- 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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- A cycloartenol synthase from the steroidal saponin biosynthesis pathway of Paris polyphylla
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Steroidal saponins named polyphyllin are the major active components of Paris polyphylla. Cycloartenol synthase (CAS) is a key enzyme that catalyzes the formation of the sterol scaffold. In this study, we cloned a putative CAS gene from Paris polyphylla. Heterologous expression in yeast indicated that PpCAS can convert 2,3-oxidosqualene into cycloartenol. qRT-PCR analysis showed that the expression of PpCAS was highest in leaves and lowest in roots. To our best knowledge, this is the first report of the functional characterization of cycloartenol synthase from Paris polyphylla, which lays the foundation for further analysis of the biosynthesis pathway of polyphyllins. (Figure presented.).
- Guo, Si-Yuan,Yin, Yan,Lei, Tao,Shi, Ying-Hui,Gao, Wei,Zhang, Xia-Nan,Li, Jia
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- Control of the 1,2-rearrangement process by oxidosqualene cyclases during triterpene biosynthesis
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Oxidosqualene cyclases (OSCs) catalyze the cyclization of an acyclic substrate into various polycyclic triterpenes through a series of cation-π cyclization and 1,2-rearrangement processes. The mechanisms by which OSCs control the fate of intermediate carbocation to generate each specific triterpene product have not yet been determined. The formation of ubiquitous sterol precursors in plants, cycloartenol and Cucurbitaceae-specific cucurbitadienol, only differs by the extent of the 1,2-rearrangement of methyl and hydride. In the present study, we identified critical residues in cycloartenol synthase and cucurbitadienol synthase that were primarily responsible for switching product specificities between the two compounds. The mutation of tyrosine 118 to leucine in cycloartenol synthase resulted in the production of cucurbitadienol as a major product, while the mutation of the corresponding residue leucine 125 to tyrosine in cucurbitadienol synthase resulted in the production of parkeol. Our discovery of this "switch" residue will open up future possibilities for the rational engineering of OSCs to produce the desired triterpenes.
- Takase, Shohei,Saga, Yusuke,Kurihara, Nozomi,Naraki, Shingo,Kuze, Kenta,Nakata, Genki,Araki, Takeshi,Kushiro, Tetsuo
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p. 7331 - 7336
(2015/07/01)
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- DRUGS, FOOD OR DRINK FOR IMPROVING PANCREATIC FUNCTIONS
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Compounds having a cyclolanostane skeleton such as 9,19-cyclolanostan-3-ol and 24-methylene-9,19-cyclolanostan-3-ol are used as an active ingredient of a drug and food or drink for improving pancreatic functions.
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Page/Page column 14
(2008/06/13)
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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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- Inhibitory effect of cycloartenol ferulate, a component of rice bran, on tumor promotion in two-stage carcinogenesis in mouse skin
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Inhibitory activity against 12-O-tetradecanoylphorbol-13-acetate (TPA)- induced inflammation in mice was observed in the methanol extract of rice bran and γ-oryzanol. The active components of rice bran, sitosterol ferulate, 24-methylcholesterol ferulate, cycloartenol ferulate and 24- methylenecycloartanol ferulate inhibited markedly the TPA-induced inflammation in mice. The 50% inhibitory dose of these compounds for TPA- induced inflammation was 0.2-0.3 mg/ear. Furthermore, cycloartenol ferulate markedly inhibited the tumor-promoting effect of TPA in 7,12- dimethylbenz[α]lanthracene-initiated mice.
- Yasukawa, Ken,Akihisa, Toshihiro,Kimura, Yumiko,Tamura, Toshitake,Takido, Michio
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p. 1072 - 1076
(2007/10/03)
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- Conformational Analysis of Cycloartenol, 24-Methylenecycloartanol and Their Derivatives
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A conformational analysis of cycloartenol, 24-methylenecycloartenol and their derivatives was carried out in the solution and the solid state by an NMR study and X-ray crystallographic analysis, respectively.Complete assignments of the 1H NMR spectra of these compounds were made in order to elucidate the conformation involving the ring system and side chain.Rings A to C had a chair-halfchair-boat conformation, and the side chain had a zig-zag conformation.
- Yoshida, Kumi,Hirose, Yoshihiko,Imai, Yutaka,Kondo, Tadao
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p. 1901 - 1912
(2007/10/02)
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- BIOSYNTHESIS OF SITOSTEROL, CYCLOARTENOL, AND 24-METHYLENECYCLOARTANOL IN TISSUE CULTURES OF HIGHER PLANTS AND ERGOSTEROL IN YEAST FROM -AND 2H3>-ACETATE AND 2H2>MVA
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The -methyl migrations postulated in the 'biogenetic isoprene rule' proposed by Ruzicka et al. have been verified by (13)C n.m.r. spectroscopy in the biosynthesis of cycloartenol (10a), 24-methylenecycloartenol (11a), and sitosterol (12a) using cultured cells of higher plants, Rabdosia japonica and Physalis peruviana, and of ergosterol (14a) in yeast fed with acetate.The -hydride shifts from C-17 to C-20, and C-13 to C-17 have also been demonstrated in the biosynthesis of sitosterol (12b) in R. japonica and of ergosterol (14b) in yeast fed with acetate.THe -hydride shift from C-9 to C-8 has also been verified in 24-methylenecycloartanol (11b) fed acetate to tissue cultures of Trichosanthes kirilowii Maxim. var. japonica.In the side-chain formation of 24-methylenecycloartanol (11b) and ergosterol (14b), a -hydride (deuteride) shift from C-24 to C-25 is observed.Conversely, no deuterium atom at C-24 or C-25 is observed in sitosterol (12b) formation.Both C-11 and C-12 of sitosterol (12c) labelled as(13)C-(2)H2 and (13)C-(2)H(1)H, biosynthesized from MVA in R. japonica suggest that squalene is released from an enzyme and the following oxidation does not distinguish a terminal double bond of one farnesyl moiety from the other to form epoxysqualenes (8A) and (8B).
- Seo, Shujiro,Uomori, Atsuko,Yoshimura, Yohko,Takeda, Ken'ichi,Seto, Haruo,et.al.
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p. 2407 - 2414
(2007/10/02)
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