Showing papers on "Lanosterol published in 2012"
TL;DR: Transgenic basidiomycete G. lucidum is a promising system to achieve metabolic engineering of the ganoderic acid pathway, and successfully conferred carboxin resistance upon transformation.
Abstract: Ganoderic acids produced by Ganoderma lucidum, a well-known traditional Chinese medicinal mushroom, exhibit antitumor and antimetastasis activities. Genetic modification of G. lucidum is difficult but critical for the enhancement of cellular accumulation of ganoderic acids. In this study, a homologous genetic transformation system for G. lucidum was developed for the first time using mutated sdhB, encoding the iron-sulfur protein subunit of succinate dehydrogenase, as a selection marker. The truncated G. lucidum gene encoding the catalytic domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) was overexpressed by using the Agrobacterium tumefaciens-mediated transformation system. The results showed that the mutated sdhB successfully conferred carboxin resistance upon transformation. Most of the integrated transfer DNA (T-DNA) appeared as a single copy in the genome. Moreover, deregulated constitutive overexpression of the HMGR gene led to a 2-fold increase in ganoderic acid content. It also increased the accumulation of intermediates (squalene and lanosterol) and the upregulation of downstream genes such as those of farnesyl pyrophosphate synthase, squalene synthase, and lanosterol synthase. This study demonstrates that transgenic basidiomycete G. lucidum is a promising system to achieve metabolic engineering of the ganoderic acid pathway.
78 citations
TL;DR: Hepatitis C virus G3, but not G2, selectively interferes with the late cholesterol synthesis pathway, evidenced by lower distal sterol metabolites and preserved lanosterol levels, which may explain why hypocholesterolemia persists in chronic HCV infection, and is not overcome by host cholesterol compensatory mechanisms.
74 citations
TL;DR: It is found that lanosterol induces mild depolarization of mitochondria and promotes autophagy, and this results highlight a novel sterol-based neuroprotective mechanism with direct relevance to PD.
Abstract: Parkinson's disease (PD) is a neurodegenerative disorder marked by the selective degeneration of dopaminergic neurons in the nigrostriatal pathway. Several lines of evidence indicate that mitochondrial dysfunction contributes to its etiology. Other studies have suggested that alterations in sterol homeostasis correlate with increased risk for PD. Whether these observations are functionally related is, however, unknown. In this study, we used a toxin-induced mouse model of PD and measured levels of nine sterol intermediates. We found that lanosterol is significantly (∼50%) and specifically reduced in the nigrostriatal regions of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice, indicative of altered lanosterol metabolism during PD pathogenesis. Remarkably, exogenous addition of lanosterol rescued dopaminergic neurons from 1-methyl-4-phenylpyridinium (MPP+)-induced cell death in culture. Furthermore, we observed a marked redistribution of lanosterol synthase from the endoplasmic reticulum to mitochondria in dopaminergic neurons exposed to MPP+, suggesting that lanosterol might exert its survival effect by regulating mitochondrial function. Consistent with this model, we find that lanosterol induces mild depolarization of mitochondria and promotes autophagy. Collectively, our results highlight a novel sterol-based neuroprotective mechanism with direct relevance to PD.
61 citations
TL;DR: These 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.
49 citations
TL;DR: It is concluded 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.
Abstract: 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.
41 citations
TL;DR: The increased fluconazole resistance conferred by the S279F and S279Y point mutations appeared to be mediated through a combination of a higher affinity for substrate and a lower affinity for flu Conazole.
Abstract: The effects of S279F and S279Y point mutations in Candida albicans CYP51 (CaCYP51) on protein activity and on substrate (lanosterol) and azole antifungal binding were investigated. Both S279F and S279Y mutants bound lanosterol with 2-fold increased affinities ( K s , 7.1 and 8.0 μM, respectively) compared to the wild-type CaCYP51 protein ( K s , 13.5 μM). The S279F and S279Y mutants and the wild-type CaCYP51 protein bound fluconazole, voriconazole, and itraconazole tightly, producing typical type II binding spectra. However, the S279F and S279Y mutants had 4- to 5-fold lower affinities for fluconazole, 3.5-fold lower affinities for voriconazole, and 3.5- to 4-fold lower affinities for itraconazole than the wild-type CaCYP51 protein. The S279F and S279Y mutants gave 2.3- and 2.8-fold higher 50% inhibitory concentrations (IC 50 s) for fluconazole in a CYP51 reconstitution assay than the wild-type protein did. The increased fluconazole resistance conferred by the S279F and S279Y point mutations appeared to be mediated through a combination of a higher affinity for substrate and a lower affinity for fluconazole. In addition, lanosterol displaced fluconazole from the S279F and S279Y mutants but not from the wild-type protein. Molecular modeling of the wild-type protein indicated that the oxygen atom of S507 interacts with the second triazole ring of fluconazole, assisting in orientating fluconazole so that a more favorable binding conformation to heme is achieved. In contrast, in the two S279 mutant proteins, this S507-fluconazole interaction is absent, providing an explanation for the higher K d values observed.
30 citations
TL;DR: The relevance of these processes in cancer underscores the interest for studying the role of cholesterol in tumorigenesis and exploring the possibility of interfering with the growth of malignant cells by manipulation of cholesterol metabolism.
24 citations
TL;DR: It is suggested that the product specificity of OSC is likely to be controlled by kinetics, rather than thermodynamics, as the precursor of lanosterol is in fact not the most stable cationic intermediate for wild-type OSC.
Abstract: Oxidosqualene-lanosterol cyclase (OSC) is a key enzyme in the biosynthesis of cholesterol. The catalytic mechanism and the product specificity of OSC have herein been studied using QM/MM calculations. According to our calculations, the protonation of the epoxide ring of oxidosqualene is rate-limiting. Wild-type OSC (which generates lanosterol), and the mutants H232S (which generates parkeol) and H232T (which generates protosta-12,24-dien-3-β-ol) were modeled, in order to explain the product specificity thereof. We show that the product specificity of OSC at the hydride/methyl-shifting stage is unlikely to be achieved by the stabilization of the cationic intermediates, as the precursor of lanosterol is in fact not the most stable cationic intermediate for wild-type OSC. The energy barriers for the product-determining conversions are instead found to be related to the product specificity of different OSC mutants, and we thus suggest that the product specificity of OSC is likely to be controlled by kinetics, rather than thermodynamics.
18 citations
Journal Article•
TL;DR: In this paper, the product specificity of OSC at the hydride/methyl-shifting stage is shown to be determined by the stabilization of the cationic intermediates, as the precursor of lanosterol is in fact not the most stable intermediate for wild-type OSC.
Abstract: Oxidosqualene-lanosterol cyclase (OSC) is a key enzyme in the biosynthesis of cholesterol. The catalytic mechanism and the product specificity of OSC have herein been studied using QM/MM calculations. According to our calculations, the protonation of the epoxide ring of oxidosqualene is rate-limiting. Wild-type OSC (which generates lanosterol), and the mutants H232S (which generates parkeol) and H232T (which generates protosta-12,24-dien-3-β-ol) were modeled, in order to explain the product specificity thereof. We show that the product specificity of OSC at the hydride/methyl-shifting stage is unlikely to be achieved by the stabilization of the cationic intermediates, as the precursor of lanosterol is in fact not the most stable cationic intermediate for wild-type OSC. The energy barriers for the product-determining conversions are instead found to be related to the product specificity of different OSC mutants, and we thus suggest that the product specificity of OSC is likely to be controlled by kinetics, rather than thermodynamics.
17 citations
TL;DR: It is shown that the methyl-29 group is critical to the correct folding of 1, with lesser contributions from the other branched methyl groups, such as methyl-26, -27, and -28.
Abstract: Lanosterol synthase catalyzes the polycyclization reaction of (3S)-2,3-oxidosqualene (1) into tetracyclic lanosterol 2 by folding 1 in a chair-boat-chair-chair conformation. 27-Nor- and 29-noroxidosqaulenes (7 and 8, respectively) were incubated with this enzyme to investigate the role of the methyl groups on 1 for the polycyclization cascade. Compound 7 afforded two enzymatic products, namely, 30-norlanosterol (12) and 26-normalabaricatriene (13; 12/13 9:1), which were produced through the normal chair-boat-chair-chair conformation and an atypical chair-chair-boat conformation, respectively. Compound 8 gave two products 14 and 15 (14/15 4:5), which were generated by the normal and the unusual polycyclization pathways through a chair-chair-boat-chair conformation, respectively. It is remarkable that the twist-boat structure for the B-ring formation was changed to an energetically favored chair structure for the generation of 15. Surprisingly, 14 and 15 consisted of a novel 6,6,6,6-fused tetracyclic ring system, thus differing from the 6,6,6,5-fused lanosterol skeleton. Together with previous results, we conclude that the methyl-29 group is critical to the correct folding of 1, with lesser contributions from the other branched methyl groups, such as methyl-26, -27, and -28. Furthermore, we demonstrate that the methyl-29 group has a crucial role in the formation of the five-membered D ring of the lanosterol scaffold.
15 citations
TL;DR: In vitro incubation of parkeol produced a product profile similar to that of the in vivo experiment, suggesting that two different metabolic pathways of postoxidosqualene cyclization may exist in S. cerevisiae.
TL;DR: CYP51–FprD fusion enzymes showed distinct spectral properties of both flavoprotein and CYP, and were fused together with designed linker sequences to construct an artificial self‐sufficient cytochrome P450 monooxygenase (CYP) with only FprD, CYP51, and iron–sulfur containing FPRD.
Abstract: CYP51, a sterol 14α-demethylase, is one of the key enzymes involved in sterol biosynthesis and requires electrons transferred from its redox partners. A unique CYP51 from Nocardia farcinica IFM10152 forms a distinct cluster with iron-sulfur containing NADPH-P450 reductase (FprD) downstream of CYP51. Previously, sequence alignment of nine reductases from N. farcinica revealed that FprC, FprD, and FprH have an additional sequence at their N-termini that has very high identity with iron-sulfur clustered ferredoxin G (FdxG). To construct an artificial self-sufficient cytochrome P450 monooxygenase (CYP) with only FprD, CYP51, and iron-sulfur containing FprD were fused together with designed linker sequences. CYP51-FprD fusion enzymes showed distinct spectral properties of both flavoprotein and CYP. CYP51-FprD F1 and F2 in recombinant Escherichia coli BL21(DE3) catalyzed demethylation of lanosterol more efficiently, with k(cat) /K(m) values of 96.91 and 105.79 nmol/min/nmol, respectively, which are about 35-fold higher compared to those of CYP51 and FprD alone.
TL;DR: A chemical complementarity involving hydrogen bonding formation of specific active site contacts to the nucleophilic C3-group of sterol is required for proper orientation of the substrate C-methyl intermediate in the activated complex.
Journal Article•
TL;DR: In this article, the authors established a determination method of lanosterol in mycelium of Trichophyton rubrum by reversed phase high performance liquid chromatography (HPLC).
Abstract: Objective To establish a determination method of lanosterol in mycelium of Trichophyton rubrum by reversed phase high performance liquid chromatography.Methods The sample was analyzed after saponifing,extracting and methanol voluming.Results The retention time of lanosterol was 12.1 min.There was excellent linearity between peak area and concentration of analyte in the concentration range of 0.1~10.0 mg/L for lanosterol.The correlation coefficient was 0.998 1.The average recovery was 93.7%.The relative standard deviation was 1.9%.The detection limit of concentration was 0.05 mg/L.Conclusion This method is accurate,simple and precise.
01 Jan 2012
TL;DR: The objective of this project is to find the new sources of phytosterols from plant found in Malaysia, the plant use is Petai (Panda speciosa), an edible legume, the seeds of which are already consumed in Malaysia as a minor vegetable.
Abstract: Steroids are found in plants, animals and fungi. All steroids are made in cells either from the sterols lanosterol (animals and fungi) or from cycloartenol (plants). Both lanosterol and cycloartenol are derived from the cyclization of the triterpene squalene. Triterpenes are terpenes consisting of six isoprene units and have the molecular formula C3017148 (e.g carotenoids). The steroids derived from animal are the cholesterol while the steroids derived from plant is the phytosterols. There are more than 100 types of phytosterols have been reported in plant species, but the more abundant are sitosterol, stigmasterol and campesterol. The importance of phytosterols is their contribution towards lowering body cholesterol. Cholesterol is transported in blood in the form of lipoprotein which includes the high density (HDL) and low density (LDL) lipoproteins where LDL is the bad cholesterol. Too much LDL cholesterol in the blood can cause cholesterol to build up in the artery walls, leading to the narrowing of the arteries, Atherosclerosis. Thus dietary cholesterol intake is reduced in patients with such disease. Another way to reduce blood cholesterol is to consume phytosterols which compete with cholesterol absorption. The objective of this project is to find the new sources of phytosterols from plant found in Malaysia. The plant use in this project is Petai (Panda speciosa). Petai is an edible legume, the seeds of which are already consumed in Malaysia as a minor vegetable. Although many legumes have been studied as sources of phytosterols very little is reported on Petai. The practical study is carry out by using extraction to extract phytosterols from Petai and chromatographic and spectrophotometric method to separate and identify the extracted phytosterols. Petai is a legume. Legumes like soybean are known to be rich in phytosterols.