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.

• Beta-amyrin synthase--cloning of oxidosqualene cyclase that catalyzes the formation of the most popular triterpene among higher plants.

  Kushiro T., Shibuya M., Ebizuka Y.

  Beta-amyrin, a typical pentacyclic triterpene having an oleanane skeleton, is one of the most commonly occuring triterpenes in nature and is biosynthesized from (3S)-2,3-oxidosqualene. The enzyme, beta-amyrin synthase, catalyzing the cyclization of oxidosqualene into beta-amyrin, generates five ri ... >> More

  Beta-amyrin, a typical pentacyclic triterpene having an oleanane skeleton, is one of the most commonly occuring triterpenes in nature and is biosynthesized from (3S)-2,3-oxidosqualene. The enzyme, beta-amyrin synthase, catalyzing the cyclization of oxidosqualene into beta-amyrin, generates five rings and eight asymmetric centers in a single transformation. A homology-based PCR method was attempted to obtain the cDNA of this enzyme from the hairy root of Panax ginseng which produces oleanane saponins together with dammarane-type saponins. Two sets of degenerate oligonucleotide primers were designed at the regions which are highly conserved among known oxidosqualene cyclases (OSCs). Nested PCRs using these primers successfully amplified the core fragment which revealed the presence of two OSC clones PNX and PNY. Specific amplification of each clone by 3'-RACE and 5'-RACE was carried out to obtain the whole sequences. The two clones exhibited 60% amino acid identity to each other. A full-length clone of PNY was ligated into the yeast expression vector pYES2 under the GAL1 promoter to give pOSC(PNY). Beta-amyrin production was observed with the mutant yeast lacking lanosterol synthase, transformed by this plasmid. The sequence of pOSC(PNY) contains an open reading frame of 2289 nucleotides which codes for 763 amino acids with a predicted molecular mass of 88 kDa. Sequence comparison with other OSCs showed a high level of similarity with lanosterol, cycloartenol and lupeol synthases. The other clone, pOSC(PNX), was shown to be cycloartenol synthase by similar expression in yeast. The present studies have revealed that distinct OSC exists for triterpene formation in higher plants, and the high level of similarity with cycloartenol synthase indicates close evolutional relationship between sterol and triterpene biosynthesis. << Less

  Eur. J. Biochem. 256:238-244(1998) [PubMed] [EuropePMC]

• Functional characterization of triterpene synthases in <i>Cibotium barometz</i>.

  Ji Z., Fan B., Chen Y., Yue J., Chen J., Zhang R., Tong Y., Liu Z., Liang J., Duan L.

  <i>Cibotium barometz</i> (Linn.) J. Sm., a tree fern in the Dicksoniaceae family, is an economically important industrial exported plant in China and widely used in Traditional Chinese Medicine. <i>C. barometz</i> produces a range of bioactive triterpenes and their metabolites. However, the biosyn ... >> More

  <i>Cibotium barometz</i> (Linn.) J. Sm., a tree fern in the Dicksoniaceae family, is an economically important industrial exported plant in China and widely used in Traditional Chinese Medicine. <i>C. barometz</i> produces a range of bioactive triterpenes and their metabolites. However, the biosynthetic pathway of triterpenes in <i>C. barometz</i> remains unknown. To clarify the origin of diverse triterpenes in <i>C. barometz</i>, we conducted <i>de novo</i> transcriptome sequencing and analysis of <i>C. barometz</i> rhizomes and leaves to identify the candidate genes involved in <i>C. barometz</i> triterpene biosynthesis. Three <i>C. barometz</i> triterpene synthases (CbTSs) candidate genes were obtained. All of them were highly expressed in <i>C. barometz</i> rhizomes, consisting of the accumulation pattern of triterpenes in <i>C. barometz</i>. To characterize the function of these CbTSs, we constructed a squalene- and oxidosqualene-overproducing yeast chassis by overexpressing all the enzymes in the MVA pathway under the control of GAL-regulated promoter and disrupted the GAL80 gene in <i>Saccharomyces cerevisiae</i> simultaneously. Heterologous expressing CbTS1, CbTS2, and CbTS3 in engineering yeast strain produced cycloartenol, dammaradiene, and diploptene, respectively. Phylogenetic analysis revealed that CbTS1 belongs to oxidosqualene cyclase, while CbTS2 and CbTS3 belong to squalene cyclase. These results decipher enzymatic mechanisms underlying the origin of diverse triterpene in <i>C. barometz</i>. << Less

  Synth Syst Biotechnol 8:437-444(2023) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• 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.