geranyl-pyrophosphate and alpha-pinene

To identify a terpene synthase that catalyzes the conversion of geranyl pyrophosphate (GPP) to α-pinene and is involved in the biosynthesis of paeoniflorin.. Two new terpene synthase genes were isolated from the transcriptome data of Peaonia lactiflora. Phylogenetic analysis and sequence characterization revealed that one gene, named PlPIN, encoded a monoterpene synthase that might be involved in the biosynthesis of paeoniflorin. In vitro enzyme assay showed that, in contrast to most monoterpene synthases, PlPIN encoded an α-pinene synthase which converted GPP into α-pinene as a single product.. This newly identified α-pinene synthase could be used for improving paeoniflorin accumulation by metabolic engineering or for producing α-pinene via synthetic biology.

Topics: Alkyl and Aryl Transferases; Bicyclic Monoterpenes; Monoterpenes; Paeonia; Polyisoprenyl Phosphates; Terpenes

Cotton plants accumulate gossypol and related sesquiterpene aldehydes, which function as phytoalexins against pathogens and feeding deterrents to herbivorous insects. However, to date little is known about the biosynthesis of volatile terpenes in this crop. Herein is reported that 5 monoterpenes and 11 sesquiterpenes from extracts of a glanded cotton cultivar, Gossypium hirsutum cv. CCRI12, were detected by gas chromatography-mass spectrometry (GC-MS). By EST data mining combined with Rapid Amplification of cDNA Ends (RACE), full-length cDNAs of three terpene synthases (TPSs), GhTPS1, GhTPS2 and GhTPS3 were isolated. By in vitro assays of the recombinant proteins, it was found that GhTPS1 and GhTPS2 are sesquiterpene synthases: the former converted farnesyl pyrophosphate (FPP) into β-caryophyllene and α-humulene in a ratio of 2:1, whereas the latter produced several sesquiterpenes with guaia-1(10),11-diene as the major product. By contrast, GhTPS3 is a monoterpene synthase, which produced α-pinene, β-pinene, β-phellandrene and trace amounts of other monoterpenes from geranyl pyrophosphate (GPP). The TPS activities were also supported by Virus Induced Gene Silencing (VIGS) in the cotton plant. GhTPS1 and GhTPS3 were highly expressed in the cotton plant overall, whereas GhTPS2 was expressed only in leaves. When stimulated by mechanical wounding, Verticillium dahliae (Vde) elicitor or methyl jasmonate (MeJA), production of terpenes and expression of the corresponding synthase genes were induced. These data demonstrate that the three genes account for the biosynthesis of volatile terpenes of cotton, at least of this Upland cotton.

Topics: Acetates; Alkyl and Aryl Transferases; Bicyclic Monoterpenes; Bridged Bicyclo Compounds; Cyclohexane Monoterpenes; Cyclohexenes; Cyclopentanes; Gas Chromatography-Mass Spectrometry; Gossypium; Intramolecular Lyases; Monocyclic Sesquiterpenes; Monoterpenes; Oxylipins; Phytoalexins; Polycyclic Sesquiterpenes; Polyisoprenyl Phosphates; Sesquiterpenes; Sesquiterpenes, Guaiane; Terpenes; Volatile Organic Compounds

Cotton (Gossypium hirsutum L.) plants damaged by insects emit a blend of volatiles, including monoterpenes and sesquiterpenes, which can directly repel herbivores and/or indirectly protect the plant by attracting natural enemies of the herbivores. To understand the molecular basis of terpene biosynthesis and regulation in cotton, two terpene synthase genes, GhTPS1 and GhTPS2, were heterologously expressed and characterized. Recombinant GhTPS1 accepted farnesyl pyrophosphate as substrate and produced (E)-β-caryophyllene and α-humulene. GhTPS2 was characterized as a monoterpene synthase which formed α-pinene and β-pinene using geranyl pyrophosphate as substrate. Quantitative real-time PCR analysis revealed that GhTPS1 and GhTPS2 gene expression was elevated after methyl jasmonate (MeJA) treatment in cotton leaves. Moreover, feeding of the green plant bug Apolygus lucorum, a major cotton pest in northern China, resulted in increased GhTPS2 expression in young leaves, suggesting that GhTPS2 might be involved in plant defense in cotton.

Topics: Acetates; Adaptation, Physiological; Alkyl and Aryl Transferases; Animals; Bicyclic Monoterpenes; Bridged Bicyclo Compounds; Carbon-Oxygen Lyases; China; Cyclopentanes; Gene Expression; Genes, Plant; Gossypium; Herbivory; Insecta; Monocyclic Sesquiterpenes; Monoterpenes; Oxylipins; Plant Diseases; Plant Leaves; Plant Proteins; Polycyclic Sesquiterpenes; Polyisoprenyl Phosphates; Sesquiterpenes; Terpenes

Based on quantum chemical studies, mechanisms to form bornyl diphosphate from geranyl diphosphate are suggested. While bornyl diphosphate is usually proposed to be generated via combination of the pyrophosphate group with a secondary bornyl cation, quantum chemical computations indicate that the bornyl cation is actually not a minimum. Instead, concerted attack of the pyrophosphate group coupled with an alkyl shift could yield bornyl diphosphate from either the pinyl cation or the camphyl cation. Hints of bifurcating pathways on the energy surfaces for such reactions were also uncovered. Of particular note is the development and validation of a large model of the pyrophosphate counterion treated entirely with quantum chemistry.

Topics: Bicyclic Monoterpenes; Bridged Bicyclo Compounds; Diphosphates; Diterpenes; Molecular Dynamics Simulation; Monoterpenes; Polyisoprenyl Phosphates; Quantum Theory; Stereoisomerism; Terpenes

The three pinene synthases (cyclases) from common sage (Salvia officinalis) catalyze the conversion of geranyl pyrophosphate to the bicyclic olefins (+)-alpha-pinene and (+)-camphene (cyclase I), (-)-alpha-pinene, (-)-beta-pinene, and (-)-camphene (cyclase II), and (+)-alpha-pinene and (+)-beta-pinene (cyclase III), in addition to smaller amounts of monocyclic and acyclic monoterpene olefins. (1R)-4-2H1- and (1S)-4-2H1-labeled geranyl pyrophosphates were prepared and used to examine the stereochemistry of the C3-proton elimination from the pinyl cation intermediates in the formation of the alpha-pinene enantiomers. Mass spectrometric analysis of the biosynthetic products derived from the chirally deuterated substrates revealed that cyclase I and cyclase III removed the C4-proR-hydrogen of the substrate (C3 proton trans to the dimethyl bridge of the pinyl nucleus) with a stereoselectivity exceeding 94% in the formation of (+)-alpha-pinene. Similarly, cyclase II removed the C4-proS-hydrogen of the substrate (C3-trans proton of the corresponding pinyl cation) with a stereoselectivity exceeding 78% in the formation of (-)-alpha-pinene. The stereoselectivity of these C3-axial hydrogen eliminations is rationalized on the basis of a stereochemical model for the electrophilic isomerization-cyclization reaction sequence catalyzed by the pinene cyclases. The changes in the overall rates of olefin biosynthesis by these enzymes and in the product ratios resulting from deuterium substitution also permitted confirmation of isotopically sensitive branching in pinene biosynthesis and allowed the observation of primary kinetic isotope effects in isolation.

Topics: Alkenes; Bicyclic Monoterpenes; Binding Sites; Deuterium; Intramolecular Lyases; Isomerases; Isotope Labeling; Magnoliopsida; Monoterpenes; Polyisoprenyl Phosphates; Stereoisomerism; Substrate Specificity; Terpenes

The conversion of geranyl pyrophosphate to (+)-alpha-pinene and to (-)-beta-pinene is considered to proceed by the initial isomerization of the substrate to (-)-(3R)- and to (+)-(3S)-linalyl pyrophosphate, respectively, and the subsequent cyclization of the anti, endo-conformer of these bound intermediates by mirror-image sequences which should result in the net retention of configuration at C1 of the geranyl precursor. Incubation of (1R)-[2-14C,1-3H]- and (1S)-[2-14C,1-3H]geranyl pyrophosphate with (+)-pinene cyclase and with (-)-pinene cyclase from common sage (Salvia officinalis) gave labeled (+)-alpha- and (-)-beta-pinene of unchanged 3H/14C ratio in all cases, and the (+)- and (-)-olefins were stereoselectively converted to (+)- and (-)-borneol, respectively, which were oxidized to the corresponding (+)- and (-)-isomers of camphor, again without change in isotope ratio. The location of the tritium was determined in each case by stereoselective, base-catalyzed exchange of the exo-alpha-hydrogens of these derived ketones. The results indicated that the configuration at C1 of the substrate was retained in the enzymatic transformations to the (+)- and (-)-pinenes, which is entirely consistent with the syn-isomerization of geranyl pyrophosphate to linalyl pyrophosphate, transoid to cisoid rotation, and anti, endo-cyclization of the latter. The absolute stereochemical elements of the antipodal reaction sequences were confirmed by the selective enzymatic conversions of (3R)- and (3S)-1Z-[1-3H]linalyl pyrophosphate to (+)-alpha-pinene and (-)-beta-pinene, respectively, and by the location of the tritium in the derived camphors as before. The summation of the results fully defines the overall stereochemistry of the coupled isomerization and cyclization of geranyl pyrophosphate to the antipodal pinenes.

Topics: Bicyclic Monoterpenes; Bridged Bicyclo Compounds; Carbon Radioisotopes; Isomerases; Isomerism; Kinetics; Molecular Structure; Monoterpenes; Polyisoprenyl Phosphates; Radioisotope Dilution Technique; Terpenes; Tritium

Topics: Bicyclic Monoterpenes; Bridged Bicyclo Compounds; Bridged-Ring Compounds; Kinetics; Monoterpenes; Plants; Polyisoprenyl Phosphates; Radioisotope Dilution Technique; Structure-Activity Relationship; Terpenes; Tritium