The Experts below are selected from a list of 504 Experts worldwide ranked by ideXlab platform
Seiichi P T Matsuda - One of the best experts on this subject based on the ideXlab platform.
-
lanosterol biosynthesis in plants
Archives of Biochemistry and Biophysics, 2006Co-Authors: Mariya D Kolesnikova, Silvia Lodeiro, Quanbo Xiong, Seiichi P T MatsudaAbstract:Abstract Plants biosynthesize sterols from Cycloartenol using a pathway distinct from the animal and fungal route through lanosterol. Described herein are genome-mining experiments revealing that Arabidopsis encodes, in addition to Cycloartenol Synthase, an accurate lanosterol Synthase ( LSS )—the first example of lanosterol Synthases cloned from a plant. The coexistence of Cycloartenol Synthase and lanosterol Synthase implies specific roles for both cyclopropyl and conventional sterols in plants. Phylogenetic reconstructions reveal that lanosterol Synthases are broadly distributed in eudicots but evolved independently from those in animals and fungi. Novel catalytic motifs establish that plant lanosterol Synthases comprise a third catalytically distinct class of lanosterol Synthase.
-
enzyme redesign two mutations cooperate to convert Cycloartenol Synthase into an accurate lanosterol Synthase
Journal of the American Chemical Society, 2005Co-Authors: Silvia Lodeiro, Tanja Schulzgasch, Seiichi P T MatsudaAbstract: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.
-
directed evolution experiments reveal mutations at Cycloartenol Synthase residue his477 that dramatically alter catalysis
Organic Letters, 2002Co-Authors: Michael J R Segura, Michelle M Meyer, Silvia Lodeiro, And Akash J Patel, Seiichi P T MatsudaAbstract:Cycloartenol Synthase cyclizes and rearranges oxidosqualene to the protosteryl cation and then specifically deprotonates from C-19. To identify mutants that deprotonate differently, randomly generated mutant Cycloartenol Synthases were selected in a yeast lanosterol Synthase mutant. A novel His477Asn mutant was uncovered that produces 88% lanosterol and 12% parkeol. The His477Gln mutant produces 73% parkeol, 22% lanosterol, and 5% Δ7-lanosterol. These are the most accurate lanosterol Synthase and parkeol Synthase that have been generated by mutagenesis.
-
directed evolution to generate Cycloartenol Synthase mutants that produce lanosterol
Organic Letters, 2002Co-Authors: Michelle M Meyer, And Ran Xu, Seiichi P T MatsudaAbstract: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.
-
steric bulk at Cycloartenol Synthase position 481 influences cyclization and deprotonation
Organic Letters, 2000Co-Authors: Seiichi P T Matsuda, Michelle M Meyer, Lisa B Darr, Elizabeth A Hart, Jennifer B R Herrera, Kelly E Mccann, Jihai Pang, Hala G SchepmannAbstract: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.
Hubert Schaller - One of the best experts on this subject based on the ideXlab platform.
-
Analysis of CFB, a cytokinin-responsive gene of Arabidopsis thaliana encoding a novel F-box protein regulating sterol biosynthesis.
Journal of Experimental Botany, 2017Co-Authors: Wolfram G. Brenner, Hubert Schaller, Jan Erik Leuendorf, Anne Cortleven, Laetitia B. B. Martin, Thomas SchmüllingAbstract:: Protein degradation by the ubiquitin-26S proteasome pathway is important for the regulation of cellular processes, but the function of most F-box proteins relevant to substrate recognition is unknown. We describe the analysis of the gene Cytokinin-induced F-box encoding (CFB, AT3G44326), identified in a meta-analysis of cytokinin-related transcriptome studies as one of the most robust cytokinin response genes. F-box domain-dependent interaction with the E3 ubiquitin ligase complex component ASK1 classifies CFB as a functional F-box protein. Apart from F-box and transmembrane domains, CFB contains no known functional domains. CFB is expressed in all plant tissues, predominantly in root tissue. A ProCFB:GFP-GUS fusion gene showed strongest expression in the lateral root cap and during lateral root formation. CFB-GFP fusion proteins were mainly localized in the nucleus and the cytosol but also at the plasma membrane. cfb mutants had no discernible phenotype, but CFB overexpressing plants showed several defects, such as a white upper inflorescence stem, similar to the hypomorphic Cycloartenol Synthase mutant cas1-1. Both CFB overexpressing plants and cas1-1 mutants accumulated the CAS1 substrate 2,3-oxidosqualene in the white stem tissue, the latter even more after cytokinin treatment, indicating impairment of CAS1 function. This suggests that CFB may link cytokinin and the sterol biosynthesis pathway.
-
Inhibition of Cycloartenol Synthase (CAS) Function in Tobacco BY-2 Cells
Lipids, 2015Co-Authors: Elisabet Gas-pascual, Hubert Schaller, Biljana Simonovik, Thomas J BachAbstract:Tobacco BY-2 cell suspensions are our preferred model for studying isoprenoid biosynthesis pathways, due to their easy genetic transformation and the efficient absorption of metabolic precursors, intermediates, and/or inhibitors. Using this model system, we have analyzed the effects of chemical and genetic blockage of Cycloartenol Synthase (CAS, EC 5.4.99.8), an oxidosqualene cyclase that catalyzes the first committed step in the sterol pathway of plants. BY-2 cells were treated with RO 48-8071, a potent inhibitor of oxidosqualene cyclization. Short-term treatments (24 h) resulted in accumulation of oxidosqualene with no changes in the final sterol products. Interestingly, long-term treatments (6 days) induced down-regulation in gene expression not only of CAS but also of the SMT2 gene coding sterol methyltransferase 2 (EC 2.1.1.41). This explains some of the increase in 24-methyl sterols at the expense of the 24-ethyl sterols stigmasterol and sitosterol. In our alternative strategy, CAS gene expression was partially blocked by using an inducible artificial microRNA. The limited effectiveness of this approach might be explained by some dependence of the machinery for RNAi formation on an operating MVA/sterol pathway. For comparison we checked the effect of RO 48-8071 on a green cell suspension of Arabidopsis and on seedlings, containing a small spectrum of triterpenes besides phytosterols. Triterpenes remained essentially unaffected, but phytosterol accumulation was clearly diminished.
-
inhibition of Cycloartenol Synthase cas function in tobacco by 2 cell suspensions a proteomic analysis
Lipids, 2015Co-Authors: Elisabet Gaspascual, Hubert Schaller, Biljana Simonovik, Dimitri Heintz, Marc Bergdoll, Thomas J BachAbstract:The effect of an inhibitor of Cycloartenol Synthase (CAS, EC 5.4.99.8) on the proteome of tobacco BY-2 cells has been examined. CAS catalyzes the first committed step in phytosterol synthesis in plants. BY-2 cells were treated with RO 48-8071, a potent inhibitor of oxidosqualene cyclization. Proteins were separated by two-dimensional electrophoresis and spots, that clearly looked differentially accumulated after visual inspection, were cut, in-gel trypsin digested, and peptides were analyzed by nano-HPLC–MS/MS. Distinct peptides were compared to sequences in the data banks and attributed to corresponding proteins and genes. Inhibition of CAS induced proteins that appear to mitigate the negative effects of the chemical exposure. However, as all enzymes that are directly involved in phytosterol biosynthesis are low-abundant proteins, significant changes in their levels could not be observed. Differences could be seen with enzymes involved in primary metabolism (glycolysis, pentose phosphate pathway etc.), in proteins of the chaperonin family, and those, like actin, that participate in formation and strengthening of the cytoskeleton and have some impact on cell growth and division.
-
plant oxidosqualene metabolism Cycloartenol Synthase dependent sterol biosynthesis in nicotiana benthamiana
PLOS ONE, 2014Co-Authors: Elisabet Gaspascual, Anne Berna, Thomas J Bach, Hubert SchallerAbstract:The plant sterol pathway exhibits a major biosynthetic difference as compared with that of metazoans. The committed sterol precursor is the pentacyclic Cycloartenol (9β,19-cyclolanost-24-en-3β-ol) and not lanosterol (lanosta-8,24-dien-3β-ol), as it was shown in the late sixties. However, plant genome mining over the last years revealed the general presence of lanosterol Synthases encoding sequences (LAS1) in the oxidosqualene cyclase repertoire, in addition to Cycloartenol Synthases (CAS1) and to non-steroidal triterpene Synthases that contribute to the metabolic diversity of C30H50O compounds on earth. Furthermore, plant LAS1 proteins have been unambiguously identified by peptidic signatures and by their capacity to complement the yeast lanosterol Synthase deficiency. A dual pathway for the synthesis of sterols through lanosterol and Cycloartenol was reported in the model Arabidopsis thaliana, though the contribution of a lanosterol pathway to the production of 24-alkyl-Δ5-sterols was quite marginal (Ohyama et al. (2009) PNAS 106, 725). To investigate further the physiological relevance of CAS1 and LAS1 genes in plants, we have silenced their expression in Nicotiana benthamiana. We used virus induced gene silencing (VIGS) based on gene specific sequences from a Nicotiana tabacum CAS1 or derived from the solgenomics initiative (http://solgenomics.net/) to challenge the respective roles of CAS1 and LAS1. In this report, we show a CAS1-specific functional sterol pathway in engineered yeast, and a strict dependence on CAS1 of tobacco sterol biosynthesis.
-
Plant Oxidosqualene Metabolism: Cycloartenol Synthase–Dependent Sterol Biosynthesis in Nicotiana benthamiana
PLOS ONE, 2014Co-Authors: Elisabet Gas-pascual, Anne Berna, Thomas J Bach, Hubert SchallerAbstract:The plant sterol pathway exhibits a major biosynthetic difference as compared with that of metazoans. The committed sterol precursor is the pentacyclic Cycloartenol (9β,19-cyclolanost-24-en-3β-ol) and not lanosterol (lanosta-8,24-dien-3β-ol), as it was shown in the late sixties. However, plant genome mining over the last years revealed the general presence of lanosterol Synthases encoding sequences (LAS1) in the oxidosqualene cyclase repertoire, in addition to Cycloartenol Synthases (CAS1) and to non-steroidal triterpene Synthases that contribute to the metabolic diversity of C30H50O compounds on earth. Furthermore, plant LAS1 proteins have been unambiguously identified by peptidic signatures and by their capacity to complement the yeast lanosterol Synthase deficiency. A dual pathway for the synthesis of sterols through lanosterol and Cycloartenol was reported in the model Arabidopsis thaliana, though the contribution of a lanosterol pathway to the production of 24-alkyl-Δ5-sterols was quite marginal (Ohyama et al. (2009) PNAS 106, 725). To investigate further the physiological relevance of CAS1 and LAS1 genes in plants, we have silenced their expression in Nicotiana benthamiana. We used virus induced gene silencing (VIGS) based on gene specific sequences from a Nicotiana tabacum CAS1 or derived from the solgenomics initiative (http://solgenomics.net/) to challenge the respective roles of CAS1 and LAS1. In this report, we show a CAS1-specific functional sterol pathway in engineered yeast, and a strict dependence on CAS1 of tobacco sterol biosynthesis.
Toshiya Muranaka - One of the best experts on this subject based on the ideXlab platform.
-
upregulation of phytosterol and triterpene biosynthesis in centella asiatica hairy roots overexpressed ginseng farnesyl diphosphate Synthase
Plant Cell Reports, 2010Co-Authors: Kiyoshi Ohyama, Yong-eui Choi, Toshiya Muranaka, Baik HwangAbstract:Farnesyl diphosphate Synthase (FPS) plays an essential role in organ development in plants. However, FPS has not previously been identified as a key regulatory enzyme in triterpene biosynthesis. To elucidate the functions of FPS in triterpene biosynthesis, C. asiatica was transformed with a construct harboring Panax ginseng FPS (PgFPS)-encoding cDNA coupled to the cauliflower mosaic virus 35S promoter. Higher levels of CaDDS (C. asiatica dammarenediol Synthase) and CaCYS (C. asiatica Cycloartenol Synthase) mRNA were detected in all hairy root lines overexpressing when compared with the controls. However, no differences were detected in any expression of the CaSQS (C. asiatica squalene Synthase) gene. In particular, the upregulation of CaDDS transcripts suggests that FPS may result in alterations in triterpene biosynthesis capacity. Squalene contents in the T17, T24, and T27 lines were increased to 1.1-, 1.3- and 1.5-fold those in the controls, respectively. The total sterol contents in the T24 line were approximately three times higher than those of the controls. Therefore, these results indicated that FPS performs a regulatory function in phytosterol biosynthesis. To evaluate the contribution of FPS to triterpene biosynthesis, we applied methyl jasmonate as an elicitor of hairy roots expressing PgFPS. The results of HPLC analysis revealed that the content of madecassoside and asiaticoside in the T24 line was transiently increased by 1.15-fold after 14 days of MJ treatment. This result may indicate that FPS performs a role not only in phytosterol regulation, but also in triterpene biosynthesis.
-
dual biosynthetic pathways to phytosterol via Cycloartenol and lanosterol in arabidopsis
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Kunio Ohyama, Jun Kikuchi, Kazuki Saito, M Suzuki, Toshiya MuranakaAbstract: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 [6-13C2H3]mevalonate with Arabidopsis seedlings. Applying 13C-{1H}{2H} 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 β-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.
-
Dual biosynthetic pathways to phytosterol via Cycloartenol and lanosterol in Arabidopsis
Proceedings of the National Academy of Sciences, 2009Co-Authors: Kunio Ohyama, Jun-ichi Kikuchi, Kazuya Saito, M Suzuki, Toshiya MuranakaAbstract: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)H(3)]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 beta-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.
-
albinism and cell viability in Cycloartenol Synthase deficient arabidopsis
Plant Signaling & Behavior, 2008Co-Authors: Elena Babiychuk, Toshiya Muranaka, Masashi Suzuki, Pierrette Bouviernave, Vincent Compagnon, Marc Van Montagu, Sergei Kushnir, Hubert SchallerAbstract:Phenotypes of Arabidopsis thaliana that carry mutations in Cycloartenol Synthase 1 (CAS1) which is required in sterol biosynthesis have been described. Knockout mutant alleles are responsible of a male-specific transmission defect. Plants carrying a weak mutant allele cas1-1 accumulate 2,3-oxidosqualene, the substrate of CAS1, in all analyzed organs. Mutant cas1-1 plants develop albino inflorescence shoots that contain low amount of carotenoids and chlorophylls. The extent of this albinism, which affects Arabidopsis stems late in development, may be modulated by the light/dark regime. The fact that chloroplast differentiation and pigment accumulation in inflorescence shoots are associated with a low CAS1 expression could suggest the involvement of 2,3-oxidosqualene in a yet unknown regulatory mechanism linking the sterol biosynthetic segment, located in the cytoplasm, and the chlorophyll and carotenoid biosynthetic segments, located in the plastids, in the highly complex terpenoid network. CAS1 loss of fu...
-
allelic mutant series reveal distinct functions for arabidopsis Cycloartenol Synthase 1 in cell viability and plastid biogenesis
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Elena Babiychuk, Toshiya Muranaka, Masashi Suzuki, Pierrette Bouviernave, Vincent Compagnon, Marc Van Montagu, Sergei Kushnir, Hubert SchallerAbstract:Sterols have multiple functions in all eukaryotes. In plants, sterol biosynthesis is initiated by the enzymatic conversion of 2,3-oxidosqualene to Cycloartenol. This reaction is catalyzed by Cycloartenol Synthase 1 (CAS1), which belongs to a family of 13 2,3-oxidosqualene cyclases in Arabidopsis thaliana. To understand the full scope of sterol biological functions in plants, we characterized allelic series of cas1 mutations. Plants carrying the weak mutant allele cas1–1 were viable but developed albino inflorescence shoots because of photooxidation of plastids in stems that contained low amounts of carotenoids and chlorophylls. Consistent with the CAS1 catalyzed reaction, mutant tissues accumulated 2,3-oxidosqualene. This triterpenoid precursor did not increase at the expense of the pathway end products. Two strong mutations, cas1–2 and cas1–3, were not transmissible through the male gametes, suggesting a role for CAS1 in male gametophyte function. To validate these findings, we analyzed a conditional CRE/loxP recombination-dependent cas1–2 mutant allele. The albino phenotype of growing leaf tissues was a typical defect observed shortly after the CRE/loxP-induced onset of CAS1 loss of function. In the induced cas1–2 seedlings, terminal phenotypes included arrest of meristematic activity, followed by necrotic death. Mutant tissues accumulated 2,3-oxidosqualene and contained low amounts of sterols. The vital role of sterols in membrane functioning most probably explains the requirement of CAS1 for plant cell viability. The observed impact of cas1 mutations on a chloroplastic function implies a previously unrecognized role of sterols or triterpenoid metabolites in plastid biogenesis.
Yutaka Ebizuka - One of the best experts on this subject based on the ideXlab platform.
-
Functional genomics approach to the study of triterpene biosynthesis
Pure and Applied Chemistry, 2020Co-Authors: Yutaka Ebizuka, Yuji Katsube, Tetsuo Kushiro, Takehiko Tsutsumi, Masaaki ShibuyaAbstract:The Arabidopsis thaliana genome-sequencing project has identified the presence of 13 oxidosqualene cyclase homologs in this plant. In addition to the already identified clones, namely, CAS1 Cycloartenol Synthase, LUP1 lupeol Synthase, and YUP8H12R.43 multifunc- tional triterpene Synthase, two new cDNAs of the putative oxidosqualene cyclase genes, F1019.4 and T30F21.16, were obtained by polymerase chain reaction (PCR) and function- ally expressed in yeast. Liquid chromatography/mass spectrometry (LC/MS) analysis led to the identification of some of their reaction products. Interestingly, except for CAS1 for sterol biosynthesis of primary metabolism, so-far-obtained all triterpene Synthases of this plant are multifunctional, producing more than one cyclization product. A feeding experiment of 13 C-labeled acetate with LUP1 lupeol Synthase transformant demonstrated the stereospecific water addition to lupenyl cation intermediate, yielding 3β,20-dihydroxylupane, which ac- counts for the multiproduct nature of this Synthase.
-
Squalene cyclase and oxidosqualene cyclase from a fern
FEBS Letters, 2007Co-Authors: Junichi Shinozaki, Masaaki Shibuya, Kazuo Masuda, Yutaka EbizukaAbstract:Ferns are the most primitive vascular plants. The phytosterols of ferns are the same as those of higher plants, but they produce characteristic triterpenes. The most distinct feature is the lack of oxygen functionality at C-3, suggesting that the triterpenes of ferns may be biosynthesized by direct cyclization of squalene. To obtain some insights into the molecular bases for the biosynthesis of triterpenes in ferns, we cloned ACX, an oxidosqualene cyclase homologue, encoding a Cycloartenol Synthase (CAS) and ACH, a squalene cyclase homologue, encoding a 22-hydroxyhopane Synthase from Adiantum capillus-veneris. Phylogenetic analysis revealed that ACH is located in the cluster of bacterial SCs, while ACX is in the cluster of higher plant CASs.
-
cucurbitadienol Synthase the first committed enzyme for cucurbitacin biosynthesis is a distinct enzyme from Cycloartenol Synthase for phytosterol biosynthesis
Tetrahedron, 2004Co-Authors: Masaaki Shibuya, Shinya Adachi, Yutaka EbizukaAbstract:Three oxidosqualene cyclase (OSC) cDNAs (CPX, CPQ, CPR) were cloned from seedlings of Cucurbita pepo by homology based PCR method. Their open reading frames were expressed in lanosterol Synthase deficient (erg7) Saccharomyces cerevisiae strain GIL77. Analyses of in vitro enzyme activities and in vivo accumulated products in the transformants demonstrated that CPQ and CPX encode cucurbitadienol and Cycloartenol Synthases, respectively. These results indicated the presence of distinct OSCs for Cycloartenol and cucurbitadienol synthesis in this plant.
-
differential expression of three oxidosqualene cyclase mrnas in glycyrrhiza glabra
Biological & Pharmaceutical Bulletin, 2004Co-Authors: Hiroaki Hayashi, Masaaki Shibuya, Pengyu Huang, Megumi Obinata, Kenichiro Inoue, Satoko Takada, Yutaka EbizukaAbstract:The cultured cells and intact plants of Glycyrrhiza glabra (Fabaceae) produce betulinic acid and oleanane-type triterpene saponins (soyasaponins and glycyrrhizin). To elucidate the regulation of triterpenoid biosynthesis in G. glabra, the cDNA of lupeol Synthase, an oxidosqualene cyclase (OSC) responsible for betulinic acid biosynthesis, was cloned, and expression patterns of lupeol Synthase and two additional OSCs, β-amyrin Synthase and Cycloartenol Synthase, were compared. The mRNA expression levels of lupeol Synthase and β-amyrin Synthase were consistent with the accumulation of betulinic acid and oleanane-type triterpene saponins, respectively. The transcript of lupeol Synthase was highly expressed in the cultured cells and root nodules. The transcript of β-amyrin Synthase, an OSC responsible for oleanane-type triterpene biosynthesis, was highly expressed in the cultured cells, root nodules and germinating seeds, where soyasaponin accumulates, and in the thickened roots where glycyrrhizin accumulates. In the cultured cells, the addition of methyl jasmonate up-regulated β-amyrin Synthase mRNA and soyasaponin biosynthesis, but down-regulated lupeol Synthase mRNA. Furthermore, the addition of gibberellin A3 down-regulated β-amyrin Synthase mRNA but not lupeol Synthase mRNA in the cultured cells. The mRNA levels of Cycloartenol Synthase, an additional OSC responsible for sterol biosynthesis, in the intact plant and cultured cells were relatively constant in these experiments.
-
Oxidosqualene cyclases from cell suspension cultures of Betula platyphylla var. japonica: molecular evolution of oxidosqualene cyclases in higher plants.
Biological & Pharmaceutical Bulletin, 2003Co-Authors: Hong Zhang, Masaaki Shibuya, Shinso Yokota, Yutaka EbizukaAbstract:Betula platyphylla var. japonica is a rich source of triterpenoid as it contains dammarane type triterpenes in the leaves, and lupane type and oleanane type triterpenes in the bark. Four oxidosqualene cyclase cDNAs (BPX, BPX2, BPW and BPY) were cloned by homology based PCR methods from cell suspension cultures of B. platyphylla var. japonica. Open reading frames consisting of 2274, 2304, 2268 and 2340 bp were ligated into yeast expression plasmid pYES2 under the control of GAL1 promoter and introduced into lanosterol Synthase deficient (erg7) Saccharomyces cerevisiae strain GIL77. Analyses of in vitro enzyme activities and/or accumulated products in the transformants demonstrated that they encode Cycloartenol Synthase (BPX and BPX2), lupeol Synthase (BPW) and β-amyrin Synthase (BPY) proteins. Phylogenetic tree was constructed for all the known oxidosqualene cyclases (OSCs) including the clones obtained in this study, revealing that OSCs having the same enzyme function form respective branches in the tree even though they derive from different plant species. Intriguing correlation was found between reaction mechanism and molecular evolution of OSCs in higher plants.
Masaaki Shibuya - One of the best experts on this subject based on the ideXlab platform.
-
Functional genomics approach to the study of triterpene biosynthesis
Pure and Applied Chemistry, 2020Co-Authors: Yutaka Ebizuka, Yuji Katsube, Tetsuo Kushiro, Takehiko Tsutsumi, Masaaki ShibuyaAbstract:The Arabidopsis thaliana genome-sequencing project has identified the presence of 13 oxidosqualene cyclase homologs in this plant. In addition to the already identified clones, namely, CAS1 Cycloartenol Synthase, LUP1 lupeol Synthase, and YUP8H12R.43 multifunc- tional triterpene Synthase, two new cDNAs of the putative oxidosqualene cyclase genes, F1019.4 and T30F21.16, were obtained by polymerase chain reaction (PCR) and function- ally expressed in yeast. Liquid chromatography/mass spectrometry (LC/MS) analysis led to the identification of some of their reaction products. Interestingly, except for CAS1 for sterol biosynthesis of primary metabolism, so-far-obtained all triterpene Synthases of this plant are multifunctional, producing more than one cyclization product. A feeding experiment of 13 C-labeled acetate with LUP1 lupeol Synthase transformant demonstrated the stereospecific water addition to lupenyl cation intermediate, yielding 3β,20-dihydroxylupane, which ac- counts for the multiproduct nature of this Synthase.
-
Squalene cyclase and oxidosqualene cyclase from a fern
FEBS Letters, 2007Co-Authors: Junichi Shinozaki, Masaaki Shibuya, Kazuo Masuda, Yutaka EbizukaAbstract:Ferns are the most primitive vascular plants. The phytosterols of ferns are the same as those of higher plants, but they produce characteristic triterpenes. The most distinct feature is the lack of oxygen functionality at C-3, suggesting that the triterpenes of ferns may be biosynthesized by direct cyclization of squalene. To obtain some insights into the molecular bases for the biosynthesis of triterpenes in ferns, we cloned ACX, an oxidosqualene cyclase homologue, encoding a Cycloartenol Synthase (CAS) and ACH, a squalene cyclase homologue, encoding a 22-hydroxyhopane Synthase from Adiantum capillus-veneris. Phylogenetic analysis revealed that ACH is located in the cluster of bacterial SCs, while ACX is in the cluster of higher plant CASs.
-
lanosterol Synthase in dicotyledonous plants
Plant and Cell Physiology, 2006Co-Authors: Masashi Suzuki, Ting Xiang, Kiyoshi Ohyama, Hikaru Seki, Hiroaki Hayashi, Yuji Katsube, Tetsuo Kushiro, Toshiya Muranaka, Kazuki Saito, Masaaki ShibuyaAbstract:: Sterols are important as structural components of plasma membranes and precursors of steroidal hormones in both animals and plants. Plant sterols show a wide structural variety and significant structural differences from those of animals. To elucidate the origin of structural diversity in plant sterols, their biosynthesis has been extensively studied [Benveniste (2004) Annu. Rev. Plant. Biol. 55: 429, Schaller (2004) Plant Physiol. Biochem. 42: 465]. The differences in the biosynthesis of sterols between plants and animals begin at the step of cyclization of 2,3-oxidosqualene, which is cyclized to lanosterol in animals and to Cycloartenol in plants. However, here we show that plants also have the ability to synthesize lanosterol directly from 2,3-oxidosqualene, which may lead to a new pathway to plant sterols. The Arabidopsis gene At3g45130, designated LAS1, encodes a functional lanosterol Synthase in plants. A phylogenetic tree showed that LAS1 belongs to the previously uncharacterized branch of oxidosqualene cyclases, which differs from the Cycloartenol Synthase branch. Panax PNZ on the same branch was also shown to be a lanosterol Synthase in a yeast heterologous expression system. The higher diversity of plant sterols may require two biosynthetic routes in steroidal backbone formation.
-
cucurbitadienol Synthase the first committed enzyme for cucurbitacin biosynthesis is a distinct enzyme from Cycloartenol Synthase for phytosterol biosynthesis
Tetrahedron, 2004Co-Authors: Masaaki Shibuya, Shinya Adachi, Yutaka EbizukaAbstract:Three oxidosqualene cyclase (OSC) cDNAs (CPX, CPQ, CPR) were cloned from seedlings of Cucurbita pepo by homology based PCR method. Their open reading frames were expressed in lanosterol Synthase deficient (erg7) Saccharomyces cerevisiae strain GIL77. Analyses of in vitro enzyme activities and in vivo accumulated products in the transformants demonstrated that CPQ and CPX encode cucurbitadienol and Cycloartenol Synthases, respectively. These results indicated the presence of distinct OSCs for Cycloartenol and cucurbitadienol synthesis in this plant.
-
differential expression of three oxidosqualene cyclase mrnas in glycyrrhiza glabra
Biological & Pharmaceutical Bulletin, 2004Co-Authors: Hiroaki Hayashi, Masaaki Shibuya, Pengyu Huang, Megumi Obinata, Kenichiro Inoue, Satoko Takada, Yutaka EbizukaAbstract:The cultured cells and intact plants of Glycyrrhiza glabra (Fabaceae) produce betulinic acid and oleanane-type triterpene saponins (soyasaponins and glycyrrhizin). To elucidate the regulation of triterpenoid biosynthesis in G. glabra, the cDNA of lupeol Synthase, an oxidosqualene cyclase (OSC) responsible for betulinic acid biosynthesis, was cloned, and expression patterns of lupeol Synthase and two additional OSCs, β-amyrin Synthase and Cycloartenol Synthase, were compared. The mRNA expression levels of lupeol Synthase and β-amyrin Synthase were consistent with the accumulation of betulinic acid and oleanane-type triterpene saponins, respectively. The transcript of lupeol Synthase was highly expressed in the cultured cells and root nodules. The transcript of β-amyrin Synthase, an OSC responsible for oleanane-type triterpene biosynthesis, was highly expressed in the cultured cells, root nodules and germinating seeds, where soyasaponin accumulates, and in the thickened roots where glycyrrhizin accumulates. In the cultured cells, the addition of methyl jasmonate up-regulated β-amyrin Synthase mRNA and soyasaponin biosynthesis, but down-regulated lupeol Synthase mRNA. Furthermore, the addition of gibberellin A3 down-regulated β-amyrin Synthase mRNA but not lupeol Synthase mRNA in the cultured cells. The mRNA levels of Cycloartenol Synthase, an additional OSC responsible for sterol biosynthesis, in the intact plant and cultured cells were relatively constant in these experiments.
