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Jorg Bohlmann - One of the best experts on this subject based on the ideXlab platform.
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characterization of four terpene Synthase cdnas from methyl jasmonate induced douglas fir pseudotsuga menziesii
Phytochemistry, 2005Co-Authors: Dezene P W Huber, Ryan N Philippe, Kimberleyann Godard, Rona N Sturrock, Jorg BohlmannAbstract:Abstract Numerous terpenoid compounds are present in copious amounts in the oleoresin produced by conifers, especially following exposure to insect or fungal pests. CDNA clones for many terpene Synthases responsible for the biosynthesis of these defense compounds have been recovered from several conifer species. Here, the use of three terpene Synthase sequences as heterologous probes for the discovery of related terpene Synthase genes in Douglas-fir, Pseudotsuga menziesii (Mirbel) Franco (Pinaceae), is reported. Four full-length terpene Synthase cDNAs were recovered from a methyl jasmonate-induced Douglas-fir bark and shoot cDNA library. These clones encode two multi-product monoterpene Synthases [a (−)-α-pinene/(−)-camphene Synthase and a terpinolene Synthase] and two single-product sesquiterpene Synthases [an ( E )-β-farnesene Synthase and a ( E )-γ-bisabolene Synthase].
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functional characterization of nine norway spruce tps genes and evolution of gymnosperm terpene Synthases of the tps d subfamily
Plant Physiology, 2004Co-Authors: Diane M Martin, Jenny Fäldt, Jorg BohlmannAbstract:Constitutive and induced terpenoids are important defense compounds for many plants against potential herbivores and pathogens. In Norway spruce (Picea abies L. Karst), treatment with methyl jasmonate induces complex chemical and biochemical terpenoid defense responses associated with traumatic resin duct development in stems and volatile terpenoid emissions in needles. The cloning of (+)-3-carene Synthase was the first step in characterizing this system at the molecular genetic level. Here we report the isolation and functional characterization of nine additional terpene Synthase (TPS) cDNAs from Norway spruce. These cDNAs encode four monoterpene Synthases, myrcene Synthase, (−)-limonene Synthase, (−)-α/β-pinene Synthase, and (−)-linalool Synthase; three sesquiterpene Synthases, longifolene Synthase, E,E-α-farnesene Synthase, and E-α-bisabolene Synthase; and two diterpene Synthases, isopimara-7,15-diene Synthase and levopimaradiene/abietadiene Synthase, each with a unique product profile. To our knowledge, genes encoding isopimara-7,15-diene Synthase and longifolene Synthase have not been previously described, and this linalool Synthase is the first described from a gymnosperm. These functionally diverse TPS account for much of the structural diversity of constitutive and methyl jasmonate-induced terpenoids in foliage, xylem, bark, and volatile emissions from needles of Norway spruce. Phylogenetic analyses based on the inclusion of these TPS into the TPS-d subfamily revealed that functional specialization of conifer TPS occurred before speciation of Pinaceae. Furthermore, based on TPS enclaves created by distinct branching patterns, the TPS-d subfamily is divided into three groups according to sequence similarities and functional assessment. Similarities of TPS evolution in angiosperms and modeling of TPS protein structures are discussed.
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e β ocimene and myrcene Synthase genes of floral scent biosynthesis in snapdragon function and expression of three terpene Synthase genes of a new terpene Synthase subfamily
The Plant Cell, 2003Co-Authors: Natalia Dudareva, Diane M Martin, Christine M Kish, Natalia Kolosova, Nina M Gorenstein, Barbara Miller, Jenny Fäldt, Jorg BohlmannAbstract:Snapdragon flowers emit two monoterpene olefins, myrcene and (E)-β-ocimene, derived from geranyl diphosphate, in ad-dition to a major phenylpropanoid floral scent component, methylbenzoate. Emission of these monoterpenes is regulated developmentally and follows diurnal rhythms controlled by a circadian clock. Using a functional genomics approach, we have isolated and characterized three closely related cDNAs from a snapdragon petal-specific library that encode two myrcene Synthases (ama1e20 and ama0c15) and an (E)-β-ocimene Synthase (ama0a23). Although the two myrcene Synthases are almost identical (98%), except for the N-terminal 13 amino acids, and are catalytically active, yielding a single monoterpene product, myrcene, only ama0c15 is expressed at a high level in flowers and contributes to floral myrcene emission. (E)-β-Ocimene Synthase is highly similar to snapdragon myrcene Synthases (92% amino acid identity) and produces predominantly (E)-β-ocimene (97% of total monoterpene olefin product) with small amounts of (Z)-β-ocimene and myrcene. These newly isolated snapdragon monoterpene Synthases, together with Arabidopsis AtTPS14 (At1g61680), define a new subfamily of the terpene Synthase (TPS) family designated the Tps-g group. Members of this new Tps-g group lack the RRx8W motif, which is a characteristic feature of the Tps-d and Tps-b monoterpene Synthases, suggesting that the reaction mechanism of Tps-g monoterpene Synthase product formation does not proceed via an RR-dependent isomerization of geranyl diphosphate to 3S-linalyl diphosphate, as shown previously for limonene cyclase. Analyses of tissue-specific, developmental, and rhythmic expression of these monoterpene Synthase genes in snapdragon flowers revealed coordinated regulation of phenylpropanoid and isoprenoid scent production.
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terpenoid secondary metabolism in arabidopsis thaliana cdna cloning characterization and functional expression of a myrcene e beta ocimene Synthase
Archives of Biochemistry and Biophysics, 2000Co-Authors: Jorg Bohlmann, Diane M Martin, Neil J Oldham, Jonathan GershenzonAbstract:Abstract The Arabidopsis genome project has recently reported sequences with similarity to members of the terpene Synthase ( TPS ) gene family of higher plants. Surprisingly, several Arabidopsis terpene Synthase-like sequences ( AtTPS ) share the most identity with TPS genes that participate in secondary metabolism in terpenoid-accumulating plant species. Expression of a putative Arabidopsis terpene Synthase gene, designated AtTPS03, was demonstrated by amplification of a 392-bp cDNA fragment using primers designed to conserved regions of plant terpene Synthases. Using the AtTPS03 fragment as a hybridization probe, a second AtTPS cDNA, designated AtTPS10, was isolated from a jasmonate-induced cDNA library. The partial AtTPS10 cDNA clone contained an open reading frame of 1665 bp encoding a protein of 555 amino acids. Functional expression of AtTPS10 in Escherichia coli yielded an active monoterpene Synthase enzyme, which converted geranyl diphosphate (C 10 ) into the acyclic monoterpenes β-myrcene and ( E )-β-ocimene and small amounts of cyclic monoterpenes. Based on sequence relatedness, AtTPS10 was classified as a member of the TPSb subfamily of angiosperm monoterpene Synthases. Sequence comparison of AtTPS10 with previously cloned monoterpene Synthases suggests independent events of functional specialization of terpene Synthases during the evolution of terpenoid secondary metabolism in gymnosperms and angiosperms. Functional characterization of the AtTPS10 gene was prompted by the availability of Arabidopsis genome sequences. Although Arabidoposis has not been reported to form terpenoid secondary metabolites, the unexpected expression of TPS genes belonging to the TPSb subfamily in this species strongly suggests that terpenoid secondary metabolism is active in the model system Arabidopsis.
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Terpenoid-based defenses in conifers: cDNA cloning, characterization, and functional expression of wound-inducible (E)-alpha-bisabolene Synthase from grand fir (Abies grandis).
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Jorg Bohlmann, John Crock, Reinhard JetterAbstract:(E)-α-Bisabolene Synthase is one of two wound-inducible sesquiterpene Synthases of grand fir (Abies grandis), and the olefin product of this cyclization reaction is considered to be the precursor in Abies species of todomatuic acid, juvabione, and related insect juvenile hormone mimics. A cDNA encoding (E)-α-bisabolene Synthase was isolated from a wound-induced grand fir stem library by a PCR-based strategy and was functionally expressed in Escherichia coli and shown to produce (E)-α-bisabolene as the sole product from farnesyl diphosphate. The expressed Synthase has a deduced size of 93.8 kDa and a pI of 5.03, exhibits other properties typical of sesquiterpene Synthases, and resembles in sequence other terpenoid Synthases with the exception of a large amino-terminal insertion corresponding to Pro81–Val296. Biosynthetically prepared (E)-α-[3H]bisabolene was converted to todomatuic acid in induced grand fir cells, and the time course of appearance of bisabolene Synthase mRNA was shown by Northern hybridization to lag behind that of mRNAs responsible for production of induced oleoresin monoterpenes. These results suggest that induced (E)-α-bisabolene biosynthesis constitutes part of a defense response targeted to insect herbivores, and possibly fungal pathogens, that is distinct from induced oleoresin monoterpene production.
Sean A. Agger - One of the best experts on this subject based on the ideXlab platform.
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identification of sesquiterpene Synthases from nostoc punctiforme pcc 73102 and nostoc sp strain pcc 7120
Journal of Bacteriology, 2008Co-Authors: Sean A. Agger, Thomas R. Hoye, Fernando Lopezgallego, Claudia SchmidtdannertAbstract:Terpenoids constitute the largest group of natural products, constituting over 20,000 described compounds (14). These compounds have a wide range of biological functions and are synthesized by plants and microbes as, for example, pigments, hormones and signaling molecules, antibiotics, antifeedants, or pollinator attractants. Many of these compounds are present in minute quantities and have been an important target for synthetic chemists (22, 52) and metabolic engineers (10, 35, 50). Terpenes are synthesized from linear isoprene diphosphate precursors with various chain lengths, and their enormous structural diversity is the result of the large number of possible enzyme-catalyzed cyclizations and rearrangements that the double-bond-containing isoprene chains can undergo (14, 49). Cyclizations of isoprene diphosphate chains are catalyzed by terpene Synthases (also known as terpene cyclases). Depending on the chain length of their isoprene diphosphate substrates, terpene Synthases can be classified as mono-, sesqui-, or diterpene Synthases, which catalyze the cyclization of geranyl diphosphate (C10), farnesyl diphosphate (FPP; C15), or geranylgeranyl diphosphate, respectively. Well-known examples of cyclic terpenes are the monoterpene 2-methylisoborneol and sesquiterpene geosmin (responsible for the earthy odor of soil and lake water), the trichothecenes (mycotoxins), the sesquiterpene artemisinin (antimalarial drug), and the diterpene paclitaxel (Taxol; anticancer drug). Sesquiterpene Synthases catalyze the cyclization of FPP into any of 300 known hydrocarbon skeletons. These enzymes are typically either monomeric or homodimeric with molecular masses ranging from 40 to 65 kDa and require Mg2+ for catalysis. The Mg2+ ions bind to two metal-binding motifs that are conserved among all terpene Synthases. These metal ions complex to the substrate pyrophosphate, thereby promoting the ionization of the leaving group of FPP, resulting in the generation of a highly reactive allylic cation. The enzyme then controls carbocation migration through the isoprene chain with concomitant C-C bond formations and alkyl-shifts to produce a terminal carbocation that is finally quenched by a base (14). Crystal structures of several sesquiterpene Synthases (11, 32, 44) have been solved to aid in the investigation of the complex cyclization mechanisms catalyzed by these enzymes (17, 47). All crystallized sesquiterpene Synthases share a conserved class I α-helical terpene Synthase fold, but their primary sequences are much less conserved, and sequences often share less then 25% identity at the amino acid level. To date, investigations have mostly focused on plant and fungal sesquiterpene Synthases, while only a small number of bacterial enzymes have been reported from Streptomyces. Germacradienol/geosmin Synthases (for convenience, structures of the most relevant sesquiterpenes mentioned throughout this paper are collected in Fig. Fig.1)1) in both Streptomyces avermitilis and Streptomyces coelicolor A3(2) have been described (7, 9, 18). This protein contains two fused complete terpene Synthase domains, which result in a bifunctional enzyme. The N-terminal domain of this fusion-type terpene Synthase is responsible for catalyzing the formation of germacradienol. Germacradienol is released from the N-terminal active site and becomes the substrate of the C-terminal active site to produce geosmin. Geosmin has an earthy, musty odor and has been implicated in the contamination of water supplies, agricultural products, and wine. Conversion of germacradienol to geosmin under in vitro conditions, however, is inefficient, resulting in the formation of only 8 to 15% geosmin among the total terpene products (26). Two other typical single-domain sesquiterpene Synthases, pentalenene Synthase and epi-isozizaene Synthase, have also been characterized from Streptomyces strains (33, 51). Pentalenene Synthase, cloned from both Streptomyces sp. strain UC5319 and S. avermitilis, has been studied the most extensively and its mechanistic details are well understood. epi-Isozizaene Synthase has been recently cloned from S. coelicolor A3(2) as part of a genome mining effort and shown to make a previously unknown sesquiterpene (33). Very recently, genes involved in the biosynthesis of the monoterpenoid alcohol 2-methylisobornoneol have been identified in actinomycetes (28). FIG. 1. Structures, common names (in bold), and chemical names in the Chemical Abstracts Service registry for the most relevant sesquiterpenes discussed in the text. Actinomycetes like Streptomyces are known to produce a range of bioactive natural products. Cyanobacteria are another phylum of bacteria that are typically associated with natural product biosynthesis. These photosynthetic bacteria are found in nearly every habitat imaginable and produce numerous secondary metabolites, including polyketides, nonribosomal peptides, and terpenes. To date, except for the biosynthesis of carotenes, little is known about the biosynthesis of other terpenes in cyanobacteria. Cyanobacteria are the major source of the musty smelling and tasting cyclic terpene geosmin and 2-methylisoborneol that are found in many natural water supplies and which are difficult to remove by conventional water treatment methods (31). Two terpene Synthase sequences with homology to the Streptomyces germacradienol/geosmin Synthase sequence have recently been amplified (but not functionally characterized) from a cyanobacterium implicated as the main producer of geosmin in a Saxonian water reservoir (34). In this study, we have identified three sesquiterpene Synthases from Nostoc punctiforme PCC 73102 and Nostoc sp. strain PCC 7120 (hereafter, N. punctiforme and Nostoc sp. refer to these two strains, respectively). One of the identified putative terpene Synthases (NP2) from N. punctiforme shows homology to germacradienol/geosmin Synthase from Streptomyces, while the other two terpene Synthase homologs found in Nostoc sp. (NS1) and Nostoc punctiforme (NP1) are typical single-domain terpene Synthases. Genes encoding the NS1 and NP1 enzymes are located in an apparent gene cluster containing open reading frames (ORFs) encoding a cytochrome P450 and a putative hybrid two-component protein.
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Identification of Sesquiterpene Synthases from Nostoc punctiforme PCC 73102 and Nostoc sp. Strain PCC 7120
Journal of Bacteriology, 2008Co-Authors: Sean A. Agger, Fernando López-gallego, Thomas R. Hoye, Claudia Schmidt-dannertAbstract:Cyanobacteria are a rich source of natural products and are known to produce terpenoids. These bacteria are the major source of the musty-smelling terpenes geosmin and 2-methylisoborneol, which are found in many natural water supplies; however, no terpene Synthases have been characterized from these organisms to date. Here, we describe the characterization of three sesquiterpene Synthases identified in Nostoc sp. strain PCC 7120 (terpene Synthase NS1) and Nostoc punctiforme PCC 73102 (terpene Synthases NP1 and NP2). The second terpene Synthase in N. punctiforme (NP2) is homologous to fusion-type sesquiterpene Synthases from Streptomyces spp. shown to produce geosmin via an intermediate germacradienol. The enzymes were functionally expressed in Escherichia coli, and their terpene products were structurally identified as germacrene A (from NS1), the eudesmadiene 8a-epi-α-selinene (from NP1), and germacradienol (from NP2). The product of NP1, 8a-epi-α-selinene, so far has been isolated only from termites, in which it functions as a defense compound. Terpene Synthases NP1 and NS1 are part of an apparent minicluster that includes a P450 and a putative hybrid two-component protein located downstream of the terpene Synthases. Coexpression of P450 genes with their adjacent located terpene Synthase genes in E. coli demonstrates that the P450 from Nostoc sp. can be functionally expressed in E. coli when coexpressed with a ferredoxin gene and a ferredoxin reductase gene from Nostoc and that the enzyme oxygenates the NS1 terpene product germacrene A. This represents to the best of our knowledge the first example of functional expression of a cyanobacterial P450 in E. coli.
Claudia Schmidt-dannert - One of the best experts on this subject based on the ideXlab platform.
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Identification of Sesquiterpene Synthases from Nostoc punctiforme PCC 73102 and Nostoc sp. Strain PCC 7120
Journal of Bacteriology, 2008Co-Authors: Sean A. Agger, Fernando López-gallego, Thomas R. Hoye, Claudia Schmidt-dannertAbstract:Cyanobacteria are a rich source of natural products and are known to produce terpenoids. These bacteria are the major source of the musty-smelling terpenes geosmin and 2-methylisoborneol, which are found in many natural water supplies; however, no terpene Synthases have been characterized from these organisms to date. Here, we describe the characterization of three sesquiterpene Synthases identified in Nostoc sp. strain PCC 7120 (terpene Synthase NS1) and Nostoc punctiforme PCC 73102 (terpene Synthases NP1 and NP2). The second terpene Synthase in N. punctiforme (NP2) is homologous to fusion-type sesquiterpene Synthases from Streptomyces spp. shown to produce geosmin via an intermediate germacradienol. The enzymes were functionally expressed in Escherichia coli, and their terpene products were structurally identified as germacrene A (from NS1), the eudesmadiene 8a-epi-α-selinene (from NP1), and germacradienol (from NP2). The product of NP1, 8a-epi-α-selinene, so far has been isolated only from termites, in which it functions as a defense compound. Terpene Synthases NP1 and NS1 are part of an apparent minicluster that includes a P450 and a putative hybrid two-component protein located downstream of the terpene Synthases. Coexpression of P450 genes with their adjacent located terpene Synthase genes in E. coli demonstrates that the P450 from Nostoc sp. can be functionally expressed in E. coli when coexpressed with a ferredoxin gene and a ferredoxin reductase gene from Nostoc and that the enzyme oxygenates the NS1 terpene product germacrene A. This represents to the best of our knowledge the first example of functional expression of a cyanobacterial P450 in E. coli.
Thomas R. Hoye - One of the best experts on this subject based on the ideXlab platform.
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identification of sesquiterpene Synthases from nostoc punctiforme pcc 73102 and nostoc sp strain pcc 7120
Journal of Bacteriology, 2008Co-Authors: Sean A. Agger, Thomas R. Hoye, Fernando Lopezgallego, Claudia SchmidtdannertAbstract:Terpenoids constitute the largest group of natural products, constituting over 20,000 described compounds (14). These compounds have a wide range of biological functions and are synthesized by plants and microbes as, for example, pigments, hormones and signaling molecules, antibiotics, antifeedants, or pollinator attractants. Many of these compounds are present in minute quantities and have been an important target for synthetic chemists (22, 52) and metabolic engineers (10, 35, 50). Terpenes are synthesized from linear isoprene diphosphate precursors with various chain lengths, and their enormous structural diversity is the result of the large number of possible enzyme-catalyzed cyclizations and rearrangements that the double-bond-containing isoprene chains can undergo (14, 49). Cyclizations of isoprene diphosphate chains are catalyzed by terpene Synthases (also known as terpene cyclases). Depending on the chain length of their isoprene diphosphate substrates, terpene Synthases can be classified as mono-, sesqui-, or diterpene Synthases, which catalyze the cyclization of geranyl diphosphate (C10), farnesyl diphosphate (FPP; C15), or geranylgeranyl diphosphate, respectively. Well-known examples of cyclic terpenes are the monoterpene 2-methylisoborneol and sesquiterpene geosmin (responsible for the earthy odor of soil and lake water), the trichothecenes (mycotoxins), the sesquiterpene artemisinin (antimalarial drug), and the diterpene paclitaxel (Taxol; anticancer drug). Sesquiterpene Synthases catalyze the cyclization of FPP into any of 300 known hydrocarbon skeletons. These enzymes are typically either monomeric or homodimeric with molecular masses ranging from 40 to 65 kDa and require Mg2+ for catalysis. The Mg2+ ions bind to two metal-binding motifs that are conserved among all terpene Synthases. These metal ions complex to the substrate pyrophosphate, thereby promoting the ionization of the leaving group of FPP, resulting in the generation of a highly reactive allylic cation. The enzyme then controls carbocation migration through the isoprene chain with concomitant C-C bond formations and alkyl-shifts to produce a terminal carbocation that is finally quenched by a base (14). Crystal structures of several sesquiterpene Synthases (11, 32, 44) have been solved to aid in the investigation of the complex cyclization mechanisms catalyzed by these enzymes (17, 47). All crystallized sesquiterpene Synthases share a conserved class I α-helical terpene Synthase fold, but their primary sequences are much less conserved, and sequences often share less then 25% identity at the amino acid level. To date, investigations have mostly focused on plant and fungal sesquiterpene Synthases, while only a small number of bacterial enzymes have been reported from Streptomyces. Germacradienol/geosmin Synthases (for convenience, structures of the most relevant sesquiterpenes mentioned throughout this paper are collected in Fig. Fig.1)1) in both Streptomyces avermitilis and Streptomyces coelicolor A3(2) have been described (7, 9, 18). This protein contains two fused complete terpene Synthase domains, which result in a bifunctional enzyme. The N-terminal domain of this fusion-type terpene Synthase is responsible for catalyzing the formation of germacradienol. Germacradienol is released from the N-terminal active site and becomes the substrate of the C-terminal active site to produce geosmin. Geosmin has an earthy, musty odor and has been implicated in the contamination of water supplies, agricultural products, and wine. Conversion of germacradienol to geosmin under in vitro conditions, however, is inefficient, resulting in the formation of only 8 to 15% geosmin among the total terpene products (26). Two other typical single-domain sesquiterpene Synthases, pentalenene Synthase and epi-isozizaene Synthase, have also been characterized from Streptomyces strains (33, 51). Pentalenene Synthase, cloned from both Streptomyces sp. strain UC5319 and S. avermitilis, has been studied the most extensively and its mechanistic details are well understood. epi-Isozizaene Synthase has been recently cloned from S. coelicolor A3(2) as part of a genome mining effort and shown to make a previously unknown sesquiterpene (33). Very recently, genes involved in the biosynthesis of the monoterpenoid alcohol 2-methylisobornoneol have been identified in actinomycetes (28). FIG. 1. Structures, common names (in bold), and chemical names in the Chemical Abstracts Service registry for the most relevant sesquiterpenes discussed in the text. Actinomycetes like Streptomyces are known to produce a range of bioactive natural products. Cyanobacteria are another phylum of bacteria that are typically associated with natural product biosynthesis. These photosynthetic bacteria are found in nearly every habitat imaginable and produce numerous secondary metabolites, including polyketides, nonribosomal peptides, and terpenes. To date, except for the biosynthesis of carotenes, little is known about the biosynthesis of other terpenes in cyanobacteria. Cyanobacteria are the major source of the musty smelling and tasting cyclic terpene geosmin and 2-methylisoborneol that are found in many natural water supplies and which are difficult to remove by conventional water treatment methods (31). Two terpene Synthase sequences with homology to the Streptomyces germacradienol/geosmin Synthase sequence have recently been amplified (but not functionally characterized) from a cyanobacterium implicated as the main producer of geosmin in a Saxonian water reservoir (34). In this study, we have identified three sesquiterpene Synthases from Nostoc punctiforme PCC 73102 and Nostoc sp. strain PCC 7120 (hereafter, N. punctiforme and Nostoc sp. refer to these two strains, respectively). One of the identified putative terpene Synthases (NP2) from N. punctiforme shows homology to germacradienol/geosmin Synthase from Streptomyces, while the other two terpene Synthase homologs found in Nostoc sp. (NS1) and Nostoc punctiforme (NP1) are typical single-domain terpene Synthases. Genes encoding the NS1 and NP1 enzymes are located in an apparent gene cluster containing open reading frames (ORFs) encoding a cytochrome P450 and a putative hybrid two-component protein.
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Identification of Sesquiterpene Synthases from Nostoc punctiforme PCC 73102 and Nostoc sp. Strain PCC 7120
Journal of Bacteriology, 2008Co-Authors: Sean A. Agger, Fernando López-gallego, Thomas R. Hoye, Claudia Schmidt-dannertAbstract:Cyanobacteria are a rich source of natural products and are known to produce terpenoids. These bacteria are the major source of the musty-smelling terpenes geosmin and 2-methylisoborneol, which are found in many natural water supplies; however, no terpene Synthases have been characterized from these organisms to date. Here, we describe the characterization of three sesquiterpene Synthases identified in Nostoc sp. strain PCC 7120 (terpene Synthase NS1) and Nostoc punctiforme PCC 73102 (terpene Synthases NP1 and NP2). The second terpene Synthase in N. punctiforme (NP2) is homologous to fusion-type sesquiterpene Synthases from Streptomyces spp. shown to produce geosmin via an intermediate germacradienol. The enzymes were functionally expressed in Escherichia coli, and their terpene products were structurally identified as germacrene A (from NS1), the eudesmadiene 8a-epi-α-selinene (from NP1), and germacradienol (from NP2). The product of NP1, 8a-epi-α-selinene, so far has been isolated only from termites, in which it functions as a defense compound. Terpene Synthases NP1 and NS1 are part of an apparent minicluster that includes a P450 and a putative hybrid two-component protein located downstream of the terpene Synthases. Coexpression of P450 genes with their adjacent located terpene Synthase genes in E. coli demonstrates that the P450 from Nostoc sp. can be functionally expressed in E. coli when coexpressed with a ferredoxin gene and a ferredoxin reductase gene from Nostoc and that the enzyme oxygenates the NS1 terpene product germacrene A. This represents to the best of our knowledge the first example of functional expression of a cyanobacterial P450 in E. coli.
Claudia Schmidtdannert - One of the best experts on this subject based on the ideXlab platform.
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identification of sesquiterpene Synthases from nostoc punctiforme pcc 73102 and nostoc sp strain pcc 7120
Journal of Bacteriology, 2008Co-Authors: Sean A. Agger, Thomas R. Hoye, Fernando Lopezgallego, Claudia SchmidtdannertAbstract:Terpenoids constitute the largest group of natural products, constituting over 20,000 described compounds (14). These compounds have a wide range of biological functions and are synthesized by plants and microbes as, for example, pigments, hormones and signaling molecules, antibiotics, antifeedants, or pollinator attractants. Many of these compounds are present in minute quantities and have been an important target for synthetic chemists (22, 52) and metabolic engineers (10, 35, 50). Terpenes are synthesized from linear isoprene diphosphate precursors with various chain lengths, and their enormous structural diversity is the result of the large number of possible enzyme-catalyzed cyclizations and rearrangements that the double-bond-containing isoprene chains can undergo (14, 49). Cyclizations of isoprene diphosphate chains are catalyzed by terpene Synthases (also known as terpene cyclases). Depending on the chain length of their isoprene diphosphate substrates, terpene Synthases can be classified as mono-, sesqui-, or diterpene Synthases, which catalyze the cyclization of geranyl diphosphate (C10), farnesyl diphosphate (FPP; C15), or geranylgeranyl diphosphate, respectively. Well-known examples of cyclic terpenes are the monoterpene 2-methylisoborneol and sesquiterpene geosmin (responsible for the earthy odor of soil and lake water), the trichothecenes (mycotoxins), the sesquiterpene artemisinin (antimalarial drug), and the diterpene paclitaxel (Taxol; anticancer drug). Sesquiterpene Synthases catalyze the cyclization of FPP into any of 300 known hydrocarbon skeletons. These enzymes are typically either monomeric or homodimeric with molecular masses ranging from 40 to 65 kDa and require Mg2+ for catalysis. The Mg2+ ions bind to two metal-binding motifs that are conserved among all terpene Synthases. These metal ions complex to the substrate pyrophosphate, thereby promoting the ionization of the leaving group of FPP, resulting in the generation of a highly reactive allylic cation. The enzyme then controls carbocation migration through the isoprene chain with concomitant C-C bond formations and alkyl-shifts to produce a terminal carbocation that is finally quenched by a base (14). Crystal structures of several sesquiterpene Synthases (11, 32, 44) have been solved to aid in the investigation of the complex cyclization mechanisms catalyzed by these enzymes (17, 47). All crystallized sesquiterpene Synthases share a conserved class I α-helical terpene Synthase fold, but their primary sequences are much less conserved, and sequences often share less then 25% identity at the amino acid level. To date, investigations have mostly focused on plant and fungal sesquiterpene Synthases, while only a small number of bacterial enzymes have been reported from Streptomyces. Germacradienol/geosmin Synthases (for convenience, structures of the most relevant sesquiterpenes mentioned throughout this paper are collected in Fig. Fig.1)1) in both Streptomyces avermitilis and Streptomyces coelicolor A3(2) have been described (7, 9, 18). This protein contains two fused complete terpene Synthase domains, which result in a bifunctional enzyme. The N-terminal domain of this fusion-type terpene Synthase is responsible for catalyzing the formation of germacradienol. Germacradienol is released from the N-terminal active site and becomes the substrate of the C-terminal active site to produce geosmin. Geosmin has an earthy, musty odor and has been implicated in the contamination of water supplies, agricultural products, and wine. Conversion of germacradienol to geosmin under in vitro conditions, however, is inefficient, resulting in the formation of only 8 to 15% geosmin among the total terpene products (26). Two other typical single-domain sesquiterpene Synthases, pentalenene Synthase and epi-isozizaene Synthase, have also been characterized from Streptomyces strains (33, 51). Pentalenene Synthase, cloned from both Streptomyces sp. strain UC5319 and S. avermitilis, has been studied the most extensively and its mechanistic details are well understood. epi-Isozizaene Synthase has been recently cloned from S. coelicolor A3(2) as part of a genome mining effort and shown to make a previously unknown sesquiterpene (33). Very recently, genes involved in the biosynthesis of the monoterpenoid alcohol 2-methylisobornoneol have been identified in actinomycetes (28). FIG. 1. Structures, common names (in bold), and chemical names in the Chemical Abstracts Service registry for the most relevant sesquiterpenes discussed in the text. Actinomycetes like Streptomyces are known to produce a range of bioactive natural products. Cyanobacteria are another phylum of bacteria that are typically associated with natural product biosynthesis. These photosynthetic bacteria are found in nearly every habitat imaginable and produce numerous secondary metabolites, including polyketides, nonribosomal peptides, and terpenes. To date, except for the biosynthesis of carotenes, little is known about the biosynthesis of other terpenes in cyanobacteria. Cyanobacteria are the major source of the musty smelling and tasting cyclic terpene geosmin and 2-methylisoborneol that are found in many natural water supplies and which are difficult to remove by conventional water treatment methods (31). Two terpene Synthase sequences with homology to the Streptomyces germacradienol/geosmin Synthase sequence have recently been amplified (but not functionally characterized) from a cyanobacterium implicated as the main producer of geosmin in a Saxonian water reservoir (34). In this study, we have identified three sesquiterpene Synthases from Nostoc punctiforme PCC 73102 and Nostoc sp. strain PCC 7120 (hereafter, N. punctiforme and Nostoc sp. refer to these two strains, respectively). One of the identified putative terpene Synthases (NP2) from N. punctiforme shows homology to germacradienol/geosmin Synthase from Streptomyces, while the other two terpene Synthase homologs found in Nostoc sp. (NS1) and Nostoc punctiforme (NP1) are typical single-domain terpene Synthases. Genes encoding the NS1 and NP1 enzymes are located in an apparent gene cluster containing open reading frames (ORFs) encoding a cytochrome P450 and a putative hybrid two-component protein.
