Molecular Cloning of aSchizosaccharomyces pombecDNA Encoding Lanosterol Synthase and Investigation of Conserved Tryptophan Residues

Corey, E. J.; Matsuda, Seiichi P. T.; Baker, Carleton H.; Ting, Amal; Cheng, Hengmiao

doi:10.1006/bbrc.1996.0232

Cited by 71 publications

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“…The differences in the biosynthesis of sterols between higher plants and yeast/mammals are generally accepted to begin at the cyclization step of 2,3-oxidosqualene, a common precursor. Ergosterol and cholesterol are biosynthesized via lanosterol, catalyzed by lanosterol synthase (LAS), in yeast and in mammals (2,3), respectively. Phytosterols, such as campesterol and sitosterol, are biosynthesized via cycloartenol and catalyzed by cycloartenol synthase (CAS) in higher plants ( Fig.…”

mentioning

confidence: 99%

Dual biosynthetic pathways to phytosterol via cycloartenol and lanosterol in Arabidopsis

Ohyama

Suzuki²,

Kikuchi³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

179

138

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The differences between the biosynthesis of sterols in higher plants and yeast/mammals are believed to originate at the cyclization step of oxidosqualene, which is cyclized to cycloartenol in higher plants and lanosterol in yeast/mammals. Recently, lanosterol synthase genes were identified from dicotyledonous plant species including Arabidopsis, suggesting that higher plants possess dual biosynthetic pathways to phytosterols via lanosterol, and through cycloartenol. To identify the biosynthetic pathway to phytosterol via lanosterol, and to reveal the contributions to phytosterol biosynthesis via each cycloartenol and lanosterol, we performed feeding experiments by using [6-13 C 2 H3]mevalonate with Arabidopsis seedlings. Applying 13 C-{ 1 H}{ 2 H} nuclear magnetic resonance (NMR) techniques, the elucidation of deuterium on C-19 behavior of phytosterol provided evidence that small amounts of phytosterol were biosynthesized via lanosterol. The levels of phytosterol increased on overexpression of LAS1, and phytosterols derived from lanosterol were not observed in a LAS1-knockout plant. This is direct evidence to indicate that the biosynthetic pathway for phytosterol via lanosterol exists in plant cells. We designate the biosynthetic pathway to phytosterols via lanosterol ''the lanosterol pathway.'' LAS1 expression is reported to be induced by the application of jasmonate and is thought to have evolved from an ancestral cycloartenol synthase to a triterpenoid synthase, such as ␤-amyrin synthase and lupeol synthase. Considering this background, the lanosterol pathway may contribute to the biosynthesis of not only phytosterols, but also steroids as secondary metabolites. metabolic diversity ͉ mevalonate ͉ sterol ͉ tracer experiment ͉ triterpene

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mentioning

confidence: 99%

Dual biosynthetic pathways to phytosterol via cycloartenol and lanosterol in Arabidopsis

Ohyama

Suzuki²,

Kikuchi³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

179

138

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“…Matsuda (Department of Chemistry and Biochemistry and Cell Biology, Rice University Houston, Texas-USA) [16]. The yeast strain was preserved in 40% glycerol at 2 808C.…”

Section: S Cerevisiae Strain and Culture Conditionsmentioning

confidence: 99%

Umbelliferone aminoalkyl derivatives as inhibitors of human oxidosqualene-lanosterol cyclase

Oliaro‐Bosso

Taramino

Viola

et al. 2009

Journal of Enzyme Inhibition and Medicinal Chemistry

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Human and murine lanosterol synthases (EC 5.4.99.7) were studied as targets of a series of umbelliferone aminoalkyl derivatives previously tested as inhibitors of oxidosqualene cyclases from other eukaryotes. Tests were carried out on cell cultures of human keratinocytes and mouse 3T3 fibroblasts incubated with radiolabeled acetate, and on homogenates prepared from yeast cells expressing human lanosterol synthase, incubated with radiolabeled oxidosqualene. In cell cultures of both human keratinocytes and mouse 3T3 fibroblasts, the observed inhibition of cholesterol biosynthesis was selective for oxidosqualene cyclase. The most active compounds bear an allylmethylamino chain in position-7 of the coumarin ring. The inhibition was critically dependent on the position and length of the inhibitor side chain, as well as on the type of aminoalkyl group inserted at the end of the same chain. Molecular docking analyses, carried out to clarify details of inhibitors/enzyme interactions, proved useful to explain the observed differences in inhibitory activities.

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“…SMY8pBJ1.21 strain, expressing the OSC of T. cruzi; SMY8pSM61.21, expressing the wild-type yeast OSC; and SMY8pSM60.21, expressing the Arabidopsis thaliana OSC obtained by transformation of lanosterol synthase mutant strain of S. cerevisiae SMY8 (MATα erg7::HIS3 hem1::TRP1 ura3-52-trpl-∆63 leu2-3.112 his3-∆200 ade2 Gal + ) were kindly provided by Professor S.P.T. Matsuda [Department of Chemistry and Biochemistry and Cell Biology, Rice University, Houston, TX (21,27,28)]. All strains were grown in a YPD culture broth (yeast extract 1%, peptone 2%, dextrose 2%) containing hemin (0.013 mg/mL) and ergosterol (0.02 mg/mL).…”

Section: Materials Substrates and Test Compoundsmentioning

confidence: 99%

“…Hemin had to be added to the growth medium since the SMY8 strains contains a mutation (hem1::TRP1) affecting heme biosynthesis. The presence of a heme mutant background is necessary for the viability of lanosterol synthase mutants in aerobic conditions (27). OSC expression was induced in YPG broth (yeast extract 1%, peptone 2%, galactose 2%) containing hemin (0.013 mg/mL).…”

Section: Materials Substrates and Test Compoundsmentioning

confidence: 99%

Analogs of squalene and oxidosqualene inhibit oxidosqualene cyclase of Trypanosoma cruzi expressed in Saccharomyces cerevisiae

Oliaro‐Bosso

Ceruti

Balliano

et al. 2005

Lipids

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ABSTRACT:Recently, a number of inhibitors of the enzyme oxidosqualene cyclase (OSC; EC 5.4.99.7), a key enzyme in sterol biosynthesis, were shown to inhibit in mammalian cells the multiplication of Trypanosoma cruzi, the parasite agent of Chagas' disease. The gene coding for the OSC of T. cruzi has been cloned and expressed in Saccharomyces cerevisiae. The expression in yeast cells could be a safe and easy model for studying the activity and the selectivity of the potential inhibitors of T. cruzi OSC. Sterols are eukaryotic membrane components that are necessary to support cell growth and differentiation. Sterol biosynthesis is a well-established chemotherapeutic target in pathogenic eukaryotes, such as fungi (1,2). The final product of the sterol biosynthetic pathway in fungi is the 24-alkyl sterol ergosterol. Ergosterol or similar 24-alkyl sterols are also the final products of the sterol synthetic pathway of some pathogenic protozoa including Trypanosoma species. These organisms cannot completely substitute these sterols with the cholesterol biosynthesized in host mammalian cells and need to synthesize at least some of their own distinctive sterols (3-5). This dependence on sterols suggests that sterol biosynthesis inhibitors may be useful antiprotozoal drugs. The sterol biosynthetic pathway ( Fig. 1) offers many potential targets for therapeutic purposes. Many effective antifungal drugs already in use act by inhibiting different enzymes of sterol biosynthesis; the allylamine antifungal drug terbinafine inhibits squalene epoxidase (6), and azole drugs such as ketoconazole and itraconazole are inhibitors of lanosterol C 14 -demethylase (7). The activity of these inhibitors can be explained as a consequence either of depletion of the ergosterol or of accumulation of toxic intermediates or side products. In addition to the antifungal activity, some of these compounds Acetyl-CoAwere shown to inhibit the growth of Trypanosoma and other kinetoplastid parasites (8). Kinetoplastid parasites have also been shown recently to be susceptible to inhibitors of squalene synthase (9,10) and oxidosqualene cyclase (11,12). The enzyme oxidosqualene cyclase (OSC; EC 5.4.99.7), which catalyzes the formation of the first cyclic precursor of sterols, is considered a good target for the inhibition of sterol biosynthesis in both humans and fungi. Many inhibitors, designed on the basis of the complex cyclization mechanism of OSC (13), are active on the enzyme and have been investigated as potential cholesterol-lowering or antifungal drugs (1,2,13). The recent crystallization of human OSC as a complex with the potent inhibitor RO48-8071 opens the way to new structurebased studies of the cyclization mechanism (14).We have designed and tested against mammalian and fungal OSC many acyclic substrate analogs modified either to mimic the high-energy carbocationic intermediates (compounds 1-3 of Fig. 2) or to bear reactive functions able to interact with the active site residues, as the methylidene (compounds 4-8), vinyl sulfide (compounds 9...

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Molecular Cloning of aSchizosaccharomyces pombecDNA Encoding Lanosterol Synthase and Investigation of Conserved Tryptophan Residues

Cited by 71 publications

References 0 publications

Dual biosynthetic pathways to phytosterol via cycloartenol and lanosterol in Arabidopsis

Dual biosynthetic pathways to phytosterol via cycloartenol and lanosterol in Arabidopsis

Umbelliferone aminoalkyl derivatives as inhibitors of human oxidosqualene-lanosterol cyclase

Analogs of squalene and oxidosqualene inhibit oxidosqualene cyclase of Trypanosoma cruzi expressed in Saccharomyces cerevisiae

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