Publications

• Oxidosqualene cyclases from cell suspension cultures of Betula platyphylla var. japonica: molecular evolution of oxidosqualene cyclases in higher plants.

  Zhang H., Shibuya M., Yokota S., Ebizuka Y.

  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 suspen ... >> More

  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 beta-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. << Less

  Biol. Pharm. Bull. 26:642-650(2003) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Arabidopsis thaliana LUP1 converts oxidosqualene to multiple triterpene alcohols and a triterpene diol.

  Segura M.J.R., Meyer M.M., Matsuda S.P.T.

  The Arabidopsis thaliana LUP1 gene encodes an enzyme that converts oxidosqualene to pentacyclic triterpenes. Lupeol and beta-amyrin were previously reported as LUP1 products. Further investigation described here uncovered the additional products germanicol, taraxasterol, psi-taraxasterol, and 3,20 ... >> More

  The Arabidopsis thaliana LUP1 gene encodes an enzyme that converts oxidosqualene to pentacyclic triterpenes. Lupeol and beta-amyrin were previously reported as LUP1 products. Further investigation described here uncovered the additional products germanicol, taraxasterol, psi-taraxasterol, and 3,20-dihydroxylupane. These results suggest that the 80 known C(30)H(50)O compounds that are structurally consistent with being oxidosqualene cyclase products may be derived from fewer than 80 enzymes and that some C(30)H(52)O(2) compounds may be direct cyclization products of oxidosqualene. << Less

  Org. Lett. 2:2257-2259(2000) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Differential expression of three oxidosqualene cyclase mRNAs in Glycyrrhiza glabra.

  Hayashi H., Huang P., Takada S., Obinata M., Inoue K., Shibuya M., Ebizuka Y.

  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) res ... >> More

  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, beta-amyrin synthase and cycloartenol synthase, were compared. The mRNA expression levels of lupeol synthase and beta-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 beta-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 beta-amyrin synthase mRNA and soyasaponin biosynthesis, but down-regulated lupeol synthase mRNA. Furthermore, the addition of gibberellin A(3) down-regulated beta-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. << Less

  Biol. Pharm. Bull. 27:1086-1092(2004) [PubMed] [EuropePMC]

• Triterpene synthases from the Okinawan mangrove tribe, Rhizophoraceae.

  Basyuni M., Oku H., Tsujimoto E., Kinjo K., Baba S., Takara K.

  Oleanane-type triterpene is one of the most widespread triterpenes found in plants, together with the lupane type, and these two types often occur together in the same plant. Bruguiera gymnorrhiza (L.) Lamk. and Rhizophora stylosa Griff. (Rhizophoraceae) are known to produce both types of triterpe ... >> More

  Oleanane-type triterpene is one of the most widespread triterpenes found in plants, together with the lupane type, and these two types often occur together in the same plant. Bruguiera gymnorrhiza (L.) Lamk. and Rhizophora stylosa Griff. (Rhizophoraceae) are known to produce both types of triterpenes. Four oxidosqualene cyclase cDNAs were cloned from the leaves of B. gymnorrhiza and R. stylosa by a homology-based PCR method. The ORFs of full-length clones termed BgbAS (2280 bp, coding for 759 amino acids), BgLUS (2286 bp, coding for 761 amino acids), RsM1 (2280 bp, coding for 759 amino acids) and RsM2 (2316 bp coding for 771 amino acids) were ligated into yeast expression plasmid pYES2 under the control of the GAL1 promoter. Expression of BgbAS and BgLUS in GIL77 resulted in the production of beta-amyrin and lupeol, suggesting that these genes encode beta-amyrin and lupeol synthase (LUS), respectively. Furthermore, RsM1 produced germanicol, beta-amyrin, and lupeol in the ratio of 63 : 33 : 4, whereas RsM2 produced taraxerol, beta-amyrin, and lupeol in the proportions 70 : 17 : 13. This result indicates that these are multifunctional triterpene synthases. Phylogenetic analysis and sequence comparisons revealed that BgbAS and RsM1 demonstrated high similarities (78-93%) to beta-amyrin synthases, and were located in the same branch as beta-amyrin synthase. BgLUS formed a new branch for lupeol synthase that was closely related to the beta-amyrin synthase cluster, whereas RsM2 was found in the first branch of the multifunctional triterpene synthase evolved from lupeol to beta-amyrin synthase. Based on these sequence comparisons and product profiles, we discuss the molecular evolution of triterpene synthases and the involvement of these genes in the formation of terpenoids in mangrove leaves. << Less

  FEBS J. 274:5028-5042(2007) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Cloning and characterization of the Arabidopsis thaliana lupeol synthase gene.

  Herrera J.B.R., Bartel B., Wilson W.K., Matsuda S.P.T.

  A 2274 bp Arabidopsis thaliana cDNA was isolated that encodes a protein 57% identical to cycloartenol synthase from the same organism. The expressed recombinant protein encodes lupeol synthase, which converts oxidosqualene to the triterpene lupeol as the major product. Lupeol synthase is a multifu ... >> More

  A 2274 bp Arabidopsis thaliana cDNA was isolated that encodes a protein 57% identical to cycloartenol synthase from the same organism. The expressed recombinant protein encodes lupeol synthase, which converts oxidosqualene to the triterpene lupeol as the major product. Lupeol synthase is a multifunctional enzyme that forms other triterpene alcohols, including beta-amyrin, as minor products. Sequence analysis suggests that lupeol synthase diverged from cycloartenol synthase after plants diverged from fungi and animals. This evolutionary order is the reason that fungi and animals do not make lupeol. << Less

  Phytochemistry 49:1905-1911(1998) [PubMed] [EuropePMC]

• Two branches of the lupeol synthase gene in the molecular evolution of plant oxidosqualene cyclases.

  Shibuya M., Zhang H., Endo A., Shishikura K., Kushiro T., Ebizuka Y.

  Two new triterpene synthase cDNAs, named as OEW and TRW, were cloned from olive leaves (Olea europaea) and from dandelion roots (Taraxacum officinale), respectively, by the PCR method with primers designed from the conserved sequences found in the known oxidosqualene cyclases. Their ORFs consisted ... >> More

  Two new triterpene synthase cDNAs, named as OEW and TRW, were cloned from olive leaves (Olea europaea) and from dandelion roots (Taraxacum officinale), respectively, by the PCR method with primers designed from the conserved sequences found in the known oxidosqualene cyclases. Their ORFs consisted of 2274 bp nucleotides and coded for 758 amino acid long polypeptides. They shared high sequence identity (78%) to each other, while they showed only about 60% identities to the known triterpene synthases LUPI (lupeol synthase clone from Arabidopsis thaliana) and PNY (beta-amyrin synthase clone from Panax ginseng) at amino acid level. To determine the enzyme functions of the translates, they were expressed in an ERG7 deficient yeast mutant. Accumulation of lupeol in the cells of yeast transformants proved both of these clones code for lupeol synthase proteins. An EST (expression sequence tag) clone isolated from Medicago truncatula roots as a homologue of cycloartenol synthase gene, exhibits high sequence identity (75-77%) to these two lupeol synthase cDNAs, suggesting it to be another lupeol synthase clone. Comparatively low identity (approximately 57%) of LUP1 from Arabidopsis thaliana to either one of these clones leaves LUP1 as a distinct clone among lupeol synthases. From these sequence comparisons, now we propose that two branches of lupeol synthase gene have been generated in higher plants during the course of evolution. << Less

  Eur J Biochem 266:302-307(1999) [PubMed] [EuropePMC]

• Molecular cloning and expression in yeast of 2,3-oxidosqualene-triterpenoid cyclases from Arabidopsis thaliana.

  Husselstein-Muller T., Schaller H., Benveniste P.

  A vast array of triterpenes are found in living organisms in addition to lanosterol and cycloartenol, which are involved in sterol biosynthesis in non-photosynthetic and photosynthetic eukaryotes respectively. The chemical structure of these triterpenes is determined by a single step catalysed by ... >> More

  A vast array of triterpenes are found in living organisms in addition to lanosterol and cycloartenol, which are involved in sterol biosynthesis in non-photosynthetic and photosynthetic eukaryotes respectively. The chemical structure of these triterpenes is determined by a single step catalysed by 2,3-oxidosqualene-triterpene cyclases. The present study describes cloning and functional expression in yeast of several OS-triterpene cyclases. Three Arabidopsis thaliana cDNAs encoding proteins (ATLUP1, ATLUP2, ATPEN1) 57%, 58% and 49% identical to cycloartenol synthase from the same plant were isolated. Expression of these cDNAs in yeast showed that the recombinant proteins catalyse the synthesis of various pentacyclic triterpenes. Whereas ATLUP1 is essentially involved in the synthesis of lupeol, ATLUP2 catalyses the production of lupeol, beta- and alpha-amyrin (in a 15:55:30 ratio). ATLUP2 is therefore a typical multifunctional enzyme. Under the same conditions, ATPEN1 did not lead to any product. Systematic sequencing of the Arabidopsis genome has led to genomic sequences encoding proteins identical to the above triterpene synthases. ATLUPI and ATLUP2 are representative of a small subfamily (A) of at least five genes, whereas ATPEN1 is representative of a subfamily (B) of at least seven genes. The number of introns is characteristic of each subfamily. Whereas genes of family A possess 17 exons and 16 introns, genes of the subfamily B contain 14 exons and 13 introns. The size of each exon is remarkably conserved within each subfamily whereas that of each intron appears to be highly variable. Organization of the genes, sequences and functions of the deduced proteins are discussed in evolutionary terms. << Less

  Plant Mol. Biol. 45:75-92(2001) [PubMed] [EuropePMC]

• Cloning and characterization of a lupeol synthase involved in the synthesis of epicuticular wax crystals on stem and hypocotyl surfaces of Ricinus communis.

  Guhling O., Hobl B., Yeats T., Jetter R.

  Pentacyclic triterpenoids are a large group of secondary metabolites found in many different plant species, either as glycoside conjugates or as aglycones. The latter in many cases accumulate to high amounts in the cuticular wax and hence at the surface of plant organs. In the present work, the cu ... >> More

  Pentacyclic triterpenoids are a large group of secondary metabolites found in many different plant species, either as glycoside conjugates or as aglycones. The latter in many cases accumulate to high amounts in the cuticular wax and hence at the surface of plant organs. In the present work, the cuticle-specific formation of triterpenoids was investigated in Ricinus communis stems, combining analytical and molecular genetic methods. Two phenotypes of castor bean could be distinguished based on the glaucous or glossy appearance of the surfaces of all stem portions including the hypocotyls, and were due to the presence or absence of thread-shaped epicuticular wax crystals, respectively. Comparative studies showed that these crystals are formed by the triperpenoid lupeol, present in high amounts on all stem surfaces. On the hypocotyl portion of stems, lupeol was found to accumulate rapidly during early development of the surface (10-15 days after emergence). Mature hypocotyls of glossy individuals were covered with 12.5 microg/cm2 of wax containing approximately 1% of lupeol, whereas the glaucous phenotype had a wax load of 51.9 microg/cm2 with 56% of lupeol. Two oxidosqualene cyclases from castor bean were cloned, functionally expressed in yeast, and characterized as a cycloartenol synthase (RcCAS) and a lupeol synthase (RcLUS). Phylogenetic analyses revealed that RcLUS is similar to two clades of known lupeol synthases, but also exhibits some similarities with beta-amyrin synthases. Both the organ-specific expression of RcLUS and the expression pattern during hypocotyl development exactly matched the accumulation of cuticular lupeol in castor bean. In contrast, RcCAS was constitutively expressed in all organs at various times. We conclude that the RcLUS enzyme is responsible for formation of the cuticular lupeol, and thus for the characteristic surface properties of R. communis stems. << Less

  Arch. Biochem. Biophys. 448:60-72(2006) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Cloning and characterization of oxidosqualene cyclases from Kalanchoe daigremontiana: enzymes catalyzing up to 10 rearrangement steps yielding friedelin and other triterpenoids.

  Wang Z., Yeats T., Han H., Jetter R.

  The first committed step in triterpenoid biosynthesis is the cyclization of oxidosqualene to polycyclic alcohols or ketones C(30)H(50)O. It is catalyzed by single oxidosqualene cyclase (OSC) enzymes that can carry out varying numbers of carbocation rearrangements and, thus, generate triterpenoids ... >> More

  The first committed step in triterpenoid biosynthesis is the cyclization of oxidosqualene to polycyclic alcohols or ketones C(30)H(50)O. It is catalyzed by single oxidosqualene cyclase (OSC) enzymes that can carry out varying numbers of carbocation rearrangements and, thus, generate triterpenoids with diverse carbon skeletons. OSCs from diverse plant species have been cloned and characterized, the large majority of them catalyzing relatively few rearrangement steps. It was recently predicted that special OSCs must exist that can form friedelin, the pentacyclic triterpenoid whose formation involves the maximum possible number of rearrangement steps. The goal of the present study, therefore, was to clone a friedelin synthase from Kalanchoe daigremontiana, a plant species known to accumulate this triterpenoid in its leaf surface waxes. Five OSC cDNAs were isolated, encoding proteins with 761-779 amino acids and sharing between 57.4 and 94.3% nucleotide sequence identity. Heterologous expression in yeast and GC-MS analyses showed that one of the OSCs generated the steroid cycloartenol together with minor side products, whereas the other four enzymes produced mixtures of pentacyclic triterpenoids dominated by lupeol (93%), taraxerol (60%), glutinol (66%), and friedelin (71%), respectively. The cycloartenol synthase was found expressed in all leaf tissues, whereas the lupeol, taraxerol, glutinol, and friedelin synthases were expressed only in the epidermis layers lining the upper and lower surfaces of the leaf blade. It is concluded that the function of these enzymes is to form respective triterpenoid aglycones destined to coat the leaf exterior, probably as defense compounds against pathogens or herbivores. << Less

  J. Biol. Chem. 285:29703-29712(2010) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.