Abstract:Terpene synthases are responsible for a large diversity of terpene carbon skeletons found in nature. The multiproduct sesquiterpene synthase MtTPS5 isolated from Medicago truncatula produces 27 products from farnesyl diphosphate (1, FDP). In this paper, we report the reaction steps involved in the formation of these products using incubation experiments with deuterium-containing substrates; we determined the absolute configuration of individual products to establish the stereochemical course of the reaction ca… Show more
“…The reaction cascade is either initiated by a metal iondependent ionization of the diphosphate moiety or a protonation of the substrate, and can be terminated by proton abstraction or water addition (11,13,15). Because the reaction cascade may be branched and termination may occur at multiple levels, many TPSs are multiproduct enzymes forming complex mixtures of compounds (16)(17)(18)(19). Moreover, some TPSs also accept multiple substrates to produce monoterpenes, sesquiterpenes, and diterpenes (11).…”
Sesquiterpenes play important roles in insect communication, for example as pheromones. However, no sesquiterpene synthases, the enzymes involved in construction of the basic carbon skeleton, have been identified in insects to date. We investigated the biosynthesis of the sesquiterpene (6R,7S)-himachala-9,11-diene in the crucifer flea beetle Phyllotreta striolata, a compound previously identified as a male-produced aggregation pheromone in several Phyllotreta species. A (6R,7S)-himachala-9,11-diene–producing sesquiterpene synthase activity was detected in crude beetle protein extracts, but only when (Z,E)-farnesyl diphosphate [(Z,E)-FPP] was offered as a substrate. No sequences resembling sesquiterpene synthases from plants, fungi, or bacteria were found in the P. striolata transcriptome, but we identified nine divergent putative trans-isoprenyl diphosphate synthase (trans-IDS) transcripts. Four of these putative trans-IDSs exhibited terpene synthase (TPS) activity when heterologously expressed. Recombinant PsTPS1 converted (Z,E)-FPP to (6R,7S)-himachala-9,11-diene and other sesquiterpenes observed in beetle extracts. RNAi-mediated knockdown of PsTPS1 mRNA in P. striolata males led to reduced emission of aggregation pheromone, confirming a significant role of PsTPS1 in pheromone biosynthesis. Two expressed enzymes showed genuine IDS activity, with PsIDS1 synthesizing (E,E)-FPP, whereas PsIDS3 produced neryl diphosphate, (Z,Z)-FPP, and (Z,E)-FPP. In a phylogenetic analysis, the PsTPS enzymes and PsIDS3 were clearly separated from a clade of known coleopteran trans-IDS enzymes including PsIDS1 and PsIDS2. However, the exon–intron structures of IDS and TPS genes in P. striolata are conserved, suggesting that this TPS gene family evolved from trans-IDS ancestors.
“…The reaction cascade is either initiated by a metal iondependent ionization of the diphosphate moiety or a protonation of the substrate, and can be terminated by proton abstraction or water addition (11,13,15). Because the reaction cascade may be branched and termination may occur at multiple levels, many TPSs are multiproduct enzymes forming complex mixtures of compounds (16)(17)(18)(19). Moreover, some TPSs also accept multiple substrates to produce monoterpenes, sesquiterpenes, and diterpenes (11).…”
Sesquiterpenes play important roles in insect communication, for example as pheromones. However, no sesquiterpene synthases, the enzymes involved in construction of the basic carbon skeleton, have been identified in insects to date. We investigated the biosynthesis of the sesquiterpene (6R,7S)-himachala-9,11-diene in the crucifer flea beetle Phyllotreta striolata, a compound previously identified as a male-produced aggregation pheromone in several Phyllotreta species. A (6R,7S)-himachala-9,11-diene–producing sesquiterpene synthase activity was detected in crude beetle protein extracts, but only when (Z,E)-farnesyl diphosphate [(Z,E)-FPP] was offered as a substrate. No sequences resembling sesquiterpene synthases from plants, fungi, or bacteria were found in the P. striolata transcriptome, but we identified nine divergent putative trans-isoprenyl diphosphate synthase (trans-IDS) transcripts. Four of these putative trans-IDSs exhibited terpene synthase (TPS) activity when heterologously expressed. Recombinant PsTPS1 converted (Z,E)-FPP to (6R,7S)-himachala-9,11-diene and other sesquiterpenes observed in beetle extracts. RNAi-mediated knockdown of PsTPS1 mRNA in P. striolata males led to reduced emission of aggregation pheromone, confirming a significant role of PsTPS1 in pheromone biosynthesis. Two expressed enzymes showed genuine IDS activity, with PsIDS1 synthesizing (E,E)-FPP, whereas PsIDS3 produced neryl diphosphate, (Z,Z)-FPP, and (Z,E)-FPP. In a phylogenetic analysis, the PsTPS enzymes and PsIDS3 were clearly separated from a clade of known coleopteran trans-IDS enzymes including PsIDS1 and PsIDS2. However, the exon–intron structures of IDS and TPS genes in P. striolata are conserved, suggesting that this TPS gene family evolved from trans-IDS ancestors.
“…This is detected and characterized by GC–MS following comparison to the standards included in the database. The product peak at 18.68 min generated the dominant mass segments at m/z 161, 119, 204 and 105 perfectly matching δ-cadinol (also torreyol) in mass spectra of the database and the authentic δ-cadinol in publications [13, 25] (Electronic supplementary material).Fig. 2Expression of BvCS /pET32a+ in E. coli and purification of recombinant fusion proteins.…”
Section: Resultsmentioning
confidence: 88%
“…For the biosynthesis of cadalane sesquiterpenes, three alternative mechanisms were proposed and elucidated in bacteria [16, 17] and plants [25, 26]. The intermediacy of nerolidyl diphosphate (NPP) was generally accepted.…”
Sesquiterpenoids are very common among natural products. A large number of sesquiterpene synthase genes have been cloned and functionally characterized. However, until now there is no report about the δ-cadinol synthase predominantly forming δ-cadinol (syn. torreyol) from farnesyl diphosphate. Sesquiterpenoids boreovibrins structurally similar to δ-cadinol were previously isolated from culture broths of the basidiomycete fungus Boreostereum vibrans. This led us to expect a corresponding gene coding for a δ-cadinol synthase that may be involved in the biosynthesis of boreovibrins in B. vibrans. Here we report the cloning and heterologous expression of a new sesquiterpene synthase gene from B. vibrans. The crude and purified recombinant enzymes, when incubating with farnesyl diphosphate as substrate, gave δ-cadinol as its principal product and thereby identified as a δ-cadinol synthase.Graphical AbstractA new sesquiterpene synthase gene was cloned from the basidiomycete fungus Boreostereum vibrans and heterologously expressed in E. coli. The purified recombinant enzyme gave δ-cadinol as its principal product from farnesyl diphosphate and thereby identified as a δ-cadinol synthase (BvCS).
Electronic supplementary materialThe online version of this article (doi:10.1007/s13659-016-0096-4) contains supplementary material, which is available to authorized users.
“…39 However, it is also possible that germacradien-4-ol is generated from a reaction intermediate, (Z, E)-germacradienyl cation (2), of the ( þ )-epicubenol synthesis ( Figure 1). 40 Cubenene can also be generated from another reaction intermediate, bicyclic cation (4) (Figure 1).…”
Recent bacterial genome sequencing projects have shown the presence of many putative sesquiterpene cyclase (SC) genes, especially in the Gram-positive, filamentous bacterial genus Streptomyces. We describe here the characterization of a SC gene (SGR6065, named gecA) from Streptomyces griseus. Overexpression of gecA in Streptomyces lividans produced a sesquiterpene, which was isolated and determined to be ( þ )-epicubenol using spectroscopic analyses. The N-terminal histidine-tagged GecA protein was produced in Escherichia coli. Incubation of the recombinant GecA protein with farnesyl diphosphate (FPP) yielded ( þ )-epicubenol as the major product. The K m value for FPP and the k cat value for ( þ )-epicubenol formation were calculated to be 254 ± 7.1 nM and 0.026 ± 0.001 s À1 , respectively. The k cat /K m value (0.10 s À1 lM À1 ) was broadly comparable to those reported for known bacterial SCs. ( þ )-Epicubenol was detected in the crude cell lysate of wild-type S. griseus, but not in a gecA-knockout mutant, indicating that GecA is a genuine ( þ )-epicubenol synthase. Although ( þ )-epicubenol synthases have been previously purified and characterized from the liverwort Heteroscyphus planus and Streptomyces sp. LL-B7, no ( þ )-epicubenol synthase gene has been cloned to date. The gecA gene is thus the first example of an ( þ )-epicubenol synthase-encoding gene. ( þ )-Epicubenol production was not controlled by the microbial hormone A-factor that induces morphological differentiation and production of several secondary metabolites in S. griseus.
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