Comparative responses of the yeast mutant strain GL7 to lanosterol,cycloartenol, and cyclolaudenol

Under anaerobic growth conditions the isomeric 4,4′,14-trimethylcholestane derivatives lanosterol and, more efficiently, cycloartenol satisfy the sterol requirement of the yeast sterol auxotroph $\text{Saccharomyces cerevisiae}$ strain GL7. Aerobic mutant growth is supported only by cycloartenol and not by lanosterol, suggesting different structural requirements for aerobic and anaerobic cells. It is proposed that the non-planar conformation imposed by the 9,19-cyclopropane ring of cycloartenol moderates the adverse membrane effects of the nuclear methyl groups at C-4 and C-14. Under both aerobic and anaerobic conditions cyclolaudenol, a C-24-methyl derivative of cycloartenol, is a significantly more effective sterol source for strain GL7 than cycloartenol. This result is in keeping with the predominance of C-24-methyl sterols (ergosterol) in wild-type yeast.     

Effect of ethanol on the sterols of the fission yeast Schizosaccharomyces pombe

Abstract Ergosterol, lanosterol and two further unidentified sterols were detected and quantified in Schizosaccharomyces pombe cell extracts. In cells grown under anaerobic conditions, the levels of these sterols were dramatically reduced with a concomitant increase of their squaline precursor as compared with cells growing under aerobic conditions. Presence of ethanol resulted in a decrease in the sterol content under aerobic conditions. On the contrary, under anaerobic conditions presence of ethanol resulted in a three-fold increase of total sterols. Lanosterol was the main constituent of this elevation. It is suggested that lanosterol in parallel with unsaturated fatty acids is responsible for maintaining membrane integrity of S. pombe cells growing in the presence of ethanol.     

Is Pneumocystis a Plant?

Pneumocystis, an AIDS-associated opportunistic pathogen of the lung has some unusual features. This article focuses on work done by my group to understand the organism's distinct sterols. Although Pneumocystis is closely related to fungi, it lacks the major fungal sterol, ergosterol. Several delta(7) 24-alkysterols synthesized by P. carinii are the same as those reported in some basidiomycete rust fungi. The 24-alkylsterols are synthesized by the action of S-adenosyl-L-methionine:C-24 sterol methyl transferase (SAM:SMT). Fungal SAM:SMT enzymes normally transfer only one methyl group to the C-24 position of the sterol side chain and the cells accumulate C28 24-alkylsterols. In contrast, the P. carinii SAM:SMT and those of some plants catalyze one or two methyl transfer reactions producing both C28 and C29 24-alkylsterols. However, unlike most fungi, plants, and the kinetoplastid flagellates Leishmania and Trypanosoma cruzi, P. carinii does not appear to form double bonds at C-5 of the sterol nucleus and C-22 of the sterol side chain. Furthermore, the P. carinii SAM:SMT substrate preference for C30 lanosterol differs from that of homologous enzymes in any other organisms studied. C31 24-Methylenelanosterol and C32 pneumocysterol, products of SAM:SMT activity on lanosterol, can accumulate in high amounts in some, but not all, human-derived Pneumocystis jiroveci populations.     

Sterol 24-C-methyltransferase: An enzymatic target for the disruption of ergosterol biosynthesis and homeostasis in Cryptococcus neoformans

Growth of Cryptococcus neoformans was inhibited by nine nitrogen and sulfur-containing sterols with a heteroatom positioned at C3, C7, C24, C25 or C32 in the lanostane frame. Analysis of the sterol composition of control and treated cells by GC-MS and ¹H NMR has proven that the C-methylation reaction catalyzed by the sterol 24-C-methyltransferase (24-SMT) is the crucial first step in a kinetically favored pathway that fails to include obtusifoliol or zymosterol as intermediates. Cultures fed [methyl-²H₃]methionine led to two deuterium atoms into each of the newly biosynthesized sterols forming a route lanosterol, eburicol (24(28)-methylene-24,25-dihydrolanosterol), 32-noreburicol and ergost-7-enol to ergosterol. Examination of the substrate specificity of a soluble 24-SMT from C. neoformans showed lanosterol to be the optimal acceptor molecule. Incubation with the test compounds generated induced amounts of lanosterol, eburicol or 32-noreburicol concurrent with a decrease of ergosterol. Among them 24(R,S),25-epiminolanosterol (inhibitor of 24-SMT) showed the most potent in vitro antifungal activity comparable to those of itraconazole (inhibitor of the 14-demethylase). Taken together, these data indicate that treatment with substrate-based inhibitors of 24-SMT, a catalyst not found in humans, can disrupt ergosterol homeostasis involved with fungal growth and therefore these compounds can provide leads for rational drug design of opportunistic pathogens.     

The introduction of the C-22–C-23 ethylenic linkage in ergosterol biosynthesis

Methods for the chemical synthesis of [23-(3)H(2)]lanosterol, [23,25-(3)H(3)]24-methyldihydrolanosterol and [24,28-(3)H(2)]24-methyldihydrolanosterol are described. It is shown that, in the biosynthesis of ergosterol from [26,27-(14)C(2),23-(3)H(2)]lanosterol by the whole cells of Saccharomyces cerevisiae, one of the original C-23 hydrogen atoms is lost and the other is retained at C-23 of ergosterol. It is also shown that 24-methyldihydrolanosterol is converted into ergosterol in good yield and without prior conversion into a 24-methylene derivative. On the basis of these results possible pathways for the formation of the ergosterol side chain from a 24-methylene side chain are discussed.     

The transfer of hydrogen from C-24 to C-25 in ergosterol biosynthesis

1. A convenient synthesis of 3-hydroxytrisnorlanost-8-en-24-al and its conversion into [24-(3)H]lanosterol and [26,27-(14)C(2)]lanosterol is described. 2. A method for the efficient incorporation of lanosterol into ergosterol by the whole cells of Saccharomyces cerevisiae is also described. 3. It is shown that in the biosynthesis of ergosterol from doubly labelled lanosterol the C-24 hydrogen atom of lanosterol is retained in ergosterol. 4. On the basis of unambiguous degradations it is shown that the C-alkylation step in ergosterol biosynthesis is accompanied by the migration of a hydrogen atom from C-24 to C-25. 5. The mechanism for the biosynthesis of the ergosterol side chain is presented. 6. Mechanisms of other C-alkylation reactions are also discussed.     

8(9),22 -Ergostadiene-3 -ol, an ergosterol precursor accumulated in wild-type and mutants of yeast

Whereas wild-type strains of Saccharomyces cerevisiae can synthesize up to 7% dry weight of ergosterol, a polyene-resistant mutant has been obtained which produces no ergosterol. Instead, a C-28 methyl sterol is produced, and it has been identified as Delta(8(9),22)-ergostadiene-3beta-ol. This sterol is converted to ergosterol by wild-type yeasts and is observed transiently in cells during aerobic adaption of anaerobically grown wild-type yeasts. The new sterol is proposed as an intermediate in ergosterol biosynthesis.     

Ergosterol biosynthesis in novel melanized fungi from hypersaline environments

Halotolerant and halophilic melanized fungi were recently described in hypersaline waters. A close study of the sterol composition of such fungi, namely Hortaea werneckii, Alternaria alternata, Cladosporium sphaerospermum, Cladosporium sp., and Aureobasidium pullulans revealed the dominance of ergosterol and the presence of 29 intermediates of its biosynthesis pathway. The presence or absence of intermediates from distinct synthesis routes gave insight into the operative synthetic pathways from 4,4,14-trimethylcholesta-8,24-dien-3 beta-ol (lanosterol) to ergosterol in melanized fungi and in Saccharomyces cerevisiae, a reference yeast cultured in parallel. In all studied melanized fungi, initial methylation at C-24 took place before C-14 and C-4 demethylation, involving a different reaction sequence from that observed in S. cerevisiae. Further transformation was observed to occur through various routes. In A. alternata, isomerization at C-7 takes place prior to desaturation at C-5 and C-22, and methylene reduction at C-24. In addition to these pathways in Cladosporium spp., H. werneckii, and A. pullulans, ergosterol may also be synthesized through reduction of the C-24 methylene group before desaturation at C-5 and C-22 or vice versa. Moreover, in all studied melanized fungi except A. alternata, ergosterol biosynthesis may also proceed through C-24 methylene reduction prior to C-4 demethylation. -- Méjanelle, L., J. F. Lòpez, N. Gunde-Cimerman, and J. O. Grimalt. Ergosterol biosynthesis in novel melanized fungi from hypersaline environments. J. Lipid Res. 2001. 42: 352--358.     

Flux of sterol intermediates in a yeast strain deleted of the lanosterol C-14 demethylase Erg11p

Lanosterol C-14 demethylase Erg11p of the yeast Saccharomyces cerevisiae catalyzes the enzymatic step following formation of lanosterol by the lanosterol synthase Erg7p in lipid particles (LP). Localization experiments employing microscopic inspection and cell fractionation revealed that Erg11p in contrast to Erg7p is associated with the endoplasmic reticulum (ER). An erg11Delta mutation in erg3Delta background, which is required to circumvent lethality of the erg11 defect, did not only change the sterol pattern but also the sterol distribution within the cell. Whereas in wild type the plasma membrane was highly enriched in ergosterol and LP harbored large amounts of sterol precursors in the form of steryl esters, sterol intermediates were more or less evenly distributed among organelles of erg11Delta erg3Delta. This distribution is not result of the erg3Delta background, because in the erg3Delta strain the major intermediate formed, ergosta-7,22-dienol, is also highly enriched in the plasma membrane similar to ergosterol in wild type. These results indicate that (i) exit of lanosterol from LP occurs independently of functional Erg11p, (ii) random supply of sterol intermediates to all organelles of erg11Delta erg3Delta appears to compensate for the lack of ergosterol in this mutant, and (iii) preferential sorting of ergosterol in wild type, but also of ergosta-7,22-dienol in erg3Delta, supplies sterol to the plasma membrane.     

Novel sterol metabolic network of Trypanosoma brucei procyclic and bloodstream forms

Trypanosoma brucei is the protozoan parasite that causes African trypanosomiasis, a neglected disease of people and animals. Co-metabolite analysis, labelling studies using [methyl-2H3]-methionine and substrate/product specificities of the cloned 24-SMT (sterol C24-methyltransferase) and 14-SDM (sterol C14demethylase) from T. brucei afforded an uncommon sterol metabolic network that proceeds from lanosterol and 31-norlanosterol to ETO [ergosta-5,7,25(27)-trien-3β-ol], 24-DTO [dimethyl ergosta-5,7,25(27)-trienol] and ergosterol [ergosta-5,7,22(23)-trienol]. To assess the possible carbon sources of ergosterol biosynthesis, specifically 13C-labelled specimens of lanosterol, acetate, leucine and glucose were administered to T. brucei and the 13C distributions found were in accord with the operation of the acetate-mevalonate pathway, with leucine as an alternative precursor, to ergostenols in either the insect or bloodstream form. In searching for metabolic signatures of procyclic cells, we observed that the 13C-labelling treatments induce fluctuations between the acetyl-CoA (mitochondrial) and sterol (cytosolic) synthetic pathways detected by the progressive increase in 13C-ergosterol production (control<[2-(13)C]leucine<[2-(13)C]acetate<[1-(13)C]glucose) and corresponding depletion of cholesta-5,7,24-trienol. We conclude that anabolic fluxes originating in mitochondrial metabolism constitute a flexible part of sterol synthesis that is further fluctuated in the cytosol, yielding distinct sterol profiles in relation to cell demands on growth.     

Stereochemical specificity for sterols in Saccharomyces cerevisiae

When sterol biosynthesis in oxygen-deprived wild type Saccharomyces cerevisiae was prevented by the presence of 2,3-iminosqualene, an inhibitor of 2,3-oxidosqualene cyclase, an absolute requirement for a sterol with a 24 beta-methyl group was found. Neither the configuration nor the size of the alkyl group at C-24 could be altered. For instance, while 24 beta-methylcholesterol (22-dihydrobrassicasterol) permitted good growth, contrary to earlier work without the inhibitor no growth at all resulted from the presence of cholesterol or its 24 alpha-methyl-, 24 alpha-ethyl-, or 24 beta-ethyl derivatives (campesterol, sitosterol, and clionasterol, respectively). The only sterol lacking a 24 beta-methyl group which allowed growth was desmosterol (24-dehydro-cholesterol), but desmosterol was metabolized to 24 beta-methylcholesterol by C1-transfer and reduction. When cholesterol supported growth in the absence of the inhibitor, small amounts of endogenously synthesized 24 beta-methylsterols (ergosterol and 22-dihydroergosterol) were identified. This previously unrecognized absolute specificity for both chirality and bulk at C-24 suggests the involvement of protein binding in at least one of the roles which sterol plays in this single-celled eukaryote.     

Identification of C31 and C32 Sterols in Pneumocystis carinii hominis-Infected Human Lungs

SUMMARY Two sterols in autopsied whole lung specimens obtained from Pneumocystis carinii pneumonia patients were detected by gas-liquid chromatography and their structures were elucidated by mass spectrometry and nuclear magnetic resonance spectrometry. Both were in the lanosterol series; the C₃₁ sterol, with a methyl group at C-24, was identified as euphorbol, and the more abundant C₃₂ sterol, with an ethyl group at C-24, is given the trivial name pnemocysterol.     

Molecular dynamics simulation of the structure of dimyristoylphosphatidylcholine bilayers with cholesterol, ergosterol, and lanosterol

Five molecular dynamics computer simulations were performed on different phospholipid:sterol membrane systems in order to study the influence of sterol structure on membrane properties. Three of these simulated bilayer systems were composed of a 1:8 sterol:phospholipid ratio, each of which employed one of the sterol molecules: cholesterol, ergosterol, and lanosterol. The two other simulations were of a bilayer with a 1:1 sterol:phospholipid ratio. These simulations employed cholesterol and lanosterol, respectively, as their sterol components. The observed differences in simulations with cholesterol and lanosterol may have their implication on the form of the phospholipid/sterol phase diagram.     

甾醇C-24甲基转移酶和甾醇C-8异构酶在酿酒酵母麦角甾醇生物合成中的调控作用

摘要：【目的】研究ERG6基因编码的甾醇C-24甲基转移酶和ERG2基因编码的甾醇C-8异构酶在酿酒酵母麦角甾醇生物合成代谢中的调控作用。【方法】通过PCR扩增克隆到酿酒酵母甾醇C-8异构酶的编码序列及其终止子序列,以大肠杆菌-酿酒酵母穿梭质粒YEp352为载体,以磷酸甘油酸激酶基因PGK1启动子为上游调控元件构建了酵母菌表达质粒pPERG2;同时,在本实验室已构建的ERG6表达质粒pPERG6的基础上,构建了ERG2和ERG6共表达的重组质粒pPERG6-2。将表达质粒转化酿酒酵母单倍体菌株YS58,依据营养缺陷互补筛选到重组菌株YS58(pPERG2)和YS58(pPERG6-2)。通过紫外分光光度法和气相色谱法分析重组菌株甾醇组分和含量。【结果】在ERG6高表达的重组酵母菌中,甾醇中间体和终产物麦角甾醇的含量均比对照菌高;而在ERG2高表达的酵母菌株中,无论甾醇中间体,还是麦角甾醇的含量均明显降低。ERG6和ERG2共表达重组菌株YS58(pPERG6-2)的麦角甾醇含量是对照菌株YS58(YEp352)的1.41倍,是ERG2单独高表达菌株YS58(pPERG2)的1.92倍,是ERG6单独高表达菌株YS58(pPERG6)的1.12倍。【结论】本研究首次证明甾醇C-24甲基转移酶催化的反应是酿酒酵母麦角甾醇合成代谢途径中的一个重要的限速步骤,该酶活性提高不但补偿了ERG2高表达对甾醇合成的负效应,而且使麦角甾醇含量进一步提高,为构建麦角甾醇高产酵母工程菌株提供了实验依据。     

Structural discrimination in the sparking function of sterols in the yeast Saccharomyces cerevisiae.

A Saccharomyces cerevisiae sterol auxotroph, SPK14 (a hem1 erg6 erg7 ura), was constructed to test the ability of selected C-5,6 unsaturated sterols at growth-limiting concentrations to spark growth on bulk cholestanol. The native sterol, ergosterol, initiated growth faster and allowed a greater cell yield than did other sterols selectively altered in one or more features of the sterol. Although the C-5,6 unsaturation is required for the sparking function, the presence of the C-22 unsaturation was found to facilitate sparking far better than did the C-7 unsaturation, whereas the C-24 methyl was the least important group. The addition of delta-aminolevulinic acid to the medium allowed the sparking of FY3 (hem1 erg7 ura) on bulk cholestanol due to the derepression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and the production of endogenous ergosterol. The optimal concentration of delta-aminolevulinic acid to spark growth was 800 ng/ml, whereas higher concentrations caused a growth inhibition. The growth yield of FY3 reached a plateau maximum at about 5 micrograms/ml when the bulk cholestanol was varied in the presence of 10 ng of sparking erogosterol per ml.     

Ergosterol depletion and 4-methyl sterols accumulation in the yeast Saccharomyces cerevisiae treated with an antifungal, 6-amino-2-n-pentylthiobenzothiazole.

In Saccharomyces cerevisiae treated with an antifungal agent, 6-amino-2-n-pentylthiobenzothiazole, levels of ergosterol and other 4-desmethylsterols were found to be significantly reduced. Major sterols in treated yeast were lanosterol, 4,4-dimethylzymosterol, 4-methylzymosterol and 4-methylfecosterol. A hypothesis is stated that the antifungal agent inhibits sterol demethylation at C-4 and forces the biosynthesis to a blind pathway ending by 4-methylfecosterol.     

Mechanism of binding of prothioconazole to Mycosphaerella graminicola CYP51 differs from that of other azole antifungals

Prothioconazole is one of the most important commercially available demethylase inhibitors (DMIs) used to treat Mycosphaerella graminicola infection of wheat, but specific information regarding its mode of action is not available in the scientific literature. Treatment of wild-type M. graminicola (strain IPO323) with 5 μg of epoxiconazole, tebuconazole, triadimenol, or prothioconazole ml(-1) resulted in inhibition of M. graminicola CYP51 (MgCYP51), as evidenced by the accumulation of 14α-methylated sterol substrates (lanosterol and eburicol) and the depletion of ergosterol in azole-treated cells. Successful expression of MgCYP51 in Escherichia coli enabled us to conduct spectrophotometric assays using purified 62-kDa MgCYP51 protein. Antifungal-binding studies revealed that epoxiconazole, tebuconazole, and triadimenol all bound tightly to MgCYP51, producing strong type II difference spectra (peak at 423 to 429 nm and trough at 406 to 409 nm) indicative of the formation of classical low-spin sixth-ligand complexes. Interaction of prothioconazole with MgCYP51 exhibited a novel spectrum with a peak and trough observed at 410 nm and 428 nm, respectively, indicating a different mechanism of inhibition. Prothioconazole bound to MgCYP51 with 840-fold less affinity than epoxiconazole and, unlike epoxiconazole, tebuconazole, and triadimenol, which are noncompetitive inhibitors, prothioconazole was found to be a competitive inhibitor of substrate binding. This represents the first study to validate the effect of prothioconazole on the sterol composition of M. graminicola and the first on the successful heterologous expression of active MgCYP51 protein. The binding affinity studies documented here provide novel insights into the interaction of MgCYP51 with DMIs, especially for the new triazolinethione derivative prothioconazole.     

Inhibition of sterol synthesis by delta 5-sterols in a sterol auxotroph of yeast defective in oxidosqualene cyclase and cytochrome P-450

Synthesis of ergosterol is demonstrated in the GL7 mutant of Saccharomyces cerevisiae. This sterol auxotroph has been thought to lack the ability to synthesize sterols due both to the absence of 2,3-oxidosqualene cyclase and to a heme deficiency eliminating cytochrome P-450 which is required in demethylation at C-14. However, when the medium sterol was 5 alpha-cholestan-3 beta-ol, 5 alpha-cholest-8(14)-en-3 beta-ol, or 24 beta-methyl-5 alpha-cholest-8(14)-en-3 beta-ol, sterol synthesis was found to proceed yielding 1-3 fg/cell of ergosterol (24 beta-methylcholesta-5,7,22E-trien-3 beta-ol). Ergosterol was identified by mass spectroscopy, gas and high performance liquid chromatography, ultraviolet spectroscopy, and radioactive labeling from [3H]acetate. Except for some cholest-5-en-3 beta-ol (cholesterol) which was derived from the 5 alpha-cholestan-3 beta-ol, the stanol and the two 8(14)-stenols were not significantly metabolized confirming the absence of an isomerase for migration of the double bond from C-8(14) to C-7. Drastic reduction of ergosterol synthesis to not more than 0.06 fg/cell was observed when the medium sterol either had a double bond at C-5, as in the case of cholesterol, or could be metabolized to a sterol with such a bond. Thus, both 5 alpha-cholest-8(9)-en-3 beta-ol and 5 alpha-cholest-7-en-3 beta-ol (lathosterol) were converted to cholesta-5,7-dien-3 beta-ol (7-dehydrocholesterol), and the presence of the latter dienol depressed the level of ergosterol. The most attractive of the possible explanations for our observations is the assumption of two genetic compartments for synthesis of sterols, one of which has and one of which has not been affected by the two mutations. The ability, despite the mutations, to synthesize small amounts of ergosterol which could act to regulate the cell cycle may also explain why this mutant can grow aerobically with cholesterol (acting in the bulk membrane role) as the sole exogenous sterol.     

76 The Pneumocystis sam:smt, an atypical fungal enzyme protein

Pneumocystis , an opportunistic fungal protist, causes a type of pneumonia in immunocompromised individuals such as AIDS patients. Rat-derived P. carinii and human-derived P. jiroveci contain a large number of sterols with C-24 alkyl groups. S-Adenosyl-L-methionine:sterol C-24 methyl transferase (SAM:SMT) is the enzyme that transfers methyl groups from SAM to the C-24 position of the sterol side chain. An alkyl group at the C-24 sterol side chain position appears to be essential for the organism to proliferate. Thus SAM:SMT, which is absent in mammals, is an attractive target for chemotherapeutic attack against the pathogen. The P. carinii erg6 gene that codes for SAM:SMT has been sequenced, cloned, and the protein expressed in E. coli . Since bacteria do not synthesize sterols, and do not have SAM:SMT, the P. carinii erg6 gene product expressed in E. coli would only transmethylate exogenously provided sterol substrates. The P. carinii recombinant SAM:SMT is unique because lanosterol, a central intermediate in sterol biosynthesis, is its preferred substrate for enzyme activity. Most SAM:SMT from other organisms do not bind lanosterol and prefer other sterol substrates produced from lanosterol. Furthermore, it appears that this unusual P. carinii SAM:SMT can also methylate cholesterol, which is readily scavenged from the lungs of its rat host. The recombinant enzyme protein is being purified by affinity chromatography techniques, which will be used to obtain definitive structural analyses of the sterol compounds formed by the enzyme reaction using different sterols substrates and allow detailed structural analysis of this unusual SAM:SMT enzyme protein.     

Sterol biosynthesis via lanosterol by the trypanosomid Crithidia fasciculata

Abstract Incorporation of [1-¹⁴ C]acetate by the trypanosomid Crithidia fasciculata showed that ergosterol biosynthesis occurs de novo in this protozoon, via lanosterol and 31-norlanosterol. No cycloartenol could be detected, indicating that this biosynthesis pathway is rather similar to those of other non-photosynthetic organisms (animals, fungi). From the point of view of sterol biosynthesis, C. fasciculata is not related to other ergosterol-synthesising protozoa, such as the hitherto examined phytoflagellates and soil amoebae, which synthesise their sterols via cycloartenol, like photosynthetic organisms (plants, algae).