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PubMed1995

Molecular order and dynamics of phosphatidylcholine bilayer membranes in the presence of cholesterol, ergosterol and lanosterol: a comparative study using 2H-, 13C- and 31P-NMR spectroscopy.

We report the results of a comparative study of the molecular order and dynamics of phosphatidylcholine (PC) bilayer membranes in the absence and presence of cholesterol, ergosterol and lanosterol, using deuterium (2H) nuclear magnetic resonance (NMR) of deuterated phospholipid molecules, in addition to solid state 13C and 31P-NMR. Using dimyristoylphosphatidylcholines (DMPCs) specifically labeled at positions 2', 3', 4', 6', 8', 10' and 12' of the sn-2 chain together with the perdeuterated 2-[2H27]DMPC derivative, the order profile for 9 of the 13 methylene groups of the sn-2 chain was established at 25 degrees C for DMPC, DMPC/cholesterol, DMPC/ergosterol and DMPC/lanosterol membranes, at a fixed sterol/phospholipid mol ratio of 30%, and in the presence of excess water. The overall ordering effects were found to be ergosterol > cholesterol >> lanosterol. Transverse relaxation (T2e) studies of these systems indicated that while for DMPC, DMPC/cholesterol and DMPC/ergosterol the relative relaxation rates were in qualitative agreement with models which assume cooperative motions of the bilayer molecules as the main relaxation mechanism, those in DMPC/lanosterol were anomalously high, suggesting alterations of lipid packing. Using dipalmitoylphosphatidylcholine (DPPC) deuterated at the trimethylammonium group of the choline moiety, we found that the differential ordering and motional effects induced by the sterols in the acyl chains were also reflected in the headgroup, both in the gel (L beta) and liquid-crystalline phases. 13C and 1H spin dynamics studies of these systems, including cross-polarization, rotating frame longitudinal relaxation and dipolar echo relaxation rates showed that the mobility of the different regions of the phospholipid molecules in the binary lipid systems were inversely correlated with the ordering effects induced by the sterols. A novel combination of C-D bond order parameters (obtained by 2H-NMR) and 13C-1H cross polarization rates confirmed these results. The effects of the same sterols at the same molar proportion on the unsaturated lipid 1-[2H31]palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (2H31-POPC) at 25 and 35 degrees C were different from those observed on DMPC and showed ordering effects which are largest for cholesterol, while ergosterol and lanosterol produced significantly smaller effects. Transverse relaxation studies indicate that while cholesterol does not perturb cooperative motions in POPC, both ergosterol and lanosterol do. Again, high-resolution solid state 13C-NMR studies support the conclusions of the 2H-NMR experiments.(ABSTRACT TRUNCATED AT 400 WORDS)

Carbon Isotopes↗

PubMed1981

Evidence for the presence of cytochrome P-450 functional in lanosterol 14 alpha-demethylation in microsomes of aerobically grown respiring yeast.

Recent studies have shown that a cytochrome P-450 present in microsomes of semi-anaerobically grown cells of Saccharomyces cerevisiae is functional in the 14 alpha-demethylation of lanosterol (4,4,14 alpha-trimethyl-5 alpha-cholesta-8,24-dien-3 beta-ol), but the occurrence of the same cytochrome P-450 in microsomes of aerobically grown yeast cells has not yet been reported. In this study, the microsomal fraction from aerobically grown cells was found to catalyze the lanosterol demethylation in the presence of NADPH and O2 and that this activity was sensitive to CO. In Ouchterlony double diffusion test, antibodies to the yeast cytochrome P-450 formed a single precipitin line with the microsomal fraction as well as with the purified yeast cytochrome P-450 and the two precipitin lines fused with each other. Furthermore, the antibodies inhibited the lanosterol demethylation activity of the microsomal fraction from aerobically grown cells. The quadratic-derivative absorption spectrum of the microsomal fraction measured in the presence of both Na2S2O4 and CO showed an absorption band at 450 nm which is attributable to the reduced CO compound of cytochrome P-450. These facts led to the conclusion that cytochrome P-450 actually exists in aerobically grown yeast and participates in the lanosterol 14 alpha-demethylation which is essential for the ergosterol (5 alpha-ergosta-5,7,22-trien-3 beta-ol) biogenesis by yeast.

Aerobiosis↗

PubMed1991

Different substrate specificities of lanosterol 14a-demethylase (P-45014DM) of Saccharomyces cerevisiae and rat liver for 24-methylene-24,25-dihydrolanosterol and 24,25-dihydrolanosterol.

The purified lanosterol 14a-demethylase (P-45014DM) of S. cerevisiae catalyzed the 14a-demethylation of 24-methylene-24,25-dihydrolanosterol (24-methylenelanost-8-en-3 beta-ol, 24-methylene-DHL), the natural substrate of the demethylase of filamentous fungi, as well as its natural substrate, lanosterol. Lanosterol 14a-demethylase of rat liver microsomes also catalyzed the 14a-demethylation of 24-methylene-DHL, but the activity was considerably lower than that for lanosterol. The activity of the rat liver enzyme for 24-methylene-DHL was also lower than that for 24,25-dihydrolanosterol (DHL), while the activity of yeast P-45014DM for 24-methylene-DHL was considerably higher than that for DHL. Since 24-substituted sterols are not found in mammals and DHL is not an intermediate of ergosterol biosynthesis by yeast, above-mentioned different substrate specificities between the yeast and the mammalian 14a-demethylases may reflect certain evolutional alteration in their active sites in relation to the difference in their sterol biosynthetic pathways.

Animals↗

PubMed1976

Lanosterol 14alpha-demethylase. Similarity of the enzyme system from yeast and rat liver.

The chemical synthesis of 24,25-dihydro[32-14C]lanosterol is described. The incubation of this material with a cell-free system from Saccharomyces cerevisiae or with a microsomal preparation from rat liver resulted in both cases in the release of [14C]formic acid. This result suggests that in the biosynthesis of ergosterol in yeast, as well as in that of cholesterol in higher animals, the 14alpha-methyl group of lanosterol is removed as formic acid. In both systems, the measurement of the rate of release of [14C]formic acid from 24,25-dihydro[32-14C]lanosterol provides a simple and direct assay of lanosterol 14alpha-demethylase. Carbon monoxide inhibited both yeast and liver 14alpha-demethylase.

Animals↗

PubMed2004

A low-toxicity method for the separation of lanosterol and dihydrolanosterol from commercial mixtures.

We describe an inexpensive, low-toxicity and high-yielding method for the production of pure lanosterol and dihydrolanosterol from the commercially available mixture. Optimum conditions are presented for the one-pot production of the intermediate 24,25 vicinal diol of lanosterol acetate (via either epoxidation or hydroxyhalogenation) which is readily separated from the unreacted dihydrolanosterol acetate. The lanosterol diol can then be converted to pure (>97%) lanosterol. Hypophosphorous acid was used for both the conversion of the epoxide to the diol, and as a catalyst for the hydroxyhalogenation by N-halosuccinimides of the olefinic bond.

Acetylation↗

PubMed1977

Differential effects of cholesterol and lanosterol on artificial membranes.

The effects of cholesterol, 4,4-dimethylcholesterol, and lanosterol (4,4',14alpha-trimethyl-delta8,24-cholestadiene-3beta-ol) on some properties of lecithin vesicles have been compared. Unlike cholesterol, lanosterol retards the exit of trapped glucose from phospholipid vesicles only slightly. The 13C nuclear magnetic resonance spectrum of cholesterol/lecithin vesicles shows no resonances attributable to the sterol. By contrast, several resonances attributable to quaternary carbon atoms or methyl groups are seen in the 13C nuclear magnetic resonance spectrum of lanosterol/lecithin vesicles, indicating that lanosterol is much less immobilized than cholesterol. Because the membrane behavior of 4,4-dimethylcholesterol is closely similar to that of cholesterol, it is concluded that the axial 14-alpha-methyl group is responsible for the lessened membrane immobilization of lanosterol. The results emphasize the importance of a planar sterol alpha-face for interaction with phospholipid acyl chains.

Cholesterol↗

PubMed2006

Lanosterol synthase in dicotyledonous plants.

Sterols are important as structural components of plasma membranes and precursors of steroidal hormones in both animals and plants. Plant sterols show a wide structural variety and significant structural differences from those of animals. To elucidate the origin of structural diversity in plant sterols, their biosynthesis has been extensively studied [Benveniste (2004) Annu. Rev. Plant. Biol. 55: 429, Schaller (2004) Plant Physiol. Biochem. 42: 465]. The differences in the biosynthesis of sterols between plants and animals begin at the step of cyclization of 2,3-oxidosqualene, which is cyclized to lanosterol in animals and to cycloartenol in plants. However, here we show that plants also have the ability to synthesize lanosterol directly from 2,3-oxidosqualene, which may lead to a new pathway to plant sterols. The Arabidopsis gene At3g45130, designated LAS1, encodes a functional lanosterol synthase in plants. A phylogenetic tree showed that LAS1 belongs to the previously uncharacterized branch of oxidosqualene cyclases, which differs from the cycloartenol synthase branch. Panax PNZ on the same branch was also shown to be a lanosterol synthase in a yeast heterologous expression system. The higher diversity of plant sterols may require two biosynthetic routes in steroidal backbone formation.

Amino Acid Sequence↗

PubMed1980

Non-specific lanosterol and hopanoid biosynthesis be a cell-free system from the bacterium Methylococcus capsulatus.

1. A cell-free system from the bacterium Methylococcus capsulatus was incubated with [12-3H]-squalene; diploptene and diplopterol, normally present in the bacterium, were labelled. 2 The same cell-free system was incubated with (RS)-2,3-epoxy-2,3-dihydro-[3-3H]squalene. Several radioactive 3-hydroxytriterpenes were purifed. Lanosterol, which is normally present in this bacterium, was found labelled as well as 3-epilanosterol. In addition, radioactive 3 alpha-hydroxy and 3 beta-hydroxydiploptene were formed. 3. These data may be explained by the coexistence of two cyclases in M. capsulatus: a squalene/hopane cyclase and a squalene epoxide/lanosterol cyclase. The squalene cyclase exhibits the same lack of substrate specificity as those of Acetobacter pasteurianum and Tetrahymena pyriformis, i.e. in addition to its normal substrate squalene, it can cyclize the two enantiomers of squalene epoxide into 3-hydroxyhopanoids. 4. The presence of a squalene epoxide/lanosterol cyclase activity, which was suspected in view of the unique 3 beta-hydroxy 4 alpha-methyl steroids of M. capsulatus, was demonstrated by the labelling of lanosterol. More surprisingly 3-epilanosterol was also present and labelled. We showed that this does not derive from lanosterol by isomerization via a 3-oxo compound. Therefore the squalene expoxide cyclase of M. capsulatus, like the one of eukaryotes cyclizes the (3S) enantiomer of squalene epoxide into lanosterol. But it is definitely less substrate-specific as it can also cyclize the (3R) enantiomer into 3-epilanosterol.

Cyclization↗

PubMed1984

Yeast cytochrome P-450 catalyzing lanosterol 14 alpha-demethylation. I. Purification and spectral properties.

A form of cytochrome P-450 catalyzing lanosterol 14 alpha-demethylation (tentatively called "P-450(14)DM") was purified from microsomes of semi-anaerobically grown cells of Saccharomyces cerevisiae to gel electrophoretic homogeneity. An apparent monomeric Mr = 58,000 was estimated for the purified cytochrome by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both optical and EPR spectra of oxidized P-450(14)DM are characteristic of low spin ferric heme proteins, and its reduced CO complex showed a Soret absorption peak at 447 nm. As in the case of hepatic microsomal cytochromes P-450, the ethyl isocyanide complex of reduced P-450(14)DM was in a pH-dependent equilibrium between two states having Soret peaks at 429 and 453 nm, the equilibrium being considerably shifted toward the 453-nm state. Oxidized P-450(14)DM was peculiar in that in its CD spectrum there was a negative shoulder at 425 nm and the 350- and 414-nm troughs possessed larger and relatively smaller [theta] values, respectively, than those reported for other low spin ferric cytochromes P-450. Lanosterol was the only compound which caused a Type I spectral change in oxidized P-450(14)DM. The lanosterol-induced low to high spin state change was, however, only slight even at saturating concentrations of the sterol, indicating that the lanosterol-P-450(14)DM adduct was in a spin state equilibrium.

Anaerobiosis↗

PubMed1983

Altered cytochrome P-450 in a yeast mutant blocked in demethylating C-32 of lanosterol.

Spectroscopic and enzymatic analysis of a Saccharomyces cerevisiae mutant in sterol biosynthesis (SG1 (erg 11); Trocha, P. J., Jasne, S. J., and Sprinson, D. B. (1977) Biochemistry 16, 4721-4726) that was blocked in demethylating C-32 of lanosterol (4,4,14 alpha-trimethyl-5 alpha-cholesta-8,24-dien-3 beta-ol) showed that it contained low levels of cytochromes P-450 and b5 compared to those present in the parent strain D-587, while NADPH-cytochrome c reductase activity was elevated. The fungicide buthiobate (S-n-butyl S'-p-tert-butylbenzyl N-3-pyridyldithiocarbonimidate), which bound specifically to yeast cytochrome P450 responsible for demethylating C-32 of lanosterol and effected a Type II spectral change, did not react with SG1 cytochrome P-450. On the other hand, cholate-solubilized microsomes from SG1 formed a single precipitin line on Ouchterlony plates with antibodies raised against purified (Yoshida, Y., Aoyama, Y., Kumaoka, H., and Kubota, S. (1977) Biochem. Biophys. Res. Commun. 78, 1005-1010) yeast cytochrome P-450 specific for demethylating C-32 of lanosterol. Hence, mutant SG1 contained an altered protein which retained the antigenicity of cytochrome P-450 responsible for demethylating C-32 of lanosterol but lost its catalytic activity.

Cytochrome P-450 Enzyme System↗

PubMed2005

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.

Biological Transport↗

PubMed1998

Construction of a model of the Candida albicans lanosterol 14-alpha-demethylase active site using the homology modelling technique.

On the basis of all hitherto known P450 X-ray structures and applying standard homology modelling procedures a three-dimensional model of the lanosterol-14 alpha-demethylase active site was constructed. The modelled active site nicely hosts the natural substrate lanosterol and the substrate-enzyme complex displayed stability in a 70 ps molecular dynamics simulation. The importance of Thr 122 of lanosterol 14 alpha-demethylase for hydrogen bond formation with the 3-hydroxyl group of lanosterol was found to be a characteristic feature of the interaction geometry.

Binding Sites↗

PubMed2005

Enzyme redesign: two mutations cooperate to convert cycloartenol synthase into an accurate lanosterol synthase.

Efforts to modify the catalytic specificity of enzymes consistently show that it is easier to broaden the substrate or product specificity of an accurate enzyme than to restrict the selectivity of one that is promiscuous. Described herein are experiments in which cycloartenol synthase was redesigned to become a highly accurate lanosterol synthase. Several single mutants have been described that modify the catalytic specificity of cycloartenol to form some lanosterol. Modeling studies were undertaken to identify combinations of mutations that cooperate to decrease the formation of products other than lanosterol. A double mutant was constructed and characterized and was shown to cyclize oxidosqualene accurately to lanosterol (99%). This catalytic change entailed both relocating polarity with a His477Asn mutation and modifying steric constraints with an Ile481Val mutation.

Animals↗

PubMed2001

Trypanosome and animal lanosterol synthases use different catalytic motifs.

[see reaction]. Animals, fungi, and some protozoa convert oxidosqualene to lanosterol in the ring-forming reaction in sterol biosynthesis. The Trypanosoma cruzi lanosterol synthase has now been cloned. The sequence shares with the T. brucei lanosterol synthase a tyrosine substitution for the catalytically important active-site threonine found in animal and fungal lanosterol synthases.

Amino Acid Motifs↗

PubMed2006

Tryptophan 232 within oxidosqualene-lanosterol cyclase from Saccharomyces cerevisiae influences rearrangement and deprotonation but not cyclization reactions.

[reaction: see text] Oxidosqualene-lanosterol cyclases convert oxidosqualene to lanosterol in yeast and mammals. Site-saturated mutants' construction of Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase, at Trp232 exchanges against proteinogenic amino acids, and product profiles are shown. All mutants, except Lys and Arg, produced protosta-12,24-dien-3beta-ol, lanosterol, and parkeol. Overall, Trp232 plays a catalytic role in the influence of rearrangement process and determination of deprotonation position but does not involve intervention in the cyclization steps.

Arginine↗

PubMed2000

Steric bulk at position 454 in Saccharomyces cerevisiae lanosterol synthase influences B-ring formation but not deprotonation.

[reaction: see text] Lanosterol synthase converts oxidosqualene to the tetracyclic sterol precursor lanosterol. The mutation experiments described here show that an active-site valine residue in lanosterol synthase contributes to cyclization control through steric effects. Mutating to smaller alanine or glycine residues allows formation of the monocyclic achilleol A, whereas the leucine and isoleucine mutants make exclusively lanosterol. The phenylalanine mutant is inactive.

Amino Acid Substitution↗

PubMed1993

Isolation of an Arabidopsis thaliana gene encoding cycloartenol synthase by functional expression in a yeast mutant lacking lanosterol synthase by the use of a chromatographic screen.

Whereas vertebrates and fungi synthesize sterols from epoxysqualene through the intermediate lanosterol, plants cyclize epoxysqualene to cycloartenol as the initial sterol. We report the cloning and characterization of CAS1, an Arabidopsis thaliana gene encoding cycloartenol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, cycloartenol forming), EC 5.4.99.8]. A yeast mutant lacking lanosterol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7] was transformed with an A. thaliana cDNA yeast expression library, and colonies were assayed for epoxysqualene mutase activity by thin-layer chromatography. One out of approximately 10,000 transformants produced a homogenate that cyclized 2,3-epoxysqualene to the plant sterol cycloartenol. This activity was shown to be plasmid dependent. The plasmid insert contains a 2277-bp open reading frame capable of encoding an 86-kDa protein with significant homology to lanosterol synthase from Candida albicans and squalene-hopene cyclase (EC 5.4.99.-) from Bacillus acidocalcarius. The method used to clone this gene should be generally applicable to genes responsible for secondary metabolite biosynthesis.

Amino Acid Sequence↗

PubMed1994

Isolation and characterization of the gene encoding 2,3-oxidosqualene-lanosterol cyclase from Saccharomyces cerevisiae.

The ERG7 gene encoding oxidosqualene-lanosterol cyclase [(S)-2,3-epoxysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7] from Saccharomyces cerevisiae has been cloned by genetic complementation of a cyclase-deficient erg7 strain. The DNA sequence of this gene has been determined and found to contain an open reading frame of 2196 nt (including stop codon) that encodes a predicted protein of 731 amino acids. The predicted molecular mass of the S. cerevisiae cyclase, 83.4 kDa, is similar to the predicted molecular masses of the oxidosqualene-lanosterol cyclase from Candida albicans and the oxidosqualene-cycloartenol cyclase from Arabidopsis thaliana, as well as to the molecular masses assigned to vertebrate oxidosqualene-lanosterol cyclases; however, it is substantially larger than the molecular mass assigned to purified S. cerevisiae cyclase. At the level of DNA and predicted amino acid sequences, the S. cerevisiae and C. albicans cyclases share 56% and 63% identity, respectively. Tryptophan and tyrosine residues are unusually abundant in the predicted amino acid sequences of (oxido)-squalene cyclases, leading to a hypothesis that electron-rich aromatic side chains from these residues are essential features of cyclase active sites.

Amino Acid Sequence↗