Demethylation Of Lanosterol Research Articles

Characterization of a novel CYP51 from Rhodococcus triatomae and its NADH-ferredoxin reductase-coupled application in lanosterol 14α-demethylation

Abstract Reconstitution of a selective demethylation system for lanosterol is desperately needed for more efficient synthesis of steroidal drugs. Sterol 14α-demethylase cytochrome P450 (CYP51) has been confirmed to catalyze sterol 14α-demethylation, an essential reaction in sterol biosynthesis. Herein, a putative CYP51 gene (RtCYP51) was mined from the complete genome sequence of Rhodococcus triatomae BKS 15-14. Its amino acid sequence showed 25–68% identity to other sterol 14α-demethylases, and contained a novel alanine-rich sequence at the C-terminus. Heterologous expression of the RtCYP51 gene in Escherichia coli (E. coli) yielded a ∼54 kDa recombination protein that exhibited a typical reduced CO-difference spectrum and a dissociation constant (Kd) of 2.93 μM for lanosterol. Furthermore, three exogenous electron donor systems, including Fdx-FdR (Acinetobacter sp.OC4 ferredoxin and ferredoxin reductase), Fld-FdR2 (E. coli flavodoxin and flavodoxin reductase) and NfFdR (Nocardia farcinica iron-sulfur containing NADPH-P450 reductase) were selected for coupling the electron-transfer from the coenzyme to RtCYP51. Fdx-FdR was found to be the most efficient electron donor and was also confirmed to support the lanosterol demethylation activity of RtCYP51 in vitro. Under the optimum molar ratio of RtCYP51/FdR/Fdx (1:2:10), RtCYP51 exhibited a relatively high turnover number of 0.63 min−1 (nmol metabolized lanosterol/min/nmol RtCYP51), compared with known bacterial CYP51s.

Relationship of Azole Resistance with the Structural Alteration of the Target Sites: Novel Synthetic Compounds for Better Antifungal Activities

Azoles have consistently been recognized as the mainstays of antifungal drugs, amongst which ergosterol prevails as an integral component of fungal plasma membrane. It is most commonly produced via demethylation of lanosterol by the cytochrome P450-dependent enzyme lanosterol 14α-demethylase. Azoles exert their antifungal activities via binding to lanosterol 14 α-demethylase and thereby preventing the demethylation of lanosterol. This leads to the depletion of demethylated lanosterol (ergosterol) with the concomitant accumulation of methylated sterol precursors (lanosterol, 4,14-dimethylzymosterol, and 24-methylenedihydrolanosterol) and deterioration of the membrane integrity, resulting in fungal growth inhibition. Resistance to azoles is a concern, particularly during the long-term treatment of fungus mediated cellular complications. To combat azole resistance and to extend the spectrum of treatable pathogens, the development of novel and more potent azoles, with alteration in active sites has attracted worldwide scientific attention. With such an insight, this review focuses on antifungal potentials of azole compounds with an emphasis on the corresponding drug resistance episodes complemented with novel strategies for the development of new generation of azole compounds. Keywords: Azole, lanosterol, ergosterol, 14-α-lanosterol-demethylase, structure-function relationship, drug resistance.

Editorial (Thematic Issue: Emerging Azoles: Structure Function Relationship and Their Therapeutic Potential)

Natural Products Journal is one of the extremely important journals of Bentham Science Publishers. The current issue has been focused on to uncover many physico-chemical interactions persisting with regards to structural-function relationships of azole compounds and more importantly their therapeutic potential. Efforts have been made to find a link between natural and synthetic azoles’ composition and structure with their physiological or therapeutic role through various articles contributed by diverse range of authors. Here is the brief summary of the articles: Kim et al., emphasized upon Pyrazoline heterocycles which occupy a distinct attention in the field of synthetic and medicinal chemistry exhibiting a wide spectrum of biological activities including anti-inflammatory, anti-microbial, cytotoxic, anticonvulsant etc. [1-3]. Authors have made a sincere effort to review the synthetic and biological significance of Pyrazoline derivatives bringing together most of the recent studies. Varughese et al., shed light on the broad-spectrum antimicrobial and therapeutic role of azole compounds in treating cancer linked fungal infections like candidiasis and aspergillosis. Authors have drawn comparisons between first First generation azoles with that of newer azoles in terms of their action potential and activity against resistant organisms. The role of azoles has been further extended to prostate cancer and breast cancer therapy through this article [4-6]. Sandhu et al., have shown promise in Azoles as effective antifungal agents. Authors are concerned about the growing resistance against azoles in fungal pathogens and side effects accompanied with the use of synthetic azoles which makes it necessary to search for some natural and effective azoles [6-10]. Authors have shown structure-function analysis of clinically important antifungal azoles derivatives through the collection of studies and also reviewed some important patents related to them. Malik et al., mainly gained insight into the mechanistic preview as to how azole compounds bind to lanosterol 14α- demethylase and prevent the demethylation of lanosterol, thereby inhibitiing the fungal growth. Authors have focused their attention on antifungal potential of azole compounds with an emphasis on the corresponding drug resistance episode complemented with novel strategies for the development of new generation of azole compounds [11-14]. Gupta et al., reviewed the antimicrobial importance of natural imidazoles, their mode of actions and structure-activity relationship. The article would certainly help scientific community to bring further modifications or developments in the synthetic methodologies to biologically active compounds based on natural products containing imidazole moiety [15]. Thus natural imidazoles could be used as effective chemotherapeutics in future. Another article by Gupta et al., emphasized upon the effective use of second and third generation azole compounds against systemic fungal infections [16]. Given their toxicity profile being better than that of the first-generation triazoles and drug interactions remain manageable; these compounds represent a true expansion of antifungal arsenal for the future. Kumar and Kaur emphasized upon the use of Triazoles and Oxadiazoles which are nitrogen and nitrogen-oxygen containing heterocycles, for the development of effective chemotherapeutic candidates. The article not only focuses on the biological importance of triazole and oxadiazole derivatives but also highlights synthetic pathways to achieve such compounds which would certainly help the scientific community to bring further developments in the isolation and synthetic methodologies for azole- based novel bioactive agents [17-21]. The continued success of the Journal is the result of a joint effort by a dedicated editorial team under the leadership of Dr. Naeem and we will continue to evolve progressively for the benefit of our contributors and readers. While thanking all the editorial members for their continued support and cohesive interactions, we reiterate our commitment for ethical and quality publications in frontier areas of Natural Product research. The lead guest editors and the executive editor would like to extend their gratitude to authors of this issue for their valuable scientific contributions. We simultaneously thank the valuable comments and suggestions of the reviewers to further improve the quality and scientific perspective of the articles. We eagerly look forward to receiving hot novel areas for contribution to The Natural Products Journal in future. In the end, if you have any questions or suggestions to further improve the journal, please feel free to drop a line to the executive editor of NPJ.

FoxO4 interacts with the sterol regulatory factor SREBP2 and the hypoxia inducible factor HIF2α at the CYP51 promoter

The late steps of cholesterol biosynthesis are oxygen demanding, requiring eleven oxygen molecules per synthesized cholesterol molecule. A key enzymatic reaction, which occurs at the top of the Bloch and Kandutsch-Russell pathways, is the demethylation of lanosterol and dihydrolanosterol (DHL). This reaction is catalyzed by lanosterol 14α demethylase (CYP51) and requires three oxygen molecules. Thus, it is the first step in the distal pathway to be susceptible to oxygen deprivation. Having previously identified that the forkhead transcription factor 4 (FoxO4) represses CYP51 expression, we aimed to characterize its role at the CYP51 promoter. Hypoxia-treated 3T3L1 cells showed decreased cholesterol biosynthesis, accumulation of lanosterol/DHL, and stimulation of FoxO4 expression and its cytoplasmic translocation to the nucleus. Transfection assays with a CYP51 promoter reporter gene revealed that FoxO4 and sterol regulatory element binding protein (SREBP)2 exert a stimulatory effect, whereas FoxO4 and the hypoxia inducible factor (HIF)2α repress CYP51 promoter activity. Electromobility shift, chromatin immunoprecipitation, pull-down, and coimmunoprecipitation assays show that FoxO4 interacts with SREBP2 and HIF2α to modulate CYP51 promoter activity. We also show an inverse correlation between FoxO4 and CYP51 in adipose tissue of ob/ob mice and mouse fetal cortical neurons exposed to hypoxia. Overall, these studies demonstrate a role for FoxO4 in the regulation of CYP51 expression.

Novel iron-sulfur containing NADPH-Reductase from Nocardia farcinica IFM10152 and fusion construction with CYP51 lanosterol demethylase

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.

The antifungal drug voriconazole is an efficient inhibitor of brain cholesterol 24S-hydroxylase in vitro and in vivo

Cholesterol 24S-hydroxylase (CYP46A1) is of key importance for cholesterol homeostasis in the brain. This enzyme seems to be resistant toward most regulatory factors and at present no drug effects on its activity have been described. The crystal structures of the substrate-free and substrate-bound CYP46A1 were recently determined (Mast et al., Crystal structures of substrate-bound and substrate-free cytochrome P450 46A1, the principal cholesterol hydroxylase in the brain. Proc. Natl. Acad. Sci. USA. 2008. 105: 9546-9551). These structural studies suggested that ligands other than sterols can bind to CYP46A1. We show here that the antifungal drug voriconazole binds to the enzyme in vitro and inhibits CYP46A1-mediated cholesterol 24-hydroxylation with a Ki of 11 nM. Mice treated with daily intraperitoneal injections of voriconazole for 5 days had high levels of voriconazole in the brain and significantly reduced brain levels of 24S-hydroxycholesterol. The levels of squalene, lathosterol, and HMG-CoA reductase mRNA were reduced in the brain of the voriconazole-treated animals as well, indicating a reduced cholesterol synthesis. Most of this effect may be due to a reduced utilization of cholesterol by CYP46A1. One of the side-effects of voriconazole is visual disturbances. Because CYP46A1 is also expressed in the neural retina, we discuss the possibility that the inhibition of CYP46A1 by voriconazole contributes to these visual disturbances.

Biosynthesis of the Sesquiterpene Antibiotic Albaflavenone in Streptomyces coelicolor A3(2)

Cytochrome P450 170A1 (CYP170A1) is encoded by the sco5223 gene of the Gram-positive, soil-dwelling bacterium Streptomyces coelicolor A3(2) as part of a two-gene cluster with the sco5222 gene. The SCO5222 protein is a sesquiterpene synthase that catalyzes the cyclization of farnesyl diphosphate to the novel tricyclic hydrocarbon, epi-isozizaene (Lin, X., Hopson, R., and Cane, D. E. (2006) J. Am. Chem. Soc. 128, 6022-6023). The presence of CYP170A1 (sco5223) suggested that epiisozizaene might be further oxidized by the transcriptionally coupled P450. We have now established that purified CYP170A1 carries out two sequential allylic oxidations to convert epi-isozizaene to an epimeric mixture of albaflavenols and thence to the sesquiterpene antibiotic albaflavenone. Gas chromatography/mass spectrometry analysis of S. coelicolor culture extracts established the presence of albaflavenone in the wild-type strain, along with its precursors epi-isozizaene and the albaflavenols. Disruption of the CYP170A1 gene abolished biosynthesis of both albaflavenone and the albaflavenols, but not epi-isozizaene. The combined results establish for the first time the presence of albaflavenone in S. coelicolor and clearly demonstrate that the biosynthesis of this antibiotic involves the coupled action of epi-isozizaene synthase and CYP170A1.

Hypoxia Stimulates Degradation of 3-Hydroxy-3-methylglutaryl-coenzyme A Reductase through Accumulation of Lanosterol and Hypoxia-Inducible Factor-mediated Induction of Insigs

Endoplasmic reticulum-associated degradation of the enzyme 3-hydroxy-3-methylglutaryl-CoA reductase represents one mechanism by which cholesterol synthesis is controlled in mammalian cells. The key reaction in this degradation is binding of reductase to Insig proteins in the endoplasmic reticulum, which is stimulated by the cholesterol precursor lanosterol. Conversion of lanosterol to cholesterol requires removal of three methyl groups, which consumes nine molecules of dioxygen. Here, we report that oxygen deprivation (hypoxia) slows demethylation of lanosterol and its metabolite 24,25-dihydrolanosterol, causing both sterols to accumulate in cells. In addition, hypoxia increases the amount of Insig-1 and Insig-2 in a response mediated by hypoxia-inducible factor (HIF)-1alpha. Accumulation of lanosterol together with increased Insigs accelerates degradation of reductase, which ultimately slows a rate-determining step in cholesterol synthesis. These results define a novel oxygen-sensing mechanism mediated by the combined actions of methylated intermediates in cholesterol synthesis and the hypoxia-activated transcription factor HIF-1alpha.

Comparison of lanosterol-14α-demethylase (CYP51) of human and Candida albicans for inhibition by different antifungal azoles

Inhibition of fungal lanosterol-14α-demethylase (CYP51) is the working principle of the antifungal activity of azoles used in agriculture and medicine. Inhibition of human CYP51 may result in endocrine disruption since follicular fluid-meiosis activating steroid (FF-MAS), the direct product of lanosterol demethylation, is involved in the control of meiosis. To investigate the specificity of antifungal agents for the fungal enzyme, assays to determine inhibitory potencies of 13 agricultural fungicides and 6 antimycotic drugs were established. FF-MAS product formation was measured by LC–MS/MS analysis in the incubations using lanosterol as substrate. Recombinant human enzyme (hCYP51) was available from BD Gentest. CYP51 of Candida albicans (cCYP51) was co-expressed with Candida tropicalis oxidoreductase in the baculovirus system. IC 50 values of 13 fungicides for cCYP51 ranged about six-fold (0.059–0.35 μM); for hCYP51 the range was about 30-fold (1.3–37.2 μM). The most favourable IC 50 ratio human to Candida was observed for imazalil (440-fold), while the specificity of epoxiconazole and tebuconazole for cCYP51 was only by a factor of 10. For the antimycotic drugs, the range of IC 50 values for cCYP51 was similar to those of fungicides (0.039–0.30 μM). For the inhibition of hCYP51, IC 50 values split into two classes: the newer drugs fluconazole and itraconazole showed little inhibition (≥30 μM) while the older drugs were even more potent than the agricultural fungicides, with miconazole being the most potent (0.057 μM). No correlation was seen between the IC 50 values determined for the two enzymes, indicating that a housekeeping gene can show significant diversity if inhibition is concerned. Our data indicate that fungicide residues in food are unlikely to exert a relevant inhibition of CYP51 in humans whereas systemic use of some antimycotic drugs, e.g. ketoconazole or miconazole, should be carefully considered regarding disturbance of human steroid biosynthesis.

Electronic and structural features of lanosterol in the 14α-demethylation

14α-demethylation is the reaction which leads directly to norlanosterol from lanosterol, and is carried out exclusively by lanosterol structure. To discover the features, which make lanosterol a unique molecule able to undergo this demethylation, the electronic and energetic parameters of lanosterol and other structurally related steroids, were calculated. Local and global parameters were analyzed, in order to insight into the reactivity and selectivity of every molecule studied. Electrostatic potential maps were used to find differences of selectivity in each molecule, along with total energy and hardness, discovering the differences in reactivity. Lanosterol shows specific orientation and unique shape of electrostatic potential map, which does not appear in other structures, except epilanosterol, because it differs only in the orientation of a hydroxyl group, therefore they present many similarities but many differences also. For this reason, epilanosterol has a similar shape of electrostatic potential map, but not its orientation. Aoyama et al. have found, three essential structural features in lanosterol to be demethylated, which generate a specific electrostatic potential map, the hydroxyl group on C-3, the position of the double bound between C8 and C9 on cycle B, and the side chain double bond. Our study agrees with some biochemical studies, which reveal that there are three key features essential for substrate recognition by the enzyme P-450 14DM. We think the present study is an alternative methodology to find features which are related with some parameters obtained via theoretical calculations.

MT FdR: a ferredoxin reductase from M. tuberculosis that couples to MT CYP51

We report the molecular cloning, expression and partial characterization of MT FdR, an FAD-associated flavoprotein, from Mycobacterium tuberculosis similar to the oxygenase-coupled NADH-dependent ferredoxin reductases (ONFR). We establish, through kinetic and spectral analysis, that MT FdR preferentially uses NADH as cofactor. Furthermore, MT FdR forms a complex with mycobacterial ferredoxin (MT Fdx) and MT CYP51, a cytochrome P450 (CYP) from M. tuberculosis that is similar to lanosterol 14α-demethylase isozymes. This reconstituted system transfers electrons from the cofactor to the heme iron of MT CYP51 and effects the demethylation of lanosterol.

A functional cytochrome P450 lanosterol 14 alpha-demethylase CYP51 enzyme in the acrosome: transport through the Golgi and synthesis of meiosis-activating sterols.

Mammalian lanosterol 14 alpha-demethylase (CYP51) is a microsomal cytochrome P450 that demethylates lanosterol to FF-MAS, an oocyte meiosis-activating sterol and late intermediate of cholesterol biosynthesis. Herein we report CYP51 unequivocally localized to acrosomal membranes of male germ cells in mouse, bull, and ram, in which it synthesizes FF-MAS in the presence of the acrosomal form of nicotinamide adenine dinucleotide phosphate reduced-P450 reductase. In the mouse, CYP51 (53 kDa) resides in endoplasmic reticulum (ER) and Golgi during all phases of acrosome development, indicating an intracellular transport from ERs through the Golgi to the acrosome. CYP51 (50 kDa) also resides on acrosomal membranes of bull- and ram-ejaculated sperm. In mouse liver, a 53-kDa CYP51 is no longer detected in trans Golgi, suggesting retrieval back to the ER and no further transport to other organelles. Glycosylated high-molecular-mass CYP51-immunoreactive proteins in acrosomal membranes of bull and ram and Golgi-enriched fractions of mouse liver indicate that mammalian CYP51s are subjected to posttranslational modifications in the Golgi. In conclusion, CYP51 is the first cytochrome P450 enzyme to be detected on acrosomal membranes. It exhibits a unique, cell-type-specific intracellular transport that is in agreement with its cell-type-specific physiological role: production of cholesterol in the liver and sterols with signaling properties in sperm. Demethylation of lanosterol to FF-MAS by the acrosomal lanosterol 14 alpha-demethylase enzyme complex demonstrates for the first time the ability of ejaculate sperm to synthesize meiosis-activating sterols.

Production of progesterone from de novo-synthesized cholesterol in cumulus cells and its physiological role during meiotic resumption of porcine oocytes.

To investigate the role of factors secreted by cumulus cells during meiotic resumption of porcine oocytes, 1, 5, 10, or 20 cumulus-oocyte complexes (COCs) were cultured in each well of a culture dish containing 300 microl of maturation medium for 20 h. There was a significant positive correlation between the rate of germinal vesicle breakdown (GVBD) and the number of COCs cultured in each well for 20 h. The level of progesterone in the medium in which COCs had been cultured for 20 h also rose significantly with an increase in the number of COCs cultured in each well. A significantly small proportion of GVBD in oocytes when one COC was cultured in each well for 20 h was improved by the addition of progesterone. This proportion of GVBD was fully comparable to that of COCs cultured in the absence of additional progesterone with 20 COCs. Thus, progesterone secreted by COCs plays a positive role in GVBD induction in porcine oocytes. Furthermore, we also examined the role of sterol biosynthesis on progesterone production by cumulus cells and in oocyte GVBD. The results showed that the addition of ketoconazole, which suppressed the sterol biosynthetic pathway produced by demethylation of lanosterol, decreased the rate of GVBD, as well as progesterone production in COCs cultured for 20 h. However, the suppression of GVBD by ketoconazole was overtaken by the addition of progesterone. These results demonstrate that a high level of progesterone produced by cumulus cells was responsible for an acceleration of GVBD in porcine oocytes.

Cascade of related endogenous MAS sterols in human follicular fluid possessing meiotic inducing activity.

Objective: Follicular fluid-meiotic activating sterol (FF-MAS) was first identified in 1995 (Byskov et al.) as an endogenous molecule in human follicular fluid (hFF) possessing the ability to induce meiosis in arrested mouse oocytes when cultured in vitro. FF-MAS is an intermediate in sterol biosynthesis formed by demethylation of lanosterol catalyzed by the enzyme sterol 14-α-demethylase (P45014DM). FF-MAS was in follicular fluid found to be accompanied by another meiosis activating sterol, T-MAS, in micromolar concentrations (Baltsen et al., 1999). T-MAS is the product formed by reduction of FF-MAS by the enzyme sterol-14-reductase. Enzymatic activity of the MAS converting enzyme P45014DM has been observed to be up-regulated by PMSG (Yoshida et al., 1996). It is hypothesized that these endogenous molecules are involved in oocyte maturation in vivo. We wanted to explore the meiotic activity in sterols in follicular fluid. Design: UV-guided fractionation of hFF followed by nuclear magnetic resonance (NMR) and mass spectrometry (MS). Materials/Methods: See design. Results: This resulted besides FF-MAS and T-MAS in isolation of four additional sterols in microgram amounts. Characterization of the sterols by ultraviolet (UV), nuclear magnetic resonance (NMR) and mass spectrometry (MS) identified them as structurally closely related to FF-MAS and T-MAS, i.e. either C4-methylated and/or C24-reduced derivatives. The sterols are all considered intermediates on the biosynthetic pathway between lanosterol and cholesterol, and are present in micromolar concentrations in follicular fluid in the range of approximately 0.5 to 3 micromolar. When hypoxanthine arrested mouse oocytes are exposed to these sterols they all induce meiotic maturation when dosed at a concentration of 10 micromolar to a similar efficacy between 60 and 80% GVB in the mouse oocytes in vitro assay as previously described (Grøndahl et al., 1998). Conclusions: The sterols were all found to possess meiotic inducing activity and constitute a prominent addition to the family of naturally occurring meiosis activating sterols, which are metabolically intimately connected. These observations expand our knowledge about the potential signaling molecules involved in oocytes maturation. The family members all possess the ability to induce resumption of meiosis at least in vitro with comparable potency and thus could be speculated to signal through the same route.

Evidence for recycling of cytochrome P450 sterol 14-demethylase from the cis-Golgi compartment to the endoplasmic reticulum (ER) upon saturation of the ER-retention mechanism.

Cytochrome P450 sterol 14-demethylase (P450-CYP51) is the enzyme that catalyzes 14alpha demethylation of lanosterol, a step in ergosterol biosynthesis, on the cytoplasmic side of the endoplasmic reticulum (ER) in Saccharomyces cerevisiae. To investigate its localization and the localization mechanism(s), we constructed a chimera by inserting a 30-residue segment, Leu(283)-Leu(312) of P450-CYP51 containing a potential N-glycosylation site in the cytoplasmic region, into the N-terminus of the same protein and tagging the C-terminus with three repeats of a hemagglutinin epitope. This chimera complements gene disruption on a single-copy vector and undergoes N-glycosylation, showing that it functions normally in vivo. Indirect immunofluorescence microscopy revealed that this chimera is localized exclusively to the ER when it is expressed on either a single-copy or multicopy vector. We carried out pulse-chase experiments and found that this chimera, when expressed on a multicopy plasmid, gradually undergoes alpha1-->6 glycosylation, a cis-Golgi-specific modification, but not alpha1-->;3 glycosylation, a medial Golgi-specific modification. In contrast, a single-copy expression of this chimera does not lead to the cis-Golgi-specific modification. These findings suggest that, when expressed on a multicopy plasmid, a fraction of this chimera is transported from the ER to the cis-Golgi compartment and subsequently recycled to the ER, but when expressed on a single-copy plasmid, no significant transport of this protein from the ER takes place. We thus suggest the possibility that cytochrome P450 is retained in the ER by a saturable static mechanism.

Cholesterol biosynthesis from lanosterol: development of a novel assay method and characterization of rat liver microsomal lanosterol delta 24-reductase.

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.

Identification of 24-methylene-24,25-dihydrolanosterol as a precursor of ergosterol in the yeasts Schizosaccharomyces pombe and Schizosaccharomyces octosporus

Study of the plasma membrane sterol composition in the yeasts Schizosaccharomyces pombe and Schizosaccharomyces octosporus revealed the presence of ergosterol, lanosterol, dehydroergosterol, fecosterol, episterol and 24-methylene-24,25-dihydrolanosterol (eburicol), a C-31 derivative. The growth of both yeasts in the presence of ketoconazole led to a decrease by 85% of the ergosterol content while the levels of lanosterol and eburicol increased. This suggests that in the biosynthetic pathway of ergosterol in Schizosaccharomyces species, the transmethylation process on the C-24 may occur directly on lanosterol and not only on zymosterol. On the other hand, it cannot be excluded that in the genus Schizosaccharomyces two routes exist from lanosterol to ergosterol: the classical one via a direct C-14, C-4 demethylation of lanosterol and the second one via the formation of a C-31 derivative followed by demethylations.

Substrate-based inhibitors of lanosterol 14.alpha.-methyl demethylase: II. Time-dependent enzyme inactivation by selected oxylanosterol analogs

Selected 15-, 32-, and 15,32-substituted lanosterol analogs are shown here to display time-dependent inactivation and lanosterol 14 alpha-methyl demethylase. These molecules are competitive with respect to substrate and require NADPH and O2 in order to display time dependence, thus supporting the premise that they are mechanism-based inactivators. Structural features required for lanosterol demethylation by the lanosterol demethylase such as nuclear double bond location and availability of an abstractable 15 alpha-proton are also essential elements for time-dependent inactivation. 32-(S)-Vinyllanost-8-en-3 beta,32-diol is a potent time-dependent inactivator (Kinact/Ki = 0.36 min-1 microM-1), while the 32-(R)-vinyllanost-8-en-3 beta,32-diol functions solely as a competitive demethylase inhibitor. These results support the premise that stereoselective oxidation occurs during lanosterol demethylation and that the 32-pro-S proton is abstracted during the demethylation reaction.

Post-transcriptional Regulation of 3-Hydroxy-3-methylglutaryl Coenzyme-A Reductase by 3β-Hydroxy-Lanost-8-en-32-Al, an Intermediate in the Conversion of Lanosterol to Cholesterol

The lanosterol demethylation intermediate 3β-hydroxy-lanost-8-en-32-al is a known suppressor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), the rate-limiting enzyme of cholesterol biosynthesis. Studies on the mechanism of action of this compound have been hampered by its rapid metabolism. As one approach to this problem, the effects of 3β-hydroxy-lanost-8-en-32-al on HMGR gene expression were examined using a mutant cell line which lacks lanosterol 14α-methyl demethylase activity. Data are presented which suggest that 3β-hydroxy-lanost-8-en-32-al inhibits HMGR gene expression by reducing the translational efficiency of the HMGR mRNA. We have recently reported that 15α-fluoro-3β-hydroxy-lanost-7-en-32-aldehyde, a compound which is structurally similar to 3β-hydroxy-lanost-8-en-32-aldehyde, suppresses HMGR activity in cultured Chinese hamster ovary cells by a post-transcriptional process, inhibiting translation without affecting either transcription or enzyme degradation (Trzaskos et al., 1993, J. Biol. Chem. 268, 22591-22599). In contrast to the results obtained with the 15α-fluorolanostenol, the lanostenol 32-aldehyde increased the rate of degradation of HMGR in a manner similar to that reported for oxycholesterols. These data suggest that 15α-fluoro-3β-hydroxy-lanost-7-en-32-aldehyde and 3β-hydroxy-lanost-8-en-32-aldehyde, although structurally similar post-transcriptional regulators of HMGR suppress enzyme activity, at least in part, by different mechanisms.

Regio- and stereo-controlled synthesis of [24,30-14C]-labeled-2,3-epoxysqualene

[24,30-14C]-(3S)-2,3-Epoxysqualene and its racemate were synthesized by two convenient routes. The products have the label in a metabolically-nonlabile position relative to the demethylation of lanosterol to cholesterol.