The Experts below are selected from a list of 210 Experts worldwide ranked by ideXlab platform
Seiichi P T Matsuda - One of the best experts on this subject based on the ideXlab platform.
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lanosterol biosynthesis in plants
Archives of Biochemistry and Biophysics, 2006Co-Authors: Mariya D Kolesnikova, Quanbo Xiong, Silvia Lodeiro, 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.
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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.
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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.
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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.
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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.
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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.
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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.
Rolf Muller - One of the best experts on this subject based on the ideXlab platform.
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Steroid biosynthesis in prokaryotes: identification of myxobacterial steroids and cloning of the first bacterial 2,3(S)‐oxidosqualene cyclase from the myxobacterium Stigmatella aurantiaca
Molecular Microbiology, 2003Co-Authors: Helge B. Bode, Silke C Wenzel, Bernd Zeggel, Barbara Silakowski, Hans Reichenbach, Rolf MullerAbstract:Summary Steroids, such as cholesterol, are synthesized in almost all eukaryotic cells, which use these triterpenoid lipids to control the fluidity and flexibility of their cell membranes. Bacteria rarely synthesize such tetracyclic compounds but frequently replace them with a different class of triterpenoids, the pentacyclic hopanoids. The intriguing mechanisms involved in triterpene biosynthesis have attracted much attention, resulting in extensive studies of squalene-hopene cyclase in bacteria and (S)-2,3-oxidosqualene cyclases in eukarya. Nevertheless, almost nothing is known about steroid biosynthesis in bacteria. Only three steroid-synthesizing bacterial species have been identified before this study. Here, we report on a variety of sterol-producing myxobacteria. Stigmatella aurantiaca is shown to produce Cycloartenol, the well-known first cyclization product of steroid biosynthesis in plants and algae. Additionally, we describe the cloning of the first bacterial steroid biosynthesis gene, cas, encoding the Cycloartenol synthase (Cas) of S. aurantiaca. Mutants of cas generated via site-directed mutagenesis do not produce the compound. They show neither growth retardation in comparison with wild type nor any increase in ethanol sensitivity. The protein encoded by cas is most similar to the Cas proteins from several plant species, indicating a close evolutionary relationship between myxobacterial and eukaryotic steroid biosynthesis.
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steroid biosynthesis in prokaryotes identification of myxobacterial steroids and cloning of the first bacterial 2 3 s oxidosqualene cyclase from the myxobacterium stigmatella aurantiaca
Molecular Microbiology, 2003Co-Authors: Helge B. Bode, Silke C Wenzel, Bernd Zeggel, Barbara Silakowski, Hans Reichenbach, Rolf MullerAbstract:Summary Steroids, such as cholesterol, are synthesized in almost all eukaryotic cells, which use these triterpenoid lipids to control the fluidity and flexibility of their cell membranes. Bacteria rarely synthesize such tetracyclic compounds but frequently replace them with a different class of triterpenoids, the pentacyclic hopanoids. The intriguing mechanisms involved in triterpene biosynthesis have attracted much attention, resulting in extensive studies of squalene-hopene cyclase in bacteria and (S)-2,3-oxidosqualene cyclases in eukarya. Nevertheless, almost nothing is known about steroid biosynthesis in bacteria. Only three steroid-synthesizing bacterial species have been identified before this study. Here, we report on a variety of sterol-producing myxobacteria. Stigmatella aurantiaca is shown to produce Cycloartenol, the well-known first cyclization product of steroid biosynthesis in plants and algae. Additionally, we describe the cloning of the first bacterial steroid biosynthesis gene, cas, encoding the Cycloartenol synthase (Cas) of S. aurantiaca. Mutants of cas generated via site-directed mutagenesis do not produce the compound. They show neither growth retardation in comparison with wild type nor any increase in ethanol sensitivity. The protein encoded by cas is most similar to the Cas proteins from several plant species, indicating a close evolutionary relationship between myxobacterial and eukaryotic steroid biosynthesis.
Norihiko Misawa - One of the best experts on this subject based on the ideXlab platform.
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Pathway engineering for the production of β-amyrin and Cycloartenol in Escherichia coli—a method to biosynthesize plant-derived triterpene skeletons in E. coli
Applied Microbiology and Biotechnology, 2017Co-Authors: Miho Takemura, Rie Tanaka, Norihiko MisawaAbstract:Cycloartenol is biosynthetically the first sterol skeleton, which is metabolized to phytosterols such as β-sitosterol and stigmasterol. β-Amyrin is the most commonly occurring aglycone skeleton for oleanane-type saponins such as glycyrrhizin and saikosaponins. It has been regarded that these cyclic triterpenes are unable to be produced in Escherichia coli , while no reports are available on their production with E. coli . Here, we describe a method to synthesize triterpene skeletons from higher plants, including Cycloartenol and β-amyrin. We introduced into E. coli the biosynthetic pathway genes from farnesyl diphosphate (FPP) to Cycloartenol or β-amyrin, which contained Arabidopsis ( Arabidopsis thaliana )-derived squalene synthase ( AtSQS ) and squalene epoxidase ( AtSQE ) genes in addition to the Arabidopsis Cycloartenol synthase ( AtCAS1 ) gene, or the β-amyrin synthase ( EtAS ) gene of the petroleum plant Euphorbia tirucalli , along with the isopentenyl diphosphate isomerase ( HpIDI ) gene from a green algae Haematococcus pluvialis . The order of genes, HpIDI , AtSQS , AtSQE , driven by transcriptional read-through from a tac promoter to an rrnB terminator, was crucial for their functional expression in E. coli to produce Cycloartenol or β-amyrin. The co-expression of a bacterial NADPH-regenerating gene ( zwf or gdh ) as well as bacterial redox partner protein genes ( camA and camB , or NsRED and NsFER ) was found to increase the amounts of these triterpenes several fold. The present study could open up opportunities not only to carry out functional analysis of a higher-plant-derived oxidosqualene cyclase ( OSC ) gene in E. coli but also to produce functional triterpenes that originate from medicinal or herbal plants.
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pathway engineering for the production of β amyrin and Cycloartenol in escherichia coli a method to biosynthesize plant derived triterpene skeletons in e coli
Applied Microbiology and Biotechnology, 2017Co-Authors: Miho Takemura, Rie Tanaka, Norihiko MisawaAbstract:Cycloartenol is biosynthetically the first sterol skeleton, which is metabolized to phytosterols such as β-sitosterol and stigmasterol. β-Amyrin is the most commonly occurring aglycone skeleton for oleanane-type saponins such as glycyrrhizin and saikosaponins. It has been regarded that these cyclic triterpenes are unable to be produced in Escherichia coli, while no reports are available on their production with E. coli. Here, we describe a method to synthesize triterpene skeletons from higher plants, including Cycloartenol and β-amyrin. We introduced into E. coli the biosynthetic pathway genes from farnesyl diphosphate (FPP) to Cycloartenol or β-amyrin, which contained Arabidopsis (Arabidopsis thaliana)-derived squalene synthase (AtSQS) and squalene epoxidase (AtSQE) genes in addition to the Arabidopsis Cycloartenol synthase (AtCAS1) gene, or the β-amyrin synthase (EtAS) gene of the petroleum plant Euphorbia tirucalli, along with the isopentenyl diphosphate isomerase (HpIDI) gene from a green algae Haematococcus pluvialis. The order of genes, HpIDI, AtSQS, AtSQE, driven by transcriptional read-through from a tac promoter to an rrnB terminator, was crucial for their functional expression in E. coli to produce Cycloartenol or β-amyrin. The co-expression of a bacterial NADPH-regenerating gene (zwf or gdh) as well as bacterial redox partner protein genes (camA and camB, or NsRED and NsFER) was found to increase the amounts of these triterpenes several fold. The present study could open up opportunities not only to carry out functional analysis of a higher-plant-derived oxidosqualene cyclase (OSC) gene in E. coli but also to produce functional triterpenes that originate from medicinal or herbal plants.
Masaaki Shibuya - One of the best experts on this subject based on the ideXlab platform.
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lanosterol synthase in dicotyledonous plants
Plant and Cell Physiology, 2006Co-Authors: Masashi Suzuki, Toshiya Muranaka, Ting Xiang, Kiyoshi Ohyama, Hikaru Seki, Hiroaki Hayashi, Yuji Katsube, Tetsuo Kushiro, 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.
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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.
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differential expression of three oxidosqualene cyclase mrnas in glycyrrhiza glabra
Biological & Pharmaceutical Bulletin, 2004Co-Authors: Hiroaki Hayashi, Masaaki Shibuya, Pengyu Huang, Satoko Takada, Megumi Obinata, Kenichiro Inoue, 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.
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molecular cloning of pea cdna encoding Cycloartenol synthase and its functional expression in yeast
Biological & Pharmaceutical Bulletin, 1997Co-Authors: Masayo Morita, Masaaki Shibuya, Ushio Sankawa, Yutaka EbizukaAbstract:The cDNA encoding Cycloartenol synthase [EC 5.4.99.8] has been isolated from pea seedling by an efficient PCR using sets of degenerate primers based on the highly conserved sequences of the known 2, 3-oxidosqualene cyclase cDNAs. The obtained cDNA contains a 2271-bp open reading frame and is encoding a predicted protein of 757 amino acids with high homology (81%) to Arabidopsis thaliana Cycloartenol synthase. The PCR-amplified open reading frame (ORF) has been inserted into pYES2, an expression vector in yeast, under the control of galactose-inducible promoter. Significant Cycloartenol synthase activity has been found in the homogenate of the yeast transformed with the plasmid containing PCR-amplified ORF.
