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.     

Directed evolution experiments reveal mutations at cycloartenol synthase residue His477 that dramatically alter catalysis

[reaction: see text] Cycloartenol synthase cyclizes and rearranges oxidosqualene to the protosteryl cation and then specifically deprotonates from C-19. To identify mutants that deprotonate differently, randomly generated mutant cycloartenol synthases were selected in a yeast lanosterol synthase mutant. A novel His477Asn mutant was uncovered that produces 88% lanosterol and 12% parkeol. The His477Gln mutant produces 73% parkeol, 22% lanosterol, and 5% Delta(7)-lanosterol. These are the most accurate lanosterol synthase and parkeol synthase that have been generated by mutagenesis.     

Directed evolution to generate cycloartenol synthase mutants that produce lanosterol

Cycloartenol synthase converts oxidosqualene to cycloartenol, a pentacyclic isomer of the animal and fungal sterol precursor lanosterol. We used directed evolution to find cycloartenol synthase residues that affect cyclopropyl ring formation, selecting randomly generated cycloartenol synthase mutants for their ability to genetically complement a yeast strain lacking lanosterol synthase. To increase the likelihood of finding novel mutations, the little-studied Dictyostelium discoideum cycloartenol synthase was used for the mutagenesis. Several catalytically important residues were identified. [reaction: see text]     

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.     

Steric bulk at cycloartenol synthase position 481 influences cyclization and deprotonation

Cycloartenol synthase converts oxidosqualene to the pentacyclic sterol precursor cycloartenol. An Arabidopsis thaliana cycloartenol synthase Ile481Val mutant was previously shown to produce lanosterol and parkeol in addition to its native product cycloartenol. Experiments are described here to construct Phe, Leu, Ala, and Gly mutants at position 481 and to determine their cyclization product profiles. The Phe mutant was inactive, and the Leu mutant produced cycloartenol and parkeol. The Ala and Gly mutants formed lanosterol, cycloartenol, parkeol, achilleol A, and camelliol C. Monocycles comprise most of the Gly mutant product, showing that an alternate cyclization route can be made the major pathway by a single nonpolar mutation.     

Nonenzymic polycyclization of analogues of oxidosqualene with a preformed C-ring

Some nonenzymic epoxide-initiated polyolefin cyclization are reported. The presented molecules are partially constrained analogues of (3S)-oxidosqualene, the natural substrate to many important cyclase enzymes. These model compounds feature a preformed C-ring with built-in stereochemical information. The experimental results allow for an instructive comparison with the enzymic processes, particularly those of the cyclases in steroid biosynthesis (i.e. lanosterol synthase).     

A putative precursor of isomalabaricane triterpenoids from lanosterol synthase mutants

[reaction: see text]. Known lanosterol synthase mutants produce monocyclic or tetracyclic byproducts from oxidosqualene. We describe Erg7 Tyr510 mutants that cause partial substrate misfolding and generate a tricyclic byproduct. This novel triterpene, (13alphaH)-isomalabarica-14(27),17E,21-trien-3beta-ol, is the likely biosynthetic precursor of isomalabaricane triterpenoids in sponges. The results suggest the facile evolution of protective triterpenoids in sessile animals.     

Lanosterol Biosynthesis: The Critical Role of the Methyl‐29 Group of 2,3‐Oxidosqualene for the Correct Folding of this Substrate and for the Construction of the Five‐Membered D Ring

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.     

Cucurbitadienol synthase, the first committed enzyme for cucurbitacin biosynthesis, is a distinct enzyme from cycloartenol synthase for phytosterol biosynthesis

Three oxidosqualene cyclase (OSC) cDNAs (CPX, CPQ, CPR) were cloned from seedlings of Cucurbita pepo by homology based PCR method. Their open reading frames were expressed in lanosterol synthase deficient (erg7) Saccharomyces cerevisiae strain GIL77. Analyses of in vitro enzyme activities and in vivo accumulated products in the transformants demonstrated that CPQ and CPX encode cucurbitadienol and cycloartenol synthases, respectively. These results indicated the presence of distinct OSCs for cycloartenol and cucurbitadienol synthesis in this plant.     

Differential effects of cholesterol, ergosterol and lanosterol on a dipalmitoyl phosphatidylcholine membrane: a molecular dynamics simulation study

Lipid raft/domain formation may arise as a result of the effects of specific sterols on the physical properties of membranes. Here, using molecular dynamics simulation, we examine the effects of three closely-related sterols, ergosterol, cholesterol, and lanosterol, at a biologically relevant concentration (40 mol %) on the structural properties of a model dipalmitoyl phosphatidylcholine (DPPC) membrane at 309 and 323 K. All three sterols are found to order the DPPC acyl tails and condense the membrane relative to the DPPC liquid-phase membrane, but each one does this to a significantly different degree. The smooth alpha-face of ergosterol, together with the presence of tail unsaturation in this sterol, leads to closer interaction of ergosterol with the lipids and closer packing of the lipids with each other, so ergosterol has a higher condensing effect on the membrane, as reflected by the area per lipid. Moreover, ergosterol induces a higher proportion of trans lipid conformers, a thicker membrane, and higher lipid order parameters and is aligned more closely with the membrane normal. Ergosterol also positions itself closer to the bilayer/water interface. In contrast, the rough alpha-face of lanosterol leads to a less close interaction of the steroid ring system with the phospholipid acyl chains, and so lanosterol orders, straightens, and packs the lipid acyl chains less well and is less closely aligned with the membrane normal. Furthermore, lanosterol lies closer to the relatively disordered membrane center than do the other sterols. The behavior of cholesterol in all the above respects is intermediate between that of lanosterol and ergosterol. The findings here may explain why ergosterol is the most efficient of the three sterols at promoting the liquid-ordered phase and lipid domain formation and may also furnish part of the explanation as to why cholesterol is evolutionarily preferred over lanosterol in higher-vertebrate plasma membranes.     

Separation and purification of lanosterol,dihydrolanosterol, and cholesterol from lanolin by high‐performance counter‐current chromatography dual‐mode elution method

Lanosterol is a potential drug for cataracts treatment, which can reverse the aggregation of intracrystalline proteins. The low concentration in lanolin calls for high‐performance separation methods. In this study, a counter‐current chromatography dual‐mode elution method was developed for the first time to separate and purify lanosterol from hexane extract of lanolin after saponification, in which the column was first eluted with the lower phase as mobile phase in head‐to‐tail mode, followed by the upper phase in the tail‐to‐head mode. High purity of lanosterol, dihydrolanosterol, and cholesterol can be obtained simultaneously. A solvent system composed of n‐heptane/acetonitrile/ethyl acetate (5:5:1, v/v/v) was selected and optimized via partition coefficient determination. Compounds such as 111 mg lanosterol, 84 mg dihydrolanosterol, and 183 mg cholesterol with high purity of 99.77, 95.71, and 91.43%, respectively, analyzed by high‐performance liquid chromatography were obtained within 80 min from 700 mg crude extract from 1.78 g lanolin. The method was also used to improve the purity of commercial lanosterol product from 66.97 to above 99%. Counter‐current chromatography could serve as a potential and powerful technique for commercial production of highly pure lanosterol.     

Compound prioritization methods increase rates of chemical probe discovery in model organisms

Preselection of compounds that are more likely to induce a phenotype can increase the efficiency and reduce the costs for model organism screening. To identify such molecules, we screened ~81,000 compounds in Saccharomyces cerevisiae and identified ~7500 that inhibit cell growth. Screening these growth-inhibitory molecules across a diverse panel of model organisms resulted in an increased phenotypic hit-rate. These data were used to build a model to predict compounds that inhibit yeast growth. Empirical and in silico application of the model enriched the discovery of bioactive compounds in diverse model organisms. To demonstrate the potential of these molecules as lead chemical probes, we used chemogenomic profiling in yeast and identified specific inhibitors of lanosterol synthase and of stearoyl-CoA 9-desaturase. As community resources, the ~7500 growth-inhibitory molecules have been made commercially available and the computational model and filter used are provided.     

Origin of structural diversity in natural triterpenes: direct synthesis of seco-triterpene skeletons by oxidosqualene cyclase

At1g78500, one of the oxidosqualene cyclase (OSC) homologues from Arabidopsis thaliana, was expressed in a lanosterol synthase-deficient yeast strain and the products were analyzed. In addition to the known triterpenes, this OSC was found to produce two new triterpenes, the structures of which were determined by NMR and MS analyses. The new triterpenes are C-ring-seco-beta-amyrin (1) and C-ring-seco-alpha-amyrin (2) and named beta-seco-amyrin and alpha-seco-amyrin, respectively. beta-seco-Amyrin is produced from the oleanyl cation through bond cleavage between C8 and C14, and alpha-seco-amyrin is produced from the ursanyl cation in the same manner. Together with Grob fragmentation catalyzed by another OSC (marneral synthase) from A. thaliana, the formation of seco-amyrins by this OSC revealed that OSCs not only catalyze carbon-carbon bond formations and Wagner-Meerwein rearrangements but also cleave preformed ring systems in cationic intermediates. Based on this information, direct production of other natural seco-triterpenes by OSCs is proposed.     

Synthesis and biological evaluation of 3-beta,20(R)-dihydroxy-protost-24-ene

The possible lanosterol precursor, 3β, 20(R)-dihydroxy-protost-24-ene ( 1 a ) has been prepared, in thirteen steps, from 3α-hydroxy-4α, 8, 14-trimethyl-18-nor-5α, 8α, 9β, 13ξ, 14β-androstan-17-one (mixture of 2 and 3 ). In vitro experiments with rat liver homogenates failed to convert 1 a to lanosterol.     

Potential of the Compounds from Bixa orellana Purified Annatto Oil and Its Granules (Chronic®) against Dyslipidemia and Inflammatory Diseases: In Silico Studies with Geranylgeraniol and Tocotrienols

Some significant compounds present in annatto are geranylgeraniol and tocotrienols. These compounds have beneficial effects against hyperlipidemia and chronic diseases, where oxidative stress and inflammation are present, but the exact mechanism of action of such activities is still a subject of research. This study aimed to evaluate possible mechanisms of action that could be underlying the activities of these molecules. For this, in silico approaches such as ligand topology (PASS and SEA servers) and molecular docking with the software GOLD were used. Additionally, we screened some pharmacokinetic and toxicological parameters using the servers PreADMET, SwissADME, and ProTox-II. The results corroborate the antidyslipidemia and anti-inflammatory activities of geranylgeraniol and tocotrienols. Notably, some new mechanisms of action were predicted to be potentially underlying the activities of these compounds, including inhibition of squalene monooxygenase, lanosterol synthase, and phospholipase A₂. These results give new insight into new mechanisms of action involved in these molecules from annatto and Chronic^®.     

Functionalisation of terpenoids at C-4 via organopalladium dimers: cyclopropane formation during oxidation of homoallylic σ-organopalladium intermediates with lead tetraacetate

The synthesis of new potential adjuvant saponin aglycons was investigated by selective palladium mediated C-H functionalisation of appropriately functionalised derivatives of lanosterol, cholesterol, and friedelin. The desired equatorial aldehyde functionality was successfully introduced into the lanosterol skeleton as expected. Cyclopalladation of a cholesterol derivative proceeded as expected, but during oxidation of the organopalladium intermediate, participation of the adjacent alkene functionality led to stereoselective formation of a cyclopropane and introduction of an acetate group into the steroid backbone at C-6. Further investigation of this unusual cyclopropane formation on a model decalin system confirmed the result, but C-H activation on a related open chain system was prevented by complexation of the alkene functionality to the palladium.     

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.     

The stereospecific synthesis of inotodiol 3β, 22R-dihydroxylanosta-8,24-diene

Inotodiol 1a has been stereospecifically synthesised from lanosterol in six steps and Horeau's method utilised to demonstrate the R-configuration at C-22.     

Stereospecific enzymic cyclization of a synthetic 2,3-oxidosqualene analogue bearing an 18Z carbon-carbon double bond

This report discloses the first successful cyclization of a squalene analogue bearing a Δ¹⁸ Z instead of E CC bond which leads to a lanosterol analogue possessing the unnatural 20S stereochemistry. The hypotheses of sterol biosynthesis are discussed in the light of these findings.     

Side Chain Functionalization of Lanosterol by Hydroboration and Allylboration: Synthesis of a Ganoderic Acid Analog

A convenient method for the introduction of 23-keto and 26-carboxyl groups (Synthesis of a genoderic acid analog) into lanosterol side chain by use of hydroboration and allylboration as key reactions is described.