Molecular cloning, characterization, and overexpression of ERG7, the Saccharomyces cerevisiae gene encoding lanosterol synthase.

Corey, E. J.; Matsuda, Seiichi P. T.; Bartel, Bonnie

doi:10.1073/pnas.91.6.2211

Cited by 116 publications

(72 citation statements)

References 44 publications

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“…This enzyme performs a similar reaction to that of oxidosqualene-lanosterol cyclases from animals and fungi and oxidosqualene-cycloartenol cyclase from plants [2]. Together with the oxidosqualene cyclases, SHC catalyzes the most complex one-step reaction in biochemistry; 12 bonds are altered, 5 cycles formed, and 9 stereocenters established [3]. This complex reaction can be dissected into four basic steps as follow: binding of the particular conformation of the substrate ; initiation by protonation and generation of the first carbocation; propagation of the reaction and stabilization of the intermediate carbocations ; termination of the reaction and final deprotonation.…”

mentioning

confidence: 99%

Site‐Directed Mutagenesis of Putative Active‐Site Residues in Squalene‐Hopene Cyclase

Feil

Süßmuth

Jung

et al. 1996

European Journal of Biochemistry

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Squalene-hopene cyclase (SHC) catalyzes the complex polycylization of squalene to hopene, similar to the cyclization of oxidosqualene to sterols. Sequence analysis of SHC revealed a highly conserved aspartate-rich motif (DDTA), comparable to the DCTA motif of oxidosqualene cyclases, which is supposed to be part of the active site. In order to determine the importance of the motif in squalene cyclization, the conserved residues Asp376 and Asp377 in the DDTA motif of SHC from Alicyclobacillus acidocaldarius were individually replaced by glutamate, glutamine, glycine, and arginine. With the exception of the [Glu376]SHC mutant, all other substitutions resulted in almost or complete loss of enzyme activity. Compared to that of the wild-type enzyme, the specific activity of the [Glu376]SHC mutant enzyme was reduced to lo%, accompanied by a significant decrease in the apparent V, , , , whereas the apparent K,,, remained unchanged. CD measurements indicated that mutations did not affect the secondary structure. It is proposed that Asp376 and Asp377 are crucial for catalysis and may act as point charges to stabilize intermediate cations. Moreover, for squalene-hopene cyclase, a high content of a-helical conformation could be found, providing the first structural information for a triterpene cyclase.Keywords: squalene-hopene cyclase ; Alicyclobacillus acidocaldarius ; mutagenesis ; active site ; circular dichroism.Hopanoids are pentacyclic triterpenoids. These membrane condensing lipids are widely distributed in the domain Bacteria [l]. Squalene-hopene cyclase (SHC) catalyzes the crucial step in the hopanoid biosynthetic pathway (Scheme 1). This enzyme performs a similar reaction to that of oxidosqualene-lanosterol cyclases from animals and fungi and oxidosqualene-cycloartenol cyclase from plants [2]. Together with the oxidosqualene cyclases, SHC catalyzes the most complex one-step reaction in biochemistry; 12 bonds are altered, 5 cycles formed, and 9 stereocenters established [3]. This complex reaction can be dissected into four basic steps as follow: binding of the particular conformation of the substrate ; initiation by protonation and generation of the first carbocation; propagation of the reaction and stabilization of the intermediate carbocations ; termination of the reaction and final deprotonation. In the case of oxidosqualenelanosterol cyclase, the termination is accompanied by a sequence of 1,2-hydride and methyl shifts prior to proton abstraction. In this context, our aim was to obtain a first hint to the role of certain amino acids of the SHC in cyclization reaction.All known triterpene cyclases are similar in their amino acid sequences. Analysis of the primary structures revealed interesting features. All cyclases contain a non-tandem repeat, the so- called QW motif [4]. This motif is discussed to be involved in carbocation stabilization by cation-71-interactions via aromatic side chains of amino acids [5, 61. Additionally, a conserved aspartate-containing domain was found with the consensus sequences DDTA in...

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mentioning

confidence: 99%

Site‐Directed Mutagenesis of Putative Active‐Site Residues in Squalene‐Hopene Cyclase

Feil

Süßmuth

Jung

et al. 1996

European Journal of Biochemistry

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“…We therefore metabolically engineered a yeast strain that mimicked an isomerase mutant. Yeast cyclizes oxidosqualene directly to lanosterol, a tetracyclic sterol precursor (27). Because the yeast lanosterol synthase mutant SMY8 (20) cannot make sterol, it requires ergosterol supplementation.…”

Section: Resultsmentioning

confidence: 99%

Functional Cloning of an Arabidopsis thalianacDNA Encoding Cycloeucalenol Cycloisomerase

Lovato¹,

Segura²,

Giner³

et al. 2000

Journal of Biological Chemistry

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Plants and certain protists use cycloeucalenol cycloisomerase (EC 5.5.1.9) to convert pentacyclic cyclopropyl sterols to conventional tetracyclic sterols. We used a novel complementation strategy to clone a cycloeucalenol cycloisomerase cDNA. Expressing an Arabidopsis thaliana cycloartenol synthase cDNA in a yeast lanosterol synthase mutant provided a sterol auxotroph that could be genetically complemented with the isomerase. We transformed this yeast strain with an Arabidopsis yeast expression library and selected sterol prototrophs to obtain a strain that accumulated biosynthetic ergosterol. The novel phenotype was conferred by an Arabidopsis cDNA that potentially encodes a 36-kDa protein.We expressed this cDNA (CPI1) in Escherichia coli and showed by gas chromatography-mass spectrometry that extracts from this strain isomerized cycloeucalenol to obtusifoliol in vitro. The cDNA will be useful for obtaining heterologously expressed protein for catalytic studies and elucidating the in vivo roles of cyclopropyl sterols.Even distantly diverged organisms generally biosynthesize shared molecules by such similar routes that bacteria and yeast have been suitable systems for deducing much of human metabolism. Sterol biosynthesis provides an exception to this trend. Eukaryotes use two distinct pathways ( Fig. 1) to make structurally similar sterols (1). Animals and fungi cyclize oxidosqualene to the tetracyclic ⌬ 8 -triterpene lanosterol (2), which they metabolize further to tetracyclic membrane sterols (3)(4)(5). Plants cyclize instead to the pentacyclic cycloartenol (6, 7) and consequently must isomerize the cyclopropane ring to form ⌬ 8 tetracycles en route to tetracyclic sterols. Why plants use this two-enzyme route (8) rather than cyclizing directly to tetracyclic sterols using lanosterol synthase remains unknown.Although the enzyme has not been purified to homogeneity, cycloeucalenol cycloisomerase (also known as cycloeucalenolobtusifoliol isomerase) activity has been observed directly in both dicots (9) and monocots (10). In addition, the presence of an isomerase has been demonstrated indirectly in diverse plants (11)(12)(13)(14) and the protozoan Acanthamoeba polyphaga (15) by identifying cyclopropyl sterols after treatment with isomerase inhibitors. In D 2 O, the isomerase adds a deuterium to C-19 and abstracts hydrogen from C-8 (16, 17). Neither ATP nor NADPH enhances activity (8), and acidic ring opening seems the most likely mechanism. Because the nonenzymatic acid-catalyzed isomerization (18) requires severe conditions (10% H 2 SO 4 in refluxing isopropyl alcohol for 24 h), it is likely that the enzyme employs a metal or an unusually acidic amino acid residue to open the ring.As a prelude to investigating its catalytic mechanism and biological function, we have cloned and heterologously expressed the Arabidopsis thaliana cDNA that encodes cycloeucalenol cycloisomerase. EXPERIMENTAL PROCEDURESCloning the Cycloeucalenol Cycloisomerase cDNA-The A. thaliana CAS1 1 cDNA (19) cloned into the integrative yeast exp...

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“…2A and B). In contrast, the spore formation efficiency of KEY503 was lower than that of wild-type cells in the absence of choline, and instead KEY503 and KUY503 were precultured in YPD medium overnight and then inoculated into 1 ml SM at an initial OD 600 of 1.0, and incubated at 25 C for 120 h. A, Spore formation was observed as in Fig. 1B.…”

Section: Resultsmentioning

confidence: 99%

“…B, C, D, and E, Repression of PE synthesis and spore formation. TKY12G and W303 were precultured in YPG medium overnight and then inoculated into 1 ml SM-Gal at an initial OD 600 of 1.0, and incubated at 25 C for 72 h. B, Cells were stained with DAPI and observed under Nomarski optics and by fluorescence microscopy. C, The percentages of cells without spores, those with incomplete spores, and with three or four spores, are indicated.…”

Section: Resultsmentioning

confidence: 99%

Manipulation of Major Membrane Lipid Synthesis and Its Effects on Sporulation inSaccharomyces cerevisiae

Deng

Nagasawa

Ono

et al. 2008

Bioscience, Biotechnology, and Biochemistry

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Molecular cloning, characterization, and overexpression of ERG7, the Saccharomyces cerevisiae gene encoding lanosterol synthase.

Abstract: We

Cited by 116 publications

References 44 publications

Site‐Directed Mutagenesis of Putative Active‐Site Residues in Squalene‐Hopene Cyclase

Site‐Directed Mutagenesis of Putative Active‐Site Residues in Squalene‐Hopene Cyclase

Functional Cloning of an Arabidopsis thalianacDNA Encoding Cycloeucalenol Cycloisomerase

Manipulation of Major Membrane Lipid Synthesis and Its Effects on Sporulation inSaccharomyces cerevisiae

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