- Efficient cyclization of squalene epoxide to lanosterol with immobilized cells of baker's yeast
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The cyclization of squalene epoxide to lanosterol with baker's yeast (Saccharomyces cerevisiae) can conveniently be carried out in aqueous solution with glass cored immobilisates of cells in calcium alginate. This enables the manifold use of the microorganism to obtain lanosterol in a single biocatalytic step using the immobilisates repeatedly.
- Rotthaus, Olaf,Demuth, Martin
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- Partial purification and characterization of oxidosqualene-lanosterol cyclase from baker's yeast
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Partial (120-fold) purification of oxidosqualene-lanosterol cyclase from yeast is described. The enzyme derived from the microsomal fraction converts 1 mM S-squalene oxide to lanosterol in 5 h and has a pH optimum of 6.2, lower than that (pH 7.2) of the hog-liver cyclase catalyzing the same reaction. Although the yeast cyclase is stimulated by high concentrations of potassium phosphate buffer, high concentrations of potassium or sodium chloride inhibit activity. The concentration range of Triton X-100 for optimal activity is 0.7-1.2%.
- Hoshino,Williams,Chung,Scott
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- NMR Biochemical Assay for Oxidosqualene Cyclase: Evaluation of Inhibitor Activities on Trypanosoma cruzi and Human Enzymes
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Oxidosqualene cyclase (OSC), a membrane-associated protein, is a key enzyme of sterol biosynthesis. Here we report a novel assay for OSC, involving reaction in aqueous solution, NMR quantification in organic solvent, and factor analysis of spectra. We evaluated one known and three novel inhibitors on OSC of Trypanosoma cruzi, a parasite causative of Chagas disease, and compared their effects on human OSC for selectivity. Among them, one novel inhibitor showed a significant parasiticidal activity.
- Tani, Osamu,Akutsu, Yukie,Ito, Shinji,Suzuki, Takayuki,Tateishi, Yukihiro,Yamaguchi, Tomohiko,Niimi, Tatsuya,Namatame, Ichiji,Chiba, Yasunori,Sakashita, Hitoshi,Kubota, Tomomi,Yanagi, Tetsuo,Mizukami, Shusaku,Hirayama, Kenji,Furukawa, Koji,Yamasaki, Kazuhiko
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p. 5047 - 5053
(2018/05/29)
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- A well-defined monomeric aluminum complex as an efficient and general catalyst in the Meerwein-Ponndorf-Verley reduction
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The metal-catalyzed Meerwein-Ponndorf-Verley (MPV) reduction allows for the mild and sustainable reduction of aldehydes and ketones but has not found widespread application in organic synthesis due to the high catalyst loading often required to obtain satisfactory yields of the reduced product. We report here on the synthesis and structure of a sterically extremely overloaded siloxide-supported aluminum isopropoxide capable of catalytically reducing a wide range of aldehydes and ketones (52 examples) in excellent yields under mild conditions and with low catalyst loadings. The unseen activity of the developed catalyst system in MPV reductions is due to its unique monomeric nature and the neutral donor isopropanol weakly coordinating to the aluminum center. The present work implies that monomeric aluminum alkoxide catalysts may be attractive alternatives to transition-metalbased systems for the selective reduction of aldehydes and ketones to primary and secondary alcohols.
- McNerney, Brian,Whittlesey, Bruce,Cordes, David B.,Krempner, Clemens
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supporting information
p. 14959 - 14964
(2015/01/08)
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- The cysteine 703 to isoleucine or histidine mutation of the oxidosqualene-lanosterol cyclase from Saccharomyces cerevisiae generates an iridal-type triterpenoid
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The Cys703 to Ile or His mutation within Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase ERG7 (ERG7C703I/H) generates an unusual truncated bicyclic rearranged intermediate, (8R,9R,10R)-polypoda-5,13E, 17E,21-tetraen-3β-ol, related to iridal-skeleton triterpenoid. Numerous oxidosqualene-cyclized truncated intermediates, including tricyclic, unrearranged tetracyclic with 17α/β exocyclic hydrocarbon side chain, rearranged tetracyclic, and chair-chair-chair tricyclic intermediates (compounds 3-9), were also isolated from the ERG7C703X site-saturated mutations or the ERG7F699T/C703I double mutation, indicating the functional role of the Cys703 residue in stabilizing the bicyclic C-8 cation and the rearranged intermediate or interacting with Phe699, and opened a new avenue of engineering ERG7 for producing biological active agents.
- Chang, Cheng-Hsiang,Chen, Yi-Chi,Tseng, Sheng-Wei,Liu, Yuan-Ting,Wen, Hao-Yu,Li, Wen-Hsuan,Huang, Chiao-Ying,Ko, Cheng-Yu,Wang, Tsai-Ting,Wu, Tung-Kung
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p. 2376 - 2381,6
(2020/08/20)
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- Sterol C24-methyltransferase: Physio- and stereo-chemical features of the sterol C3 group required for catalytic competence
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Sterol C24-methyltransferases (24-SMTs) catalyze the electrophilic alkylation of Δ24-sterols to a variety of sterol side chain constructions, and the C3- moiety is the primary determinant for substrate binding by these enzymes. To determine what specific structural features of the C3-polar group ensure sterol catalysis, a series of structurally related C3-analogs of lanosterol that differed in stereochemistry, bulk and electronic properties were examined against the fungal 24-SMT from Paracoccidioides brasiliensis (Pb) which recognize lanosterol as the natural substrate. Analysis of the magnitude of sterol C24-methylation activity (based on the kinetic constants of Vmax/Km and product distributions determined by GC-MS) resulting from changes at the C3-position in which the 3β-OH was replaced by 3α-OH, 3β-acetyl, 3-oxo, 3-OMe, 3β-F, 3β-NH2 (protonated species) or 3H group revealed that lanosterol and five substrate analogs were catalyzed and yielded identical side chain products whereas neither the 3H- or 3α-OH lanosterol derivatives were productively bound. Taken together, our results demonstrate 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.
- Howard, Alicia L.,Liu, Jialin,Elmegeed, Gamal A.,Collins, Emily K.,Ganatra, Kalgi S.,Nwogwugwu, Chizaram A.,Nes, W. David
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body text
p. 43 - 50
(2012/08/07)
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- Alteration of the substrate's prefolded conformation and cyclization stereochemistry of oxidosqualene-lanosterol cyclase of saccharomyces cerevisiae by substitution at Phenylalanine 699
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[Chemical equaction presented] The Saccharomyces cerevisiae ERG7 Phe699 mutants produced one chair-chair-chair (C-C-C) and two chair-boat-chair (C-B-C) truncated tricyclic compounds, one tetracyclic 17α-exocyclic unrearranged intermediate, and
- Wu, Tung-Kung,Chang, Cheng-Hsiang,Wen, Hao-Yu,Liu, Yuan-Ting,Li, Wen-Hsuan,Wang, Tsai-Ting,Shie, Wen-Shiang
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scheme or table
p. 500 - 503
(2010/05/18)
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- PROCESS FOR THE PREPARATION OF LANOSTA-8-ENE COMPOU
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The present invention concerns a process for the preparation of lanosta-8-ene compounds having lanosta-8-ene-7-one or lanosta-8-ene-7-ol compounds as starting material.
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Page/Page column 4-5
(2010/04/03)
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- Protostadienol synthase from Aspergillus fumigatus: Functional conversion into lanosterol synthase
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Oxidosqualene:protostadienol cyclase (OSPC) from the fungus Aspergillus fumigatus, catalyzes the cyclization of (3S)-2,3-oxidosqualene into protosta-17(20)Z,24-dien-3β-ol which is the precursor of the steroidal antibiotic helvolic acid. To shed light on the structure-function relationship between OSPC and oxidosqualene:lanosterol cyclase (OSLC), we constructed an OSPC mutant in which the C-terminal residues 702APPGGMR708 were replaced with 702NKSCAIS708, as in human OSLC. As a result, the mutant no longer produced the protostadienol, but instead efficiently produced a 1:1 mixture of lanosterol and parkeol. This is the first report of the functional conversion of OSPC into OSLC, which resulted in a 14-fold decrease in the Vmax/KM value, whereas the binding affinity for the substrate did not change significantly. Homology modeling suggested that stabilization of the C-20 protosteryl cation by the active-site Phe701 through cation-π interactions is important for the product outcome between protostadienol and lanosterol.
- Kimura, Miki,Kushiro, Tetsuo,Shibuya, Masaaki,Ebizuka, Yutaka,Abe, Ikuro
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experimental part
p. 899 - 902
(2011/04/16)
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- Differential stereocontrolled formation of tricyclic triterpenes by mutation of tyrosine 99 of the oxidosqualene-lanosterol cyclase from saccharomyces cerevisiae
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The function of the Tyr99 residue from Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase (ERG7) was analyzed by constructing deletion, and site-saturated mutants. Two truncated intermediates, (13αH)- isomalabarica-14Z,17E,21-trien-3βol and. (13αH)
- Wu, Tung-Kung,Li, Wen-Hsuan,Chang, Cheng-Hsiang,Wen, Hao-Yu,Liu, Yuan-Ting,Chang, Yi-Chun
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experimental part
p. 5731 - 5737
(2010/03/01)
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- Importance of Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase tyrosine 707 residue for chair-boat bicyclic ring formation and deprotonation reactions
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(Chemical Equation Presented) A contact mapping strategy was applied to identify putative amino acid residues that influence the oxidosqualene- lanosterol B-ring cyclization reaction. A bicyclic intermediate with two altered deprotonation products, in conjunction with lanosterol, were isolated from the ERG7Y707X mutants, indicating that the Tyr707 residue may play a functional role in stabilizing the chair-boat bicyclic C-8 cation and the lanosteryl C-8/C-9 cation intermediates.
- Wu, Tung-Kung,Wang, Tsai-Ting,Chang, Cheng-Hsiang,Liu, Yuan-Ting,Shie, Wen-Shiang
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scheme or table
p. 4959 - 4962
(2009/06/05)
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- A putative precursor of isomalabaricane triterpenoids from lanosterol synthase mutants
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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, (13αH)-isomalabarica-14(27),17E,21-trien-3β-ol, is the likely biosynthetic precursor of isomalabaricane triterpenoids in sponges. The results suggest the facile evolution of protective triterpenoids in sessile animals.
- Lodeiro, Silvia,Wilson, William K.,Shan, Hui,Matsuda, Seiichi P. T.
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p. 439 - 442
(2007/10/03)
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- Enzyme redesign: Two mutations cooperate to convert cycloartenol synthase into an accurate lanosterol synthase
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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. Copyright
- Lodeiro, Silvia,Schulz-Gasch, Tanja,Matsuda, Seiichi P. T.
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p. 14132 - 14133
(2007/10/03)
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- A low-toxicity method for the separation of lanosterol and dihydrolanosterol from commercial mixtures
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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.
- Kavtaradze, Levan K.,Manley-Harris, Merilyn,Nicholson, Brian K.
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p. 697 - 700
(2007/10/03)
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- Directed Evolution to Generate Cycloartenol Synthase Mutants that Produce Lanosterol
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(Matrix Presented) 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.
- Meyer, Michelle M.,Xu, Ran,Matsuda, Seiichi P. T.
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p. 1395 - 1398
(2007/10/03)
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- Trypanosome and animal lanosterol synthases use different catalytic motifs.
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[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.
- Joubert,Buckner,Matsuda
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p. 1957 - 1960
(2007/10/03)
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- Steric bulk at cycloartenol synthase position 481 influences cyclization and deprotonation.
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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.
- Matsuda,Darr,Hart,Herrera,McCann,Meyer,Pang,Schepmann
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p. 2261 - 2263
(2007/10/03)
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- Steric bulk at position 454 in Saccharomyces cerevisiae lanosterol synthase influences B-ring formation but not deprotonation.
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[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.
- Joubert,Hua,Matsuda
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p. 339 - 341
(2007/10/03)
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- 1,8-Diazabicycloundec-7-ene as a Mild Deprotective Agent for Acetyl Groups
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A simple acetyl cleavage is described.The method showed selective acetyl deprotection of polyfunctionalized molecules.
- Baptistella, Lucia Helena B.,Santos, Jose Fernando dos,Ballabio, Karin Cristina,Marsaioli, Anita Jocelyne
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p. 436 - 438
(2007/10/02)
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- Une voie d'acces a la trimethyl-4,4,7a tetrahydro-3aα,4,7,7aα (3H)-benzofurannone-2 a partir des alcoxy-5 trimethyl-4,4,7a hexahydro-3aα,4,5,6,7,7aα (3H)-benzofurannones-2
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The 5-hydroxy 4,4,7a-trimethyl 3aα, 4,5,6,7,7aα-hexahydro (3H)-benzofuran-2-ones are obtained by cleavage of the corresponding methyl or ethyl ethers with iodotrimethylsilane.The two hydroxylactones 5 and 6 are transformed into sulfonic esters as a step towards the dehydrated lactone 7, without squeletal rearrangement.Solvolysis of the tosylate and the mesylate of 5, a cis-fused ring lactone, proceeds by completely different ways under analogous experimental procedures.Results are explained and compared to the solvolysis of some triterpenes.Experimental procedures for ether cleavage are discussed and the intermediates or by-products of these reactions are identified.
- Rouessac, Annick,Rouessac, Francis,Zamarlik, Henri
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p. 199 - 203
(2007/10/02)
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