Showing papers on "Lanosterol published in 1997"
TL;DR: Biochemical analysis demonstrates that Erg16p became less susceptible to fluconazole in isolate 13 in a series of isolates from an HIV-infected patient, demonstrating azole resistance in the pathogenic yeast Candida albicans.
Abstract: Azole resistance in the pathogenic yeast Candida albicans is an emerging problem in the human immunodeficiency virus (HIV)-infected population. The target enzyme of the azole drugs is lanosterol 14alpha demethylase (Erg16p), a cytochrome P-450 enzyme in the biosynthetic pathway of ergosterol. Biochemical analysis demonstrates that Erg16p became less susceptible to fluconazole in isolate 13 in a series of isolates from an HIV-infected patient. PCR-single-strand conformation polymorphism (PCR-SSCP) analysis was used to scan for genomic alterations of ERG16 in the isolates that would cause this change in the enzyme in isolate 13. Alterations near the 3' end of the gene that were identified by PCR-SSCP were confirmed by DNA sequencing. A single amino acid substitution (R467K) that occurred in isolate 13 was identified in both alleles of ERG16. Allelic differences within the ERG16 gene, in the ERG16 promoter, and in the downstream THR1 gene were eliminated in isolate 13. The loss of allelic variation in this region of the genome is most likely the result of mitotic recombination or gene conversion. The R467K mutation and loss of allelic variation that occur in isolate 13 are likely responsible for the azole-resistant enzyme activity seen in this and subsequent isolates. The description of R467K represents the first point mutation to be identified within ERG16 of a clinical isolate of C. albicans that alters the fluconazole sensitivity of the enzyme.
283 citations
TL;DR: Findings clearly differentiate the OSC inhibitor Ro 48-8.071 from simvastatin, and support the view that OSC is a distinct key component in the regulation of the cholesterol synthesis pathway.
152 citations
TL;DR: It is suggested that C-24 reduction of sterols takes place straight after sterol delta 8-->7 isomerization of zymosterol, which occurs several steps after C-32 demethylation of lanosterol in the 19-step pathway of cholesterol biosynthesis from Lanosterol.
Abstract: The membrane-bound sterol delta 24-reductase (24-reductase) catalyses anaerobic reduction of the 24(25)-enes of lanosterol and other obligatory intermediates of cholesterol biosynthesis from lanosterol. A novel assay method and properties of the 24-reductase are described. More than a 120-fold induction of the 24-reductase activity was achieved by feeding rats a diet containing 5% cholestyramine plus 0.1% lovastatin in chow and by modulating diurnal variation. With this enzyme induction condition, lanosterol was converted efficiently into dihydrolanosterol in both intact hepatic microsomes and freshly isolated hepatocytes only when either miconazole or CO was added to inhibit 14 alpha-demethylation of lanosterol. AR45 cells, which are deficient in 14 alpha-methyl demethylase (14 alpha-DM), exhibit lanosterol 24-reductase activity without addition of either CO or miconazole. Conversely, inhibition of the 24-reductase was not required for the expression of 14 alpha-DM activity. Studies on the substrate specificities for the 24-reductase using different 24(25)-enes showed that the most reactive substrate was 5 alpha-cholesta-7,24-dien-3 beta-ol, which exhibited a maximal 18-fold higher kcat than that of lanosterol without the aid of the 14 alpha-DM inhibitor. In addition, both the kinetic behaviour of lanosterol substrate in relation to the 24-reductase and a non-competitive inhibition mode of U18666A (Ki 0. 157 microM) as well as Triparanol (Ki 0.523 microM), two well-known 24-reductase inhibitors, were determined. On the basis of our new findings on the preferred substrate and on the negative effect of 14 alpha-DM on the 24-reductase, we suggest that C-24 reduction of sterols takes place straight after sterol delta 8-->7 isomerization of zymosterol, which occurs several steps after C-32 demethylation of lanosterol in the 19-step pathway of cholesterol biosynthesis from lanosterol.
93 citations
TL;DR: Lanosterol synthase, the enzyme from Saccharomyces cerevisiae which catalyzes the complex cyclization/rearrangement step in sterol biosynthesis, was overexpressed in baculovirus-infected cells and purified to homogeneity in three steps.
Abstract: Lanosterol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7], the enzyme from Saccharomyces cerevisiae which catalyzes the complex cyclization/rearrangement step in sterol biosynthesis, was overexpressed in baculovirus-infected cells and purified to homogeneity in three steps. Using pure enzyme the kinetics of cyclization were determined using Michaelis−Menten analysis for 2,3-oxidosqualene (1) and two analogs in which the C−6 methyl was replaced by H (3) or Cl (4). The measured Vmax/KM ratios for 1, 3, and 4 were found to be 138, 9.4, and 21.9, respectively, a clear indication that oxirane cleavage and cyclization to form the A-ring are concerted, since the nucleophilicity of the proximate double bond influences the rate of oxirane cleavage. No catalytic metal ions could be detected in purified lanosterol synthase by atomic absorption analysis. Site-directed mutagenesis studies of each of the six strongly conserved aspartic acid residues (D → N mutation) and each of the...
76 citations
TL;DR: It is demonstrated that treatment of the sterol auxotrophs erg25 slu1 or erg11 slu2 with azole antibiotics paradoxically restores viability to these strains in the absence of sterol supplementation via the suppression system described.
Abstract: Genetic disruption of the Saccharomyces cerevisiae C-4 sterol methyl oxidase ERG25 gene leads to sterol auxotrophy. We have characterized a suppression system that requires two mutations to restore viability to this disrupted strain. One suppressor mutation is erg11, which is blocked in 14α-demethylation of lanosterol and is itself an auxotroph. The second suppressor mutation required is either slu1 or slu2 (suppressor of lanosterol utilization). These mutations are leaky versions of HEM2 and HEM4, respectively; addition of exogenous hemin reverses the suppressing effects of slu1 and slu2. Suppression of erg25 by erg11 slu1 (or erg11 slu2) results in a slow-growing strain in which lanosterol, the first sterol in the pathway, accumulates. This result indicates that endogenously synthesized lanosterol can substitute for ergosterol and support growth. In the triple mutants, all but 1 (ERG6) of the 13 subsequent reactions of the ergosterol pathway are inactive. Azole antibiotics (clotrimazole, ketoconazole, and itraconazole) widely used to combat fungal infections are known to do so by inhibiting the ERG11 gene product, the 14α-demethylase. In this investigation, we demonstrate that treatment of the sterol auxotrophs erg25 slu1 or erg25 slu2 with azole antibiotics paradoxically restores viability to these strains in the absence of sterol supplementation via the suppression system we have described.
59 citations
Book•
14 Mar 1997
TL;DR: Inhibitors of 2,3-Oxidosqualene Cyclase as Tools for Studying the Mechanism and Function of the Enzyme, L.A. Cattel and M.D. Ceruti Aromatase Inhibitors.
Abstract: Inhibitors of 2,3-Oxidosqualene Cyclase as Tools for Studying the Mechanism and Function of the Enzyme, L. Cattel and M. Ceruti Aromatase Inhibitors, J. O'Neal Johnston Variability of Metabolism and Function of Sterols in Insects, J.A. Svoboda Oxysterols and Apoptosis: Evidence for Gene Regulation Outside the Cholesterol Pathway, E.B. Thompson and S. Ayala-Torres Tracing Steroid Synthesis in Plants, H.W. Groeneveld Biochemistry and Molecular Biology of Sterol Synthesis in Saccharomyces cerevisiae, N.D. Lees, M. Bard, and D.R. Kirsch 19-Noraldosterone, Y. Takeda Enzymology of Phytosterol Transformations, W.D. Nes and M. Venkatramesh Isoprenoid Biosynthesis in Plants: Carbon Partitioning Within the Cytoplasmic Pathway, J.D. Newman and J. Chappell Mevalonate Biosynthesis in Plants, T.J. Bach, A. Boronat, N. Campos, A. Ferrer, and K.-U. Vollack Lanosterol Analogs: Dual-Action Inhibitors of Cholesterol Biosynthesis, L.L. Frye and D.A. Leonard Biochemistry and Function of Nematode Steroids, D.J. Chitwood Occurrence, Biosynthesis, and Putative Role of Ecdysteroids in Plants, J.H. Adler and R.J. Grebenok Regulation of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Activity by Side-Chain Oxysterols and Their Derivatives, E.J. Parish, S.C. Parish, and S. Li Biological Effects of Brassinosteroids, C. Brosa Vitamin D: A Hormonal Regulator of the cAMP Signaling Pathway, J.P. Berg and E. Haug Protein Isoprenylation in Plants, S.K. Randall and D.N. Crowell Chemical Modulation of Activity in Steroidal Estrogens, E. Palomino Use of Sterol Mutants as Probes for Sterol Functions in the Yeast, Saccharomyces cerevisiae, L.W. Parks, J.H. Crowley, F.W. Leak, S.J. Smith, and M.E. Tomeo Integration of the Metabolic Pathways of Steroids, Carotenoids, and Retinoids, A.M. Gawienowski Index
59 citations
TL;DR: The degree and kinetic pattern of methylation inhibition were found to be influenced by the position and nature of the variant functional group introduced into the side chain, and new insights are provided into the topology of the ternary complex involving sterol–AdoMet–enzyme.
41 citations
TL;DR: Sterol and fatty acid composition were combined for comparison of five industrially important Aspergillus species as discussed by the authors, and the most outstanding were the differences in the amounts of linolenic acid, ergosterol, and lanosterol.
Abstract: Sterol and fatty acid composition were combined for comparison of five industrially important Aspergillus species. Quantitative amounts of individual fatty acids, especially unsaturated ones, and the composition of sterols showed the differences among fungi examined. Besides ergosterol, six other sterols were present in detectable amounts in fungal mycelia. The most outstanding were the differences in the amounts of linolenic acid, ergosterol, and lanosterol, as well as in a sterol with M+376 identified as ergosterol peroxide.
33 citations
TL;DR: In this paper, a wheat cDNA library with an heterologous CYP81B1probe fromHelianthus tuberosus was used to isolate a protein with all the characteristics of a typical P450 with high homology (32-39% identity) to the fungal and mammalian CYP51s.
33 citations
TL;DR: The synthesis and biological evaluation of three new sulfur-substituted oxidosqualene (OS) analogues are presented, and the S-18 analogue 3 showed the most potent inhibition toward the rat liver enzyme and showed potent, selective inhibition against the fungal enzyme, making it the most powerful OSC inhibitor known to date.
Abstract: The synthesis and biological evaluation of three new sulfur-substituted oxidosqualene (OS) analogues (1-3) are presented. In these analogues, C-11, C-15, or C-18 in the OS skeleton was replaced by sulfur. The sulfur position in the OS skeleton was chosen to disrupt one or more key processes involved in cyclization: (a) the folding of the B-ring into a boat conformation, (b) the anti-Markovnikov cyclization leading to the C-ring, or (c) the formation of the D-ring during the lanosterol biosynthesis. Enzyme inhibition kinetics using homogeneous mammalian oxidosqualene cyclases (OSC) were also examined for the previously reported S-19 analogue 4. The four analogues were potent inhibitors of mammalian OSCs (IC50 = 0.05-2.3 microM for pig and rat liver OSC) and fungal cell-free Candida albicans OSC (submicromolar IC50 values). In particular, the S-18 analogue 3 showed the most potent inhibition toward the rat liver enzyme (IC50 = 50 nM) and showed potent, selective inhibition against the fungal enzyme (IC50 = 0.22 nM, 10-fold more potent than the S-19 analogue 4). Thus, 3 is the most potent OSC inhibitor known to date. The Ki values ranged from 0.5 to 4.5 microM for pig OSC, with 3 and 4 showing about 10-fold higher potency for rat liver OSC. Interestingly, the S-18 analogue 3 showed time-dependent irreversible inhibition with homogeneous pig liver OSC (kinact = 0.06 min-1) but not with rat OSC.
32 citations
Journal Article•
TL;DR: St sterol -reductase (-SR) was found to be the most sensitive enzyme with a noncompetitive inhibition of this compound andstrate specificity studies of the microsomal -SR indicate that the relative reaction rate for 7-dehydrocholesterol and ergosterol are 5.6-fold and 1.8-fold higher than that for lathosterol.
Abstract: A novel assay method is described for rapid quantitation of reaction rate of sterol -reductase (-SR) which catalyzes reduction of the -double bond of sterols. Of six different organ tissues-liver, small intestine, brain, lung, kidney, and testis-. -SR activity was detected only in liver (2.30 nmol/min/mg protein) and testis (0.11 nmol/min/mg protein). Using a newly developed method which employs diet-induced enzyme proteins and ergosterol as substrate, we assessed both kinetics (, ) and inhibition of the rat hepatic -SR against well-studied cholesterol lowering agents such as triparanol (). 3--[2-(diethylamino)ethoxy]androst-5-en-17-one (U18666A) (), and trans-1.4-bis(2-chlorobenzylaminomethyl)cyclohexane dihydrochloride (AY-9944) (). Of the three well-known AY-9944-sensitive cholesterogenic enzymes (i.e., -SR, sterol -isomerase, and sterol -reductase). -SR was found to be the most sensitive enzyme with a noncompetitive inhibition of this compound (). Substrate specificity studies of the microsomal -SR indicate that the relative reaction rate for 7-dehydrocholesterol and ergosterol are 5.6-fold and 1.6-fold higher than that for lathosterol. -SR activity was also modulated by feeding rats a diet supplemented with 0.5% ergosterol (>2.6-fold) in addition to 5.0% cholestyramine plus 0.1% lovastatin (5.0-fold). Finally, microsomal -SR was solubilized by 1.5% 3-[3-(cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) and enriched on PEG (0~10%) precipitation, which should be suitable for further purification of the enzyme.
Journal Article•
TL;DR: Results were in complete agreement with the proposed mechanism of action of azoles, which act by inhibiting ergosterol biosynthesis with a consequent accumulation of lanosterol.
Abstract: The comparative effects of ketoconazole, itraconazole, and fluconazole on the lipids of five Sporothrix schenckii strains were investigated. Quantitative analysis of lipids and sterols was completed, as well as qualitative analysis of sterols, by thin-layer chromatography and ultraviolet spectrophotometry. Growth of the S. schenckii isolates in the presence of azole derivative concentrations below the minimum inhibitory concentration (MIC) resulted in significant alterations in the lipid and sterol contents as compared with the control values. Furthermore, lanosterol was detected in these azole-treated cells. These results were in complete agreement with the proposed mechanism of action of azoles, which act by inhibiting ergosterol biosynthesis with a consequent accumulation of lanosterol. Concerning the MIC values, fluconazole was found to be the least effective drug. On the other hand, as determined from a comparison of the effects of the three azoles on the sterol content of the strains studied, no significant differences in efficacy were found among the tested drugs.
TL;DR: Four non-azole inhibitors of lanosterol 14a-demethylase in Candida albicans CCH442 demonstrated broad in-vitro antifungal activity against several Candida species as well as against Cryptococcus albidus and Aspergillus niger.
Abstract: We have identified four non-azole inhibitors of lanosterol 14a-demethylase in Candida albicans CCH442. The most potent compound, A-39806, had IC50 values for ergosterol inhibition of 0.9 microM (0.3 mg/L) and 1.9 microM (0.6 mg/L) in whole cell and cell-free extract assays, respectively. A-39806 demonstrated broad in-vitro antifungal activity against several Candida species as well as against Cryptococcus albidus and Aspergillus niger. In-vitro antifungal activity was also demonstrated against a fluconazole-resistant clinical isolate of C. albicans.
TL;DR: The reaction of the S-methyl dithiocarbonate of lanosterol with the excess of tributyltin deuteride and azoisobutyronitrile in benzene======aligned lanosteryl methyl ether in which all hydrogen in the methoxy======group originated from the organotin reagent as mentioned in this paper.
01 Jan 1997
TL;DR: In this article, a suppression system that requires two mutations to restore viability to a disrupted strain of sterol methyl oxidase ERG25 gene was characterized, and it was shown that endogenously synthesized lanosterol can substitute for ergosterol and support growth.
Abstract: Genetic disruption of the Saccharomyces cerevi- siae C-4 sterol methyl oxidase ERG25 gene leads to sterol auxot- rophy. We have characterized a suppression system that requires two mutations to restore viability to this disrupted strain. One suppressor mutation is erg11, which is blocked in 14a- demethylation of lanosterol and is itself an auxotroph. The second suppressor mutation required is either slu1 or slu2 (suppressor of lanosterol utilization). These mutations are leaky versions of HEM2 and HEM4, respectively; addition of exogenous hemin reverses the suppressing effects of slu1 and slu2. Suppression of erg25 by erg11 slu1 (or erg11 slu2) results in a slow-growing strain in which lanosterol, the first sterol in the pathway, accumulates. This result indicates that endogenously synthesized lanosterol can substitute for ergosterol and support growth. In the triple mu- tants, all but 1 (ERG6) of the 13 subsequent reactions of the ergosterol pathway are inactive. Azole antibiotics (clotrimazole, ketoconazole, and itraconazole) widely used to combat fungal infections are known to do so by inhibiting the ERG11 gene product, the 14a-demethylase. In this investigation, we demon- strate that treatment of the sterol auxotrophs erg25 slu1 or erg25 slu2 with azole antibiotics paradoxically restores viability to these strains in the absence of sterol supplementation via the suppres- sion system we have described. The yeast Saccharomyces cerevisiae has long served as a model system for studies in sterol biosynthesis. The sterol pathway is a branch of the isoprenoid pathway that also gives rise to several other essential compounds in the cell. Mutations in the sterol pathway prior to lanosterol, the first sterol molecule, lead to sterol auxotrophy. Most genes in yeast ergosterol synthesis have been