Cloning of a Sesquiterpene Cyclase and Its Functional Expression by Domain Swapping Strategy

Sesquiterpene cyclase, the first committed step enzyme from the general isoprenoid building block farnesyl pyrophosphate (FPP) for the synthesis of phytoalexin capsidiol, was isolated from the UV-C treated leaves of Capsicum annuum. This sesquiterpene cyclase, termed as CASC2 showing 77% amino acid identity with the previously cloned sesquiterpene cyclase CASC1, was composed of 560 amino acids with a calculated molecular mass of 64,907. The mRNA expression pattern of CASC2 was very similar to that of CASC1 during the time course of UV-C irradiated leaves of pepper on RNA blot analysis by using each specific probe. The heterologous expression in Escherichia coli using the CASC2 full length failed; however the chimeric construct of CASC2 in which the amino terminal 164 amino acid substituted by the equivalent portion of either CASC1 or tobacco sesquiterpene cyclase was capable of expressing the functional sesquiterpene cyclase activities. The radio-labeled enzymatic products catalyzed by the partially purified chimeric CASC2 were comigrated with authentic radio-labeled sesquiterpene on thin layer chromatography.     

牛樟芝倍半萜合酶基因克隆及在不同培养基中的表达

牛樟芝(Antrodia camphorata)是一种珍稀食药用菌,能产生具有抗癌活性的倍半萜类化合物。对牛樟芝基因组进行分析,获得倍半萜合酶基因序列并设计特异引物,提取在Glu培养基(麦芽浸粉6 g/L,酵母提取物3 g/L,葡萄糖40 g/L)上生长的牛樟芝菌丝体的RNA,利用RT-PCR技术克隆得到倍半萜合酶基因AcTPS1。AcTPS1基因c DNA全长为969 bp,编码323个氨基酸,根据系统进化树可知AcTPS1氨基酸序列与其他9种真菌倍半萜合酶聚为一类。AcTPS1拥有典型倍半萜合酶的结构域(RRSRSATAEAYACFIW),之后检测AcTPS1在不同培养基上的菌丝中的表达结果显示不同碳源中,只有葡萄糖作为碳源时该基因表达,不同氮源中,以番茄浸粉和酪蛋白胨为氮源时该基因表达。说明AcTPS1是一类诱导型表达的基因。为利用发酵培养以及异源表达手段获得牛樟芝活性化合物提供参考。     

Sesquiterpene Glucosides from Chloranthus japonicus Sieb

A new lindenane sesquiterpene glucoside named yinxiancaoside A (1), a new, rare bidesmosidic megastigmane sesquiterpene glucoside named yinxiancaoside B (5), and three known sesquiterpene glucosides, chloranoside A (2), pisumionoside (3), and sarcaglaboside A (4), were isolated from the whole plant of Chloranthus japonicus Sieb. The structures of the new compounds were established by an extensive study of their spectral data, especially 1D- and 2D-NMR. The cytotoxic activity of the isolated compounds against human hepatoma (Hepg-2), human ovarian carcinoma (OV420), and human breast cancer (MCF-7) cells was investigated.     

A non-synonymous nucleotide substitution can account for one evolutionary route to sesquiterpene synthase activity in the TPS-b subgroup

Plant sesquiterpene and hemiterpene synthases in the monoterpene synthase dominated TPS-b subgroup are thought to have evolved independently from a monoterpene synthase ancestor. A TPS-b sesquiterpene synthase from apple (MdAFS1), which predominantly produces α-farnesene, can also synthesize the monoterpene (E)-β-ocimene. The dual activity offered a functional link to an ancestral MdAFS1 enzyme and a rational basis for investigation of the evolution of TPS-b sesquiterpene enzymes. Protein modelling and mutagenesis analysis of the MdAFS1 active site identified a non-synonymous nucleotide substitution that could account for the requisite shift in substrate specificity necessary for the emergence of its sesquiterpene activity during the evolution of the TPS-b enzymes.     

朝鲜蓟叶中一个新的倍半萜内酯

从朝鲜蓟（Cynarascolyrnus）叶中分离得到2个倍半萜内酯,其中一个是新化合物,通过波谱学方法确定其结构为3β,8α,11α,13-四羟基-10（14）-愈创木烯-1α,4β,5α,6β氢-6α,12-内酯（1）。     

Chemotaxonomy of Pseudowintera: Sesquiterpene dialdehyde variants are species markers

Two sesquiterpene dialdehydes, the 1β-E-coumaroyl-5α-hydroxypolygodial plus the known 1β-E-cinnamoyl-5α-hydroxypolygodial, were isolated from the recently described species Pseudowintera insperata. This discovery is a further example of the rare sesquiterpene dialdehyde coumarate/cinnamate combination being found exclusively in the family Winteraceae. Another sesquiterpene dialdehyde, isopaxidal, with the rare rearranged drimane skeleton, was isolated from Pseudowintera axillaris. The sesquiterpene dialdehyde contents of leaves of 25 individual plants of the four Pseudowintera species, all endemic to New Zealand, were measured by HPLC. P. insperata individuals all had high levels (3.0-6.9% of leaf dry wt.) of the coumarate, P. axillaris had high levels (2.2-6.9%) of paxidal, and Pseudowintera colorata from different areas of New Zealand contained varying levels of polygodial (1.4-2.9%) and 9-deoxymuzigadial (0-2.9%). Therefore the sesquiterpene dialdehydes are good species markers.     

Sesquiterpene lactones in a hairy root culture of Cichorium intybus

A transformed root culture of Cichorium intybus L. (Asteraceae) was found to produce sesquiterpene lactones of guaiane and germacrane type. Lactucopicrin, 8-desoxylactucin and three sesquiterpene lactone glycosides: crepidiaside B, sonchuside A and ixerisoside D were isolated from the roots. The yield of 8-desoxylactucin reached 0.03 g l(-1) at the early stationary phase of the culture.     

Inhibition of inosine monophosphate dehydrogenase by sesquiterpene lactones

Inosine monophosphate (IMP) dehydrogenase had previously been determined to be a likely target enzyme for the sesquiterpene lactones, a class of potential anti-neoplastic drugs. IMP dehydrogenase was purified approx. 770-fold from the P-388 lymphocytic leukemia tumor cell line. The Km values for the substrates, IMP and NAD, were determined to be 12 microM and 25 microM, respectively. Xanthine monophosphate (XMP) was shown to be a competitive inhibitor with a Ki of 67 microM. Mycophenolic acid gave mixed-type inhibition with a Ki of 8 nM for the noncompetitive component and a Ki of 2 nM for the competitive component. Dissociation constants (Kd) and rate constants for inhibition of IMP dehydrogenase by nine different sesquiterpene lactones were determined. The highest Kd was seen with 2,3-dihydrohelenalin while the lowest Kd was observed with bis-helenalinyl malonate. Binding of the drugs by IMP dehydrogenase increased as the size of the drug increased. Also, changes in structure at position 6 had a relatively large effect on the Kd. There was no correlation with hydrophobicity, as determined by octanol/water partition. The first-order rate constants for the reaction of the sesquiterpene lactones with IMP dehydrogenase (k1) and the second-order rate constants for the reaction of the sesquiterpene lactones with glutathione (k2) were also determined. The rate constants for most of the sesquiterpene lactones with the alpha-methylene-gamma-lactone moiety were similar and were approximately twice as great as the rate constants for those sesquiterpene lactones with only the alpha, beta-unsaturated cyclopentenone ring. Microlenin had approximately 5-times the reactivity of the other sesquiterpene lactones towards IMP dehydrogenase, but had approximately the same reactivity towards glutathione, suggesting that it was bound to the enzyme in a way which facilitated its reaction with one or more essential sulfhydryls. The same procedure was used for a series of N-substituted maleimide compounds with the N-substituent ranging in size from a methyl group to a benzyl group. The binding of the maleimide compounds was generally tighter than for the sesquiterpene lactones and there was an increase in binding with size.     

Dynamic evolution of herbivore-induced sesquiterpene biosynthesis in sorghum and related grass crops

Sorghum (Sorghum bicolor) plants damaged by insects emit a blend of volatiles, predominantly sesquiterpenes, that are implicated in attracting natural enemies of the attacking insects. To characterize sesquiterpene biosynthesis in sorghum, seven terpene synthase (TPS) genes, SbTPS1 through SbTPS7, were identified based on their evolutionary relatedness to known sesquiterpene synthase genes from maize and rice. While SbTPS6 and SbTPS7 encode truncated proteins, all other TPS genes were determined to encode functional sesquiterpene synthases. Both SbTPS1 and SbTPS2 produced the major products zingiberene, β-bisabolene and β-sesquiphellandrene, but with opposite ratios of zingiberene to β-sesquiphellandrene. SbTPS3 produced (E)-α-bergamotene and (E)-β-farnesene. SbTPS4 formed (E)-β-caryophyllene as the major product. SbTPS5 produced mostly (E)-α-bergamotene and (Z)-γ-bisabolene. Based on the genome sequences of sorghum, maize and rice and the sesquiterpene synthase genes they contain, collinearity analysis identified the orthologs of sorghum sesquiterpene synthase genes, except for SbTPS4, in maize and rice. Phylogenetic analysis implied that SbTPS1, SbTPS2 and SbTPS3, which exist as tandem repeats, evolved as a consequence of local gene duplication in a lineage-specific manner. Structural modeling and site-directed mutagenesis experiments revealed that three amino acids in the active site play critical roles in defining product specificity of SbTPS1, SbTPS2, SbTPS3 and their orthologs in maize and rice. The naturally occurring functional variations of sesquiterpene synthases within and between species suggest that multiple mechanisms, including lineage-specific gene duplication, subfunctionalization, neofunctionalization and pseudogenization of duplicated genes, have all played a role in the dynamic evolution of insect-induced sesquiterpene biosynthesis in grasses.     

Analysis of sesquiterpene lactones in Eupatorium lindleyanum by HPLC‐PDA‐ESI‐MS/MS

Introduction – The aerial part Eupatorium lindleyanum is commonly used as an antipyretic and detoxicant clinically in traditional Chinese medicine. Our previous research showed that germacrane sesquiterpene lactones were its main active constituents, so the development of rapid and accurate methods for the identification of the sesquiterpene lactones is of great significance. Objective – To develop an HPLC‐PDA‐ESI‐MS/MS method capable for simple and rapid analysis of germacrane sesquiterpene lactones in the aerial part E. lindleyanum. Methodology – High‐performance liquid chromatography‐photodiode array detection‐electrospray ionization‐tandem mass spectrometry was used to analyze germacrane sesquiterpene lactones of Eupatorium lindleyanum. The fragmentation behavior of germacrane sesquiterpene lactones in a Micromass Q/TOF Mass Spectrometer was discussed, and 9 germacrane sesquiterpene lactones were identified by comparison of their characteristic data of HPLC and MS analyses with those obtained from reference compounds. Results – The investigated germacrane sesquiterpene lactones were identified as eupalinolides C (1), 3β‐acetoxy‐8β‐(4′‐hydroxy‐tigloyloxy)‐14‐hydroxy‐costunolide (2), eupalinolides A (3), eupalinolides B (4), eupalinolides E (5), 3β‐acetoxy‐8β‐(4′‐oxo‐tigloyloxy)‐14‐hydroxy‐heliangolide (6), 3β‐acetoxy‐8β‐(4′‐oxo‐ tigloyloxy)‐14‐hydroxy‐costunolide (7), hiyodorilactone B (8), and 3β‐acetoxy‐8β‐(4′‐hydroxy‐tigloyloxy)‐ costunolide (9). Compounds 6, 7 and 9 were reported for the first time. Conclusion – HPLC‐PDA‐ESI‐MS/MS provides a new powerful approach to identify germacrane sesquiterpene lactones in E. lindleyanum rapidly and accurately. Copyright © 2009 John Wiley & Sons, Ltd.     

Sesquiterpene constituents in Petasites hybridus

The essential oil of the rhizomes of Petasites hybridus (Asteraceae) was investigated by gas chromatography, mass spectrometry, 1- and 2-dimensional NMR techniques and chemical correlations. Two new sesquiterpene hydrocarbons, petasitene and pethybrene, could be identified. Petasitene is the parent sesquiterpene hydrocarbon to the known norsesquiterpene albene. The absolute configuration of petasitene could be assigned by conversion of natural albene to petasitene by partial synthesis. Pethybrene is a tricyclic sesquiterpene hydrocarbon, which rearranges to the structurally related alpha-isocomene under acidic conditions. Several sesquiterpenes were isolated from the hydrodistillation products of Petasites hybridus and investigated by spectroscopic methods and chemical correlations     

Nuphar alkaloids with immediately apoptosis-inducing activity from Nuphar pumilum and their structural requirements for the activity

The methanolic extract and its alkaloid fraction from the rhizomes of Nuphar pumilum showed cytotoxic effects on human leukemia cell (U937), mouse melanoma cell (B16F10), and human fibroblast (HT1080). Dimeric sesquiterpene thioalkaloids with the 6-hydroxyl group (6-hydroxythiobinupharidine, 6,6'-dihydroxythiobinupharidine, 6-hydroxythionuphlutine B) showed substantial cytotoxic activity at a concentration of 10 microM, but dimeric sesquiterpene thioalkaloids lacking the 6-hydroxyl group (thiobinupharidine, thionuphlutine B, 6'-hydroxythionuphlutine B, neothiobinupharidine, thionuphlutine B beta-sulfoxide, and neothiobinupharidine beta-sulfoxide) and monomeric sesquiterpene alkaloids (nupharidine, 7-epideoxynupharidine, and nupharolutine) showed weak activity. Next, apoptosis-inducing activity of a principal active constituent, 6-hydroxythiobinupharidine, on U937 was examined using morphological observation and DNA fragmentation assay (TUNEL method). Apoptosis of U937 was immediately observed within 1 h after treatment of 6-hydroxythiobinupharidine at 2.5-10 microM.     

Unusual features of a recombinant apple alpha-farnesene synthase

A recombinant alpha-farnesene synthase from apple (Malus x domestica), expressed in Escherichia coli, showed features not previously reported. Activity was enhanced 5-fold by K(+) and all four isomers of alpha-farnesene, as well as beta-farnesene, were produced from an isomeric mixture of farnesyl diphosphate (FDP). Monoterpenes, linalool, (Z)- and (E)-beta-ocimene and beta-myrcene, were synthesised from geranyl diphosphate (GDP), but at 18% of the optimised rate for alpha-farnesene synthesis from FDP. Addition of K(+) reduced monoterpene synthase activity. The enzyme also produced alpha-farnesene by a reaction involving coupling of GDP and isoprenyl diphosphate but at <1% of the rate with FDP. Mutagenesis of active site aspartate residues removed sesquiterpene, monoterpene and prenyltransferase activities suggesting catalysis through the same active site. Phylogenetic analysis clusters this enzyme with isoprene synthases rather than with other sesquiterpene synthases, suggesting that it has evolved differently from other plant sesquiterpene synthases. This is the first demonstration of a sesquiterpene synthase possessing prenyltransferase activity.     

Characterization of a novel sesquiterpene cyclase involved in (+)-caryolan-1-ol biosynthesis in Streptomyces griseus

Most terpenoids have been isolated from plants and fungi and only a few from bacteria. However, an increasing number of genome sequences indicate that bacteria possess a variety of terpenoid cyclase genes. We characterized a sesquiterpene cyclase gene (SGR2079, named gcoA) found in Streptomyces griseus. When expressed in Streptomyces lividans, gcoA directed production of a sesquiterpene, isolated and determined to be (+)-caryolan-1-ol using spectroscopic analyses. (+)-Caryolan-1-ol was also detected in the crude cell lysate of wild-type S. griseus but not in a gcoA knockout mutant, indicating that GcoA is a genuine (+)-caryolan-1-ol synthase. Enzymatic properties were characterized using N-terminally histidine-tagged GcoA, produced in Escherichia coli. As expected, incubation of the recombinant GcoA protein with farnesyl diphosphate yielded (+)-caryolan-1-ol. However, a small amount of another sesquiterpene was also detected. This was identified as the bicyclic sesquiterpene hydrocarbon (+)-β-caryophyllene by comparison with an authentic sample using GC-MS. Incorporation of a deuterium atom into the C-9 methylene of (+)-caryolan-1-ol in an in vitro GcoA reaction in deuterium oxide indicated that (+)-caryolan-1-ol was synthesized by a proton attack on the C-8/C-9 double bond of (+)-β-caryophyllene. Several β-caryophyllene synthases have been identified from plants, but these cannot synthesize caryolan-1-ol. Although caryolan-1-ol has been isolated previously from several plants, the enzyme responsible for its biosynthesis has not been identified previously. GcoA is thus the first known caryolan-1-ol synthase. Isolation of caryolan-1-ol from microorganisms is unprecedented.     

Volatile components of selected species of the liverwort genera Frullania and Schusterella (Frullaniaceae) from New Zealand,Australia and South America: a chemosystematic approach

The volatile components of 25 taxa of the liverwort family Frullaniaceae from New Zealand, Australia and South America have been analyzed by GC-MS. The present Frullania species are chemically divided into five major types: (1) sesquiterpene lactones, (2) sesquiterpene lactones-bibenzyls, (3) bibenzyls, (4) 2-alkanone and (5) triterpene types; the latter two chemo-types are newly proposed for the genus. Schusterella chevalierii, belonging to the Frullaniaceae, is closely related chemically to the sesquiterpene lactone type of the Frullania species since it elaborates two eudesmanolides, beta-cyclocostunolide and dihydro-beta-cyclocostunolide as major components.     

Functional characterization of four sesquiterpene synthases from Ricinus communis (Castor bean)

Genome sequence analysis of Ricinus communis has indicated the presence of at least 22 putative terpene synthase (TPS) genes, 13 of which appear to encode sesquiterpene synthases (SeTPSs); however, no SeTPS genes have been isolated from this plant to date. cDNAs were recovered for six SeTPS candidates, and these were subjected to characterization in vivo and in vitro. The RcSeTPS candidates were expressed in either Escherichia coli or Saccharomyces cerevisiae strains with engineered sesquiterpene biosynthetic pathways, but only two (RcSeTPS1 and RcSeTPS7) produced detectable levels of product. In order to check whether the engineered microbial hosts were adequately engineered for sesquiterpene production, a selection of SeTPS genes was chosen from other plant species and demonstrated consistently high sesquiterpene titers. Activity could be demonstrated in vitro for two of the RcSeTPS candidates (RcSeTPS5 and RcSeTPS10) that were not observed to be functional in our microbial hosts. RcSeTPS1 produced two products, (−)-α-copaene and (+)-δ-cadinene, while RcSeTPS7 produced a single product, (E, E)-α-farnesene. Both RcSeTPS5 and RcSeTPS10 produced multiple sesquiterpenes.