Molecular cloning, characterization, and functional expression of rat oxidosqualene cyclase cDNA.

Abe, Ikuro; Prestwich, Glenn D.

doi:10.1073/pnas.92.20.9274

Cited by 85 publications

(43 citation statements)

References 45 publications

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“…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 SHCs or DCTA in oxidosqualene cyclases, similar to the known consensus sequence DDXXD of other terpene cyclases [7]. The high degree of sequence conservation of this motif within the triterpene cyclases supports a catalytic or structural function of this domain.…”

mentioning

confidence: 73%

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: 73%

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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“…The LSS-deficient yeast strain, SGL9, was transformed with the constructs, and LSS expression was induced as previously described (42). LSS activity was assessed as the conversion rate from [ 3 H]2,3-oxidosqualene to lanosterol (42). Three separate assays were performed for each construct.…”

Section: Methodsmentioning

confidence: 99%

“…Nucleotide substitutions were introduced into the pYES-Lss A and pYES-Lss S using the GeneEditor In Vitro Site-Directed Mutagenesis System (Promega), resulting in pYES-Lss A-139N , pYESLss A-189A , pYES-Lss A-481R , pYES-Lss S-139D , pYES-Lss S-189G , and pYES-Lss S-481Q . The LSS-deficient yeast strain, SGL9, was transformed with the constructs, and LSS expression was induced as previously described (42). LSS activity was assessed as the conversion rate from [ 3 H]2,3-oxidosqualene to lanosterol (42).…”

Section: Methodsmentioning

confidence: 99%

Lanosterol synthase mutations cause cholesterol deficiency-associated cataracts in the Shumiya cataract rat

Mori

Li²,

Abe³

et al. 2006

Journal of Clinical Investigation

103

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The Shumiya cataract rat (SCR) is a hereditary cataractous strain. It is thought that the continuous occurrence of poorly differentiated epithelial cells at the bow area of the lens forms the pathophysiological basis for cataract formation in SCRs. In this study, we attempted to identify the genes associated with cataract formation in SCRs by positional cloning. Genetic linkage analysis revealed the presence of a major cataract locus on chromosome 20 as well as a locus on chromosome 15 that partially suppressed cataract onset. Hypomorphic mutations were identified in genes for lanosterol synthase (Lss) on chromosome 20 and farnesyl diphosphate farnesyl transferase 1 (Fdft1) on chromosome 15, both of which function in the cholesterol biosynthesis pathway. A null mutation for Lss was also identified. Cataract onset was associated with the specific combination of Lss and Fdft1 mutant alleles that decreased cholesterol levels in cataractous lenses to about 57% of normal. Thus, cholesterol insufficiency may underlie the deficient proliferation of lens epithelial cells in SCRs, which results in the loss of homeostatic epithelial cell control of the underlying fiber cells and eventually leads to cataractogenesis. These findings may have some relevance to other types of cataracts, inborn defects of cholesterol synthesis, and the effects of cholesterol-lowering medication.

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“…2), which are thought to be involved in the stabilization of carbocationic intermediates formed during the cyclization of OSCs; and one Asp-Cys-Thr-Ala-Glu (DCTAE) motif (Fig. 2), a sequence common to all known OSCs [23][24][25] including b-amyrin synthase. On this basis, we were able to conclude that GsAS1 is a b-amyrin synthase gene.…”

Section: Isolation Of G Straminea B B-amyrinmentioning

confidence: 99%

Cloning and Functional Analysis of a β-Amyrin Synthase Gene Associated with Oleanolic Acid Biosynthesis in <i>Gentiana straminea</i> M<small>AXIM.</small>

Liu

Cai²,

Zhao

et al. 2009

Biological & Pharmaceutical Bulletin

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Gentiana straminea MAXIM. (Mahuaqinjiao) belongs to theGentianaceae family, and is an important medicinal plant in China. It is found on the Qinghai-Tibetan plateau at altitudes between 2000 and 5000 m asl, 1) and is a rich source of secoiridoid glucosides, including gentiopicrosidel, loganic acid, swertiamarin, oleanolic acid and roburic acid, 2) all of which have been associated with medicinal properties. The triterpenoid oleanolic acid has been used to treat hepatitis and a number of diseases of the immune system. 3,4) Wild populations of G. straminea have been threatened by excessive excavation, and its seeds germinate poorly in low altitude environments.5) Hence there is a need to enhance the production of oleanolic acid by secondary metabolic engineering in the condition of raw G. straminea limited.b-amyrin is thought to act as a precursor for oleanolic acid, and is associated with oleanolic acid production.6) It is synthesized within the isoprenoid pathway by the cyclization of 2,3-oxidosqualene, catalysed by b-amyrin synthase (an oxidosqualene cyclase, or OSC) (Fig. 1). A number of OSCs have been cloned to date, and their enzyme functions identified by heterologous expression in yeast. 7,8) These enzymes can have either mono-or multifunctional activity in higher plants, and yield various triterpene products.9-11) Furthermore, some research shows that OSCs are key enzymes in the isoprenoid pathway to form different products.12-16) Thus b-amyrin synthase in G. straminea may represent a key enzyme for oleanolic acid synthesis, and the cloning of its encoding gene is required for any effort to raise the content of a secondary metabolite such as oleanolic acid using metabolic engineering.Methyl jasmonate (MeJA) is an elicitor which enhances the content of triterpene saponins. [17][18][19][20] The indications are that the accumulation of triterpene saponins is related to OSC over-expression, mediated by MeJA. Thus, the relationship between OSC gene expression and oleanolic acid production in the presence of MeJA may be critical to our understanding of the mechanism of secondary product accumulation. Here, we describe the characteristics of G. straminea bamyrin synthase, and show how manipulation of its expression level affects the quantity of oleanolic acid in the presence of MeJA. MATERIALS AND METHODSPlant Materials G. straminea samples were collected from Yushu Country, Qinghai Province, China. Voucher specimens have been deposited in Qinghai Normal University.Bacterial and Yeast Strains and Vectors Escherichia coli strain BL21(DE3) and the pET28(a) vector provided a prokayotic expression system. Bacterial cells were grown at 37°C in LB medium containing 0.5% (w/v) yeast extract, 1% (w/v) tryptone, 1% (w/v) NaCl and 100mg/ml kanamycin. Pichia pastoris strain X-33 and the pPICZA vector provided an eukaryotic expression system. Yeast cells were grown at 30°C in YPD medium containing 1% (w/v) yeast extract, 2% (w/v) peptone and 2% (w/v) glucose supplemented with 100 mg/ml Zeocin. Media were solidified with 2% ...

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Molecular cloning, characterization, and functional expression of rat oxidosqualene cyclase cDNA.

Cited by 85 publications

References 45 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

Lanosterol synthase mutations cause cholesterol deficiency-associated cataracts in the Shumiya cataract rat

Cloning and Functional Analysis of a β-Amyrin Synthase Gene Associated with Oleanolic Acid Biosynthesis in <i>Gentiana straminea</i> M<small>AXIM.</small>

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Molecular cloning, characterization, and functional expression of rat oxidosqualene cyclase cDNA.

Cited by 85 publications

References 45 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

Lanosterol synthase mutations cause cholesterol deficiency-associated cataracts in the Shumiya cataract rat

Cloning and Functional Analysis of a &beta;-Amyrin Synthase Gene Associated with Oleanolic Acid Biosynthesis in <i>Gentiana straminea</i> M<small>AXIM.</small>

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Cloning and Functional Analysis of a β-Amyrin Synthase Gene Associated with Oleanolic Acid Biosynthesis in <i>Gentiana straminea</i> M<small>AXIM.</small>