Germacrene D (original) (raw)
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An analysis of characterized plant sesquiterpene synthases
Phytochemistry, 2019
Plants exhibit a vast array of sesquiterpenes, C15 hydrocarbons which often function as herbivore-repellents or pollinator-attractants. These in turn are produced by a diverse range of sesquiterpene synthases. A comprehensive analysis of these enzymes in terms of product specificity has been hampered by the lack of a centralized resource of sufficient functionally annotated sequence data. To address this, we have gathered 262 plant sesquiterpene synthase sequences with experimentally characterized products. The annotated enzyme sequences allowed for an analysis of terpene synthase motifs, leading to the extension of one motif and recognition of a variant of another. In addition, putative terpene synthase sequences were obtained from various resources and compared with the annotated sesquiterpene synthases. This analysis indicated regions of terpene synthase sequence space which so far are unexplored experimentally. Finally, we present a case describing mutational studies on residues...
Isolation of Sesquiterpene Synthase Homolog from Panax ginseng C.A. Meyer
Journal of Ginseng Research, 2010
Sesquiterpenes are found naturally in plants and insects as defensive agents or pheromones. They are produced in the cytosolic acetate/mevalonate pathway for isoprenoid biosynthesis. The inducible sesquiterpene synthases (STS), which are responsible for the transformation of the precursor farnesyl diphosphate, appear to generate very few olefinic products that are converted to biologically active metabolites. In this study, we isolated the STS gene from Panax ginseng C.A. Meyer, designated PgSTS, and investigated the correlation between its expression and various abiotic stresses using real-time PCR. PgSTS cDNA was observed to be 1,883 nucleotides long with an open reading frame of 1,707 bp, encoding a protein of 568 amino acids. The molecular mass of the mature protein was determined to be 65.5 kDa, with a predicted isoelectric point of 5.98. A GenBank BlastX search revealed the deduced amino acid sequence of PgSTS to be homologous to STS from other plants, with the highest similarity to an STS from Lycopersicon hirsutum (55% identity, 51% similarity). Real-time PCR analysis showed that different abiotic stresses triggered significant induction of PgSTS expression at different time points.
Surrogate Splicing for Functional Analysis of Sesquiterpene Synthase Genes1(w
A method for the recovery of full-length cDNAs from predicted terpene synthase genes containing introns is described. The approach utilizes Agrobacterium-mediated transient expression coupled with a reverse transcription-polydeoxyribonucleotide chain reaction assay to facilitate expression cloning of processed transcripts. Subsequent expression of intronless cDNAs in a suitable prokaryotic host provides for direct functional testing of the encoded gene product. The method was optimized by examining the expression of an intron-containing b-glucuronidase gene agroinfiltrated into petunia (Petunia hybrida) leaves, and its utility was demonstrated by defining the function of two previously uncharacterized terpene synthases. A tobacco (Nicotiana tabacum) terpene synthase-like gene containing six predicted introns was characterized as having 5-epi-aristolochene synthase activity, while an Arabidopsis (Arabidopsis thaliana) gene previously annotated as a terpene synthase was shown to possess a novel sesquiterpene synthase activity for a-barbatene, thujopsene, and b-chamigrene biosynthesis. ; fax 859-257-7125.
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, (À)-a-copaene and (+)-d-cadinene, while RcSeTPS7 produced a single product, (E, E)-a-farnesene. Both RcSeTPS5 and RcSeTPS10 produced multiple sesquiterpenes.
Proceedings of the National Academy of Sciences of the United States of America, 1998
Germacrene C was found by GC-MS and NMR analysis to be the most abundant sesquiterpene in the leaf oil of Lycopersicon esculentum cv. VFNT Cherry, with lesser amounts of germacrene A, guaia-6,9-diene, germacrene B, beta-caryophyllene, alpha-humulene, and germacrene D. Soluble enzyme preparations from leaves catalyzed the divalent metal ion-dependent cyclization of [1-3H]farnesyl diphosphate to these same sesquiterpene olefins, as determined by radio-GC. To obtain a germacrene synthase cDNA, a set of degenerate primers was constructed based on conserved amino acid sequences of related terpenoid cyclases. With cDNA prepared from leaf epidermis-enriched mRNA, these primers amplified a 767-bp fragment that was used as a hybridization probe to screen the cDNA library. Thirty-one clones were evaluated for functional expression of terpenoid cyclase activity in Escherichia coli by using labeled geranyl, farnesyl, and geranylgeranyl diphosphates as substrates. Nine cDNA isolates expressed se...
Cloning of a sesquiterpene synthase from Lavandula x intermedia glandular trichomes
Planta, 2013
The essential oil (EO) of Lavandula is dominated by monoterpenes, but can also contain small amounts of sesquiterpenes, depending on species and environmental conditions. For example, the sesquiterpene 9-epi-caryophyllene can make up to 8 % of the EO in a few species, including those commercially propagated for EO production. Here, we report the cloning and functional characterization of 9-epi-caryophyllene synthase (LiCPS) from the glandular trichomes of Lavandula x intermedia, cv. Grosso. The 1,617 bp open reading frame of LiCPS, which did not encode a transit peptide, was expressed in Escherichia coli and the recombinant protein purified by Ni-NTA agarose affinity chromatography. The ca. 60 kDa recombinant protein specifically converted farnesyl diphosphate to 9-epi-caryophyllene. LiCPS also produced a few monoterpenes when assayed with the monoterpene precursor geranyl diphosphate (GPP), but--unlike most monoterpene synthases--was not able to derive detectable amounts of any pro...
Planta, 2000
The cotton (+)-d-cadinene synthase, a sesquiterpene cyclase, is encoded by a complex gene family which, based on homology, can be divided into two subfamilies: cad1-A and cad1-C. Southern blots revealed several members of the cad1-C subfamily, and a single member of the cad1-A subfamily, in the diploid Gossypium arboreum genome. One of the cad1-C genes, cad1-C3, was isolated from this species. According to reverse transcriptase-polymerase chain reaction, transcripts of both cad1-C and cad1-A genes appeared in roots from the second day post germination and in 1-dold cotyledons, whereas the transcription levels were too low to be detected in the hypocotyls. Initially, sesquiterpene cyclase activities were found to be high in the seedlings, then dropped in aerial organs but increased in roots during development. Sesquiterpene aldehyde contents followed the same pattern. In fully developed plants, the transcripts of cad1-C were detected in stems, leaves and pericarps, as well as in the sepals and petals 3 d before anthesis, but not at the day of anthesis. In contrast, cad1-A transcripts were not detected in any of these aerial organs. The sesquiterpene aldehyde contents increased in petals but decreased in sepals after anthesis. Treatment of G. arboreum stems with a Verticillium dahliae elicitor-preparation activated cad1-A transcription, but a signi®cant level of cad1-C transcripts was detected both before and after elicitation. In G. hirsutum cv. GL-5, a glandless cultivar, the cad1-C gene was activated by the same fungal elicitor, followed by the synthesis of the sesquiterpene cyclase, and accumulation of sesquiterpene aldehydes. The cad1 gene expression during development and in response to elicitation, as well as the spatial and temporal pattern of sesquiterpene biosynthesis, constitute a chemical defense machinery in cotton plants.
2013
Plants have evolved the capacity to synthesize a myriad of specialized metabolites which enhance their fitness in specific living conditions. These compounds are also widely utilized for human purposes. Elucidating the enzymes in plant specialized metabolism has been one of the main forces driving plant biochemistry. The more intriguing question, however, is how these enzymes evolved to acquire their existent functions. The Asteraceae, the largest flowering plant family, is well-known for its enormously diverse and lineage-characteristic contents of sesquiterpene lactones (STLs). Thousands of compounds in this subclass of specialized metabolites have been studied extensively for their structures and valuable bioactivities. However, the details of their metabolism are poorly understood. Studying STLs in the Asteraceae thus improves our knowledge of the biosynthesis of these compounds. Furthermore, the tight links between STLs and the Asteraceae family provide an excellent model to explore enzyme adaptive evolution. My thesis aims to advance our understanding of STL metabolism by focusing on elucidating the enzyme that is responsible for the oxidation of sesquiterpene to sesquiterpene carboxylic acid in the general STL biosynthetic route of the Asteraceae. In lettuce, two cytochrome P450dependent monooxygenase (P450) isoforms responsible for oxidizing the three consecutive oxidations of germacrene A to germacrene A carboxylic acid in the biosynthesis of costunolide were characterized. This was achieved using a combination of genomic and biochemical approaches, and the aid of a metabolically-engineered yeast system. Furthermore, this germacrene A oxidase (GAO) activity was demonstrated to be highly conserved throughout the Asteraceae, even in the phylogenetically basal subfamily Barnadesioideae, which split from the rest of the family at least 50 million years go. Previous studies have characterized an Artemisia annua-specific sesquiterpene oxidase, amorphadiene oxidase (AMO), which is considered to have diverged from an ancestral GAO. The substrate specificity/promiscuity of AMO and GAOs towards each other's natural substrates and seven other non-natural substrates was investigated to test the general hypothesis of enzyme evolution from ancestral promiscuity. The results from these combinatorial biochemistry studies and phylogenetic relations of AMO and GAOs provided deep insights into the evolution of these P450s in the context of the chemical diversity of the Asteraceae. This work would never be possible without the kind supports of many people. First of all, I would like to express my gratitude to my supervisor, Dr. Dae-Kyun Ro, for introducing me to the exciting world of the sunflower family and terpenoids, and for mentoring and inspiring me throughout the years. I would like to thank Dr. Chendanda C. Chinnappa and Mrs. Janaki Chinnappa for encouraging me to get my education at the University of Calgary and making me feel like home in Canada. I want to thank my two Supervisory Committee members, Dr. Sui
Identification, Functional Characterization, and Evolution of Terpene Synthases from a Basal Dicot
Plant Physiology, 2015
Bay laurel (Laurus nobilis) is an agriculturally and economically important dioecious tree in the basal dicot family Lauraceae used in food and drugs and in the cosmetics industry. Bay leaves, with their abundant monoterpenes and sesquiterpenes, are used to impart flavor and aroma to food, and have also drawn attention in recent years because of their potential pharmaceutical applications. To identify terpene synthases (TPSs) involved in the production of these volatile terpenes, we performed RNA sequencing to profile the transcriptome of L. nobilis leaves. Bioinformatic analysis led to the identification of eight TPS complementary DNAs. We characterized the enzymes encoded by three of these complementary DNAs: a monoterpene synthase that belongs to the TPS-b clade catalyzes the formation of mostly 1,8-cineole; a sesquiterpene synthase belonging to the TPS-a clade catalyzes the formation of mainly cadinenes; and a diterpene synthase of the TPS-e/f clade catalyzes the formation of geranyllinalool. Comparison of the sequences of these three TPSs indicated that the TPS-a and TPS-b clades of the TPS gene family evolved early in the evolution of the angiosperm lineage, and that geranyllinalool synthase activity is the likely ancestral function in angiosperms of genes belonging to an ancient TPS-e/f subclade that diverged from the kaurene synthase gene lineages before the split of angiosperms and gymnosperms.