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Specialized methylketone-containing metabolites accumulate in certain plants, in particular wild tomatoes in which they serve as toxic compounds against chewing insects. In Solanum habrochaites f. glabratum , methylketone biosynthesis occurs in the plastids of glandular trichomes and begins with intermediates of de novo fatty acid synthesis. These fatty-acyl intermediates are converted via sequential reactions catalyzed by Methylketone Synthase2 (MKS2) and MKS1 to produce the n-1 methylketone. We report crystal structures of S. habrochaites MKS1, an atypical member of the α/β-hydrolase superfamily. Sequence comparisons revealed the MKS1 catalytic triad, Ala-His-Asn, as divergent to the traditional α/β-hydrolase triad, Ser-His-Asp. Determination of the MKS1 structure points to a novel enzymatic mechanism dependent upon residues Thr-18 and His-243, confirmed by biochemical assays. Structural analysis further reveals a tunnel leading from the active site consisting mostly of hydrophobic residues, an environment well suited for fatty-acyl chain binding. We confirmed the importance of this substrate binding mode by substituting several amino acids leading to an alteration in the acyl-chain length preference of MKS1. Furthermore, we employ structure-guided mutagenesis and functional assays to demonstrate that MKS1, unlike enzymes from this hydrolase superfamily, is not an efficient hydrolase but instead catalyzes the decarboxylation of 3-keto acids. [ABSTRACT FROM AUTHOR]

Methylcinnamate, which is widely distributed throughout the plant kingdom, is a significant component of many floral scents and an important signaling molecule between plants and insects. Comparison of an EST database obtained from the glandular trichomes of a basil (Ocimum basilicum) variety that produces high levels of methylcinnamate (line MC) with other varieties producing little or no methylcinnamate identified several very closely related genes belonging to the SABATH family of carboxyl methyltransferases that are highly and almost exclusively expressed in line MC. Biochemical characterization of the corresponding recombinant proteins showed that cinnamate and p-coumarate are their best substrates for methylation, thus designating these enzymes as cinnamate/p-coumarate carboxyl methyltransferases (CCMTs). Gene expression, enzyme activity, protein profiling, and metabolite content analyses demonstrated that CCMTs are responsible for the formation of methylcinnamate in sweet basil. A phylogenetic analysis of the entire SABATH family placed these CCMTs into a clade that includes indole-3-acetic acid carboxyl methyltransferases and a large number of uncharacterized carboxyl methyltransferase-like proteins from monocots and lower plants. Structural modeling and ligand docking suggested active site residues that appear to contribute to the substrate preference of CCMTs relative to other members of the SABATH family. Site-directed mutagenesis of specific residues confirmed these findings. [ABSTRACT FROM AUTHOR]

Plants have evolved a plethora of chemical solutions to help them cope with dynamic biotic and abiotic environments, and much of this diversity was used throughout human history for medicinal and hedonistic purposes (seasoning, preservatives, etc.). Despite the enormous progress made in the last decade in elucidating the genes and enzymes involved in these metabolic pathways using classical biochemical approaches and biochemical genomic methods, very few studies applied targeted and non-targeted mutagenesis for isolating novel genes to study their hitherto unknown function. This minireview will discuss recent examples of forward and reverse genetics approaches taken in some medicinal plants and herbs, and will present data that we obtained from an ethylmethane sulfate mutagenesis project in Ocimum basilicum (sweet basil), aiming to identify new genes that are involved in the biosynthesis of aroma compounds. [ABSTRACT FROM AUTHOR]

Surface glandular trichomes distributed throughout the aerial parts of sweet basil (Ocimum basilicum) produce and store monoterpene, sesquiterpene, and phenylpropene volatiles. Three distinct basil chemotypes were used to examine the molecular mechanisms underlying the divergence in their monoterpene and sesquiterpene content. The relative levels of specific terpenes in the glandular trichomes of each cultivar were correlated with the levels of transcripts for eight genes encoding distinct terpene synthases. In a cultivar that produces mostly (R)-linalool, transcripts of (R)-linalool synthase (US) were the most abundant of these eight. In a cultivar that synthesizes mostly geraniol, transcripts of geraniol synthase were the most abundant, but the glands of this cultivar also contained a transcript of an (R)-LIS gene with a 1-base insertion that caused a frame shift mutation. A geraniol synthase-LIS hybrid gene was constructed and expressed in Escherichia coli, and the protein catalyzed the formation of both geraniol and (R)-linalool from geranyl diphosphate. The total amounts of terpenes were correlated with total levels of terpene synthase activities, and negatively correlated with levels of phenylpropanoids and phenylalanine ammonia lyase activity. The relative levels of geranyl diphosphate synthase and farnesyl diphosphate synthase activities did not correlate with the total amount of terpenes produced, but showed some correlation with the ratio of monoterpenes to sesquiterpenes. [ABSTRACT FROM AUTHOR]

Abstract: Wild strawberry (Fragaria vesca) fruit contains several important phenylpropene aroma compounds such as eugenol, but cultivated varieties are mostly devoid of them. We have redirected the carbon flux in cultivated strawberry (Fragaria×ananassa) fruit from anthocyanin pigment biosynthesis to the production of acetates of hydroxycinnamyl alcohols, which serve as the precursors of the phenylpropenes, by downregulating the strawberry chalcone synthase (CHS) via RNAi-mediated gene silencing and, alternatively, by an antisense CHS construct. Simultaneous heterologous overexpression of a eugenol (EGS) and isoeugenol synthase (IGS) gene in the same cultivated strawberry fruits boosted the formation of eugenol, isoeugenol, and the related phenylpropenes chavicol and anol to concentrations orders of magnitude greater than their odor thresholds. The results show that Fragaria×ananassa still bears a phenylpropene biosynthetic pathway but the carbon flux is primarily directed to the formation of pigments. Thus, partial restoration of wild strawberry flavor in cultivated varieties is feasible by diverting the flavonoid pathway to phenylpropene synthesis through metabolic engineering. [Copyright &y& Elsevier]