sterol mutants in Chlamydomonas reinhardtii: chromatographic analysis.Lipids. 1978; 13: 533-539Crossref PubMed Scopus (11) Google Scholar) and two minor C4-methyl sterols that were detected in the wild-type cells, 4α,14α-dimethylergosta-8,25(27)-dienol and obtusifoliol (Table 2). In similar fashion, we investigated the sterol composition of KD21 reported previously to synthesize C28-7-ene, C28-5,7-diene, C29-7-ene, and C29-5,7-diene sterols, and in our studies we detected the same major sterols as well as two minor sterols obtusifoliol and cycloartenol, which were detected in wild-type cells (Table 2). Using HPLC, we purified four sterols from KD7 and two sterols from KD21. The structures of these compounds were confirmed by 1HNMR (supplementary Table I). Based on spectra of reference materials (31Zhou W. Cross G.A.M. Nes W.D. Cholesterol import fails to prevent catalyst-based inhibition of ergosterol synthesis and cell proliferation in Trypanosoma brucei.J. Lipid Res. 2007; 48: 665-673Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 37Dennis A.L. Nes W.D. Sterol methyltransferase. Evidence for successive C-methyl transfer reactions generating Δ24(28)- and Δ25(27)-olefins by a single plant enzyme.Tetrahedron Lett. 2002; 43: 7017-7021Crossref Scopus (13) Google Scholar, 38Zhou W. Lepesheva G.I. Waterman M.R. Nes W.D. Mechanistic analysis of a multiple product sterol methyltransferase implicated in ergosterol biosynthesis in Trypanosoma brucei.J. Biol. Chem. 2006; 281: 6290-6296Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), the sterols from the different cell types possessed one or more functional groups of a Δ22E double bond, a C24β-methyl/ethyl stereochemistry, and Δ25(27)-double bond in the sterol side chain. In addition, the combination of UV, MS, and 1HNMR analyses shows sterols from KD7 and KD21 to be populated by Δ7 and Δ5,7 nuclei. Analysis of a strain (KD7PY) derived by selection on nystatin of cells produced in a cross of KD7 and wild-type C. reinhardtii yielded 13 cometabolites, of which three were new C4-sterol intermediates not detected in wild-type, KD7, or KD21 cells. KD7PY cells accumulated trace amounts of cyclolaudenol and 24(28)-methylenecycloartanol along with minor amounts of 4α,14α-dimethylporifersta-8,25(27)-dienol (Table 2). The mass spectra of the structural isomers cyclolaudenol and 24(28)-methylenecycloartanol are almost identical to each other and, depending on the nature of the GC column, they coelute as we reported previously (13Nes W.D. Norton R.A. Crumley F.G. Madigan S.J. Katz E.R. Sterol phylogenesis and algal evolution.Proc. Natl. Acad. Sci. USA. 1990; 87: 7565-7569Crossref PubMed Scopus (76) Google Scholar) (Fig. 5). Indeed, Bard et al. (24Bard M. Wilson K.J. Thompson R.M. Isolation of sterol mutants in Chlamydomonas reinhardtii: chromatographic analysis.Lipids. 1978; 13: 533-539Crossref PubMed Scopus (11) Google Scholar) also noted the coelution of Δ25(27)- and Δ24(28)-sterols in their investigation of KD7 sterols, making structure identification equivocal based on GC-MS analysis. Our use of capillary GC column allows us to separate these structural isomers such that the Δ25(27)-olefin elutes before the Δ24(28)-olefin by a retention factor Δ25(27)/Δ24(28) of approximately 0.99 (Table 1); this chromato graphic technique was developed from studies on 24-SMT action (37Dennis A.L. Nes W.D. Sterol methyltransferase. Evidence for successive C-methyl transfer reactions generating Δ24(28)- and Δ25(27)-olefins by a single plant enzyme.Tetrahedron Lett. 2002; 43: 7017-7021Crossref Scopus (13) Google Scholar, 38Zhou W. Lepesheva G.I. Waterman M.R. Nes W.D. Mechanistic analysis of a multiple product sterol methyltransferase implicated in ergosterol biosynthesis in Trypanosoma brucei.J. Biol. Chem. 2006; 281: 6290-6296Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). A final incubation of C. reinhardtii with 25-thialanosterol salt, designed to block sterol C24-methyltransferase activity (27Kanagasabai R. Zhou W. Liu J. Nguyen T.T. Veeramachaneni P. Nes W.D. Disruption of ergosterol biosynthesis, growth and the morphological transition in Candida albicans by sterol methyltransferase inhibitors containing sulfur at C-25 in the sterol side chain.Lipids. 2004; 39: 737-746Crossref PubMed Scopus (18) Google Scholar, 39Nes W.D. Zhou W. Ganapathy K. Liu J. Vatsyayan R. Chamala S. Herandez K. Miranda M. Sterol C24-methyltransferase: an enzymatic target for the disruption of ergosterol biosynthesis and homeostasis in Cryptococcus neoformans.Arch. Biochem. Biophys. 2009; 481: 210-218Crossref PubMed Scopus (51) Google Scholar), led to growth inhibition after progressive addition of 25-thialanosterol to the medium (1–10 µM). At 1 µM, 10 sterols were detected by GC-MS analysis (Table 2); three of them were previously unidentified in the other cells studied and were determined to be 4α,14α-dimethylcholesta-8,24-dienol (31-norlanosterol), 14α-methylcholesta-8,24-dienol (14α-methylzymosterol), and cholesta-7,24-dienol, all sterols lacking a C24-alkyl group in the side chain. The wild-type C. reinhardtii strain contains two major sterols, ergosterol and 7-dehydroporiferasterol. The corresponding mass spectra for these 24β-alkyl sterols in the high mass end revealed ions at M+, M+-CH3, M+-H2O, and M+-CH3-H2O. Relevant ions for ergosterol appeared at m/- 396, 381, 378, and 363 amu and for 7-dehydroporiferasterol at m/z 410, 395, 392, and 377 amu (Fig. 3). When the alga was administered [methyl-2H3]methionine, three deuterium atoms were incorporated into the side chain of ergosterol (M+ m/z 399), and three or five ato
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