rotundone and guaiene

Four terpene synthases for the biosynthesis of volatile terpenoids were identified from the transcriptome of

Topics: Alkyl and Aryl Transferases; Azulenes; Biosynthetic Pathways; Flowers; Gene Expression; Plant Proteins; Saccharomyces cerevisiae; Sesquiterpenes; Sesquiterpenes, Guaiane; Thymelaeaceae

(-)-Rotundone is a potent odorant molecule with a characteristic spicy aroma existing in various plants including grapevines (Vitis vinifera). It is considered to be a significant compound in wines and grapes because of its low sensory threshold and aroma properties. (-)-Rotundone was first identified in red wine made from the grape cultivar Syrah and here we report the identification of VvSTO2 as a α-guaiene 2-oxidase which can transform α-guaiene to (-)-rotundone in the grape cultivar Syrah. It is a cytochrome P450 (CYP) enzyme belonging to the CYP 71BE subfamily, which overlaps with the very large CYP71D family and, to the best of our knowledge, this is the first functional characterization of an enzyme from this family. VvSTO2 was expressed at a higher level in the Syrah grape exocarp (skin) in accord with the localization of (-)-rotundone accumulation in grape berries. α-Guaiene was also detected in the Syrah grape exocarp at an extremely high concentration. These findings suggest that (-)-rotundone accumulation is regulated by the VvSTO2 expression along with the availability of α-guaiene as a precursor. VvSTO2 expression during grape maturation was considerably higher in Syrah grape exocarp compared to Merlot grape exocarp, consistent with the patterns of α-guaiene and (-)-rotundone accumulation. On the basis of these findings, we propose that VvSTO2 may be a key enzyme in the biosynthesis of (-)-rotundone in grapevines by acting as a α-guaiene 2-oxidase.

Topics: Amino Acid Sequence; Azulenes; Biocatalysis; Cytochrome P-450 Enzyme System; Enzyme Assays; Fruit; Gas Chromatography-Mass Spectrometry; Gene Expression Regulation, Plant; Genes, Plant; Kinetics; Molecular Sequence Data; Odorants; Phylogeny; Plant Proteins; Recombination, Genetic; RNA, Messenger; Sequence Alignment; Sesquiterpenes; Sesquiterpenes, Guaiane; Substrate Specificity; Vitis

Rotundone was initially identified as a grape-derived compound responsible for the peppery aroma of Shiraz wine varieties. It has subsequently been found in black and white pepper and several other spices. Because of its potent aroma, the molecular basis for rotundone formation is of particular relevance to grape and wine scientists and industry. We have identified and functionally characterized in planta a sesquiterpene synthase, VvGuaS, from developing grape berries, and have demonstrated that it produces the precursor of rotundone, α-guaiene, as its main product. The VvGuaS enzyme is a novel allele of the sesquiterpene synthase gene, VvTPS24, which has previously been reported to encode VvPNSeInt, an enzyme that produces a variety of selinene-type sesquiterpenes. This newly discovered VvTPS24 allele encodes an enzyme 99.5% identical to VvPNSeInt, with the differences comprising just 6 out of the 561 amino acid residues. Molecular modelling of the enzymes revealed that two of these residues, T414 and V530, are located in the active site of VvGuaS within 4 Å of the binding-site of the substrate, farnesyl pyrophosphate. Mutation of these two residues of VvGuaS into the corresponding polymorphisms in VvPNSeInt results in a complete functional conversion of one enzyme into the other, while mutation of each residue individually produces an intermediate change in the product profile. We have therefore demonstrated that VvGuaS, an enzyme responsible for production of the rotundone precursor, α-guaiene, is encoded by a novel allele of the previously characterized grapevine gene VvTPS24 and that two specific polymorphisms are responsible for functional differences between VvTPS24 alleles.

Topics: Alleles; Azulenes; Gas Chromatography-Mass Spectrometry; Genes, Plant; Models, Molecular; Mutagenesis, Site-Directed; Plant Proteins; Polymorphism, Genetic; Sesquiterpenes; Sesquiterpenes, Guaiane; Structural Homology, Protein; Vitis; Volatile Organic Compounds

Two unstable hydroperoxides, 6b and 10a, and 13 downstream sesquiterpenoids have been isolated from the autoxidation mixture of the bicyclic sesquiterpene α-guaiene (1) on cellulose filter paper. One of the significant natural products isolated was rotundone (2), which is the only known impact odorant displaying a peppery aroma. Other products included corymbolone (4a) and its C-6 epimer 4b, the (2R)- and (2S)-rotundols (7a/b), and several hitherto unknown epimers of natural chabrolidione A, namely, 7-epi-chabrolidione A (3a) and 1,7-epi-chabrolidione A (3b). Two 4-hydroxyrotundones (8a/b) and a range of epoxides (9a/b and 5a/b) were also formed in significant amounts after autoxidation. Their structures were elucidated on the basis of spectroscopic data and X-ray crystallography, and a number of them were confirmed through total synthesis. The mechanisms of formation of the majority of the products may be accounted for by initial formation of the 2- and 4-hydroperoxyguaienes (6a/b and 10a/b) followed by various fragmentation or degradation pathways. Given that α-guaiene (1) is well known to exist in the essential oils of numerous plants, coupled with the fact that aerial oxidation to form this myriad of downstream oxidation products occurs readily at ambient temperature, suggests that many of them have been overlooked during previous isolation studies from natural sources.

Topics: Azulenes; Crystallography, X-Ray; Hydrogen Peroxide; Molecular Structure; Oxidation-Reduction; Sesquiterpenes; Sesquiterpenes, Guaiane; Stereoisomerism

The aroma link between pepper and wine has recently been elucidated to be due to the important aroma compound rotundone. To date, rotundone is the only known impact odorant with a peppery aroma. Although the concentration found in products of natural origin is small, the odor detection threshold is among the lowest of any natural product yet discovered. We report herein the identification of the first known precursor to rotundone, namely, α-guaiene, and that one mechanism of transformation is simple aerial oxidation.

Topics: Azulenes; Molecular Structure; Odorants; Oxidation-Reduction; Piper nigrum; Plant Extracts; Sesquiterpenes; Sesquiterpenes, Guaiane