Funktionelle Charakterisierung von Enzymen des Sekundärstoffwechsels in Lavendel (Lavandula angustifolia) und Erdbeere (Fragaria x ananassa) (English shop)

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Plants synthesise an immense number of secondary metabolites such as terpenes, alkaloids and phenolic substances. The biosynthesis of these compounds is ensured by a correspondingly large number of different enzymes. Three groups of enzymes whose reactions play a major role in secondary metabolism have been examined in this work: glucosyltransferases, terpene synthases and acyltransferases.

Recently, a UDP-glucose:cinnamate glucosyltransferase of the cultivated strawberry (Fragaria x ananassa) (FaGT2) was described, which catalyses the formation of cinnamic acid and p-coumaric acid glucose esters during fruit ripening. This enzyme, which is expressed in the strawberry fruit in a ripening- and stress-induced manner, however also converts further, structurally diverse substrates of natural and anthropogenic origin in vitro. Their reaction kinetics were compared in order to elucidate additional biological functions of the enzyme. The spectrum of accepted substrates ranged from cinnamic acid and benzoic acid derivatives to heterocyclic and aliphatic structures and resulted in the formation of O- and S-glucose esters as well as O-glucosides. The synthesis of the glucosidic compounds was confirmed in planta after the substrates had been injected into ripe strawberries. The easy deprotonation of the glucosylation site and the conjugation of the anions formed thereby with π-electrons were identified as common chemical and structural properties required for enzymatic activity. Sorbic acid represents the substrate with the simplest structure, which was converted with very high efficiency. In addition to cinnamic acid, anthranilic acid, (E)-2-hexenoic acid, nicotinic acid and 2,5-dimethyl-4-hydroxy-3[2H]-furanone were also glucosylated as natural substrates. The glucosidic products possibly serve as precursors of important aroma compounds or as storage forms. However, FaGT2 also converted xenobiotics highly efficiently, such as the herbicide 2,4,5-trichlorophenol or 3,5-dichloro-4-hydroxybenzoic acid, which is analogous to a herbicide metabolite. These results, in conjunction with the stress-induced expression of FaGT2, suggest that the enzyme is involved in the detoxification of xenobiotics.

In the fruits of the cultivated strawberry (Fragaria x ananassa) the monoterpene alcohol linalool, which is predominantly present as the (S)-enantiomer, contributes to the aroma. The biosynthesis of (S)-linalool in strawberry fruits was recently elucidated and is catalysed by the terpene synthase FaNES1. In the leaves of Fragaria x ananassa and above all in those of the wild strawberry (Fragaria vesca), a higher proportion of®-linalool is present besides (S)-linalool. By means of a polymerase chain reaction strategy (PCR strategy) exploiting the homology of terpene synthase genes, two partial sequences showing very high identities with FaNES1 were extracted from complementary DNA (cDNA) of leaves of the species Fragaria vesca and Fragaria x ananassa. FaLINS was cloned in full length from Fragaria x ananassa, the encoded protein was expressed in Escherichia coli and incubated with the substrate geranyl diphosphate (GPP). By means of multidimensional gas chromatography-mass spectrometry it was demonstrated that FaLINS, despite some sequence differences from FaNES1, likewise exclusively forms (S)-linalool. Apparently the two sequences are different alleles or paralogous genes that are differentially expressed in leaves and fruits. In Fragaria vesca a linalool synthase (FvNES) with unknown stereospecificity has already been detected, which is likewise homologous to FaNES1 but contains an additional N-terminal sequence element. This enzyme was heterologously expressed like FaLINS and investigated in an enzyme assay. Since it exclusively forms (S)-linalool, it can be excluded that the N-terminal sequence section influences the stereospecificity of the enzymatic reaction.

The economically important essential oil of true lavender (Lavandula angustifolia) contains a large number of mono- and sesquiterpenes. Similarly as in strawberry, the three new terpene synthases LaLIMS, LaLINS and LaBERS were cloned by means of a homology-based strategy. Phylogenetic analysis showed that their sequences resemble the terpene synthases from other members of the family Lamiaceae. The enzymes were heterologously expressed in Escherichia coli and biochemically characterised with respect to optimal reaction conditions (pH value, temperature and cofactor concentrations) and kinetic parameters.

LaLIMS forms the six monoterpenes limonene (39 %), terpinolene (22 %), camphene (16 %), α-pinene (14 %), β-myrcene (8 %) and α-phellandrene (1 %). Analysis by chiral-phase gas chromatography showed that the enantiomers (1R,5R)-()-α-pinene, (1R,4S)-()-camphene and®-(+)-limonene are synthesised in large excess. The assays furthermore revealed a different product composition when manganese and magnesium cations were used, which underlines the influence of these cofactors on the structure and reaction of the terpene synthases. Multiproduct synthases such as LaLIMS explain how the many different terpenes in the essential oil arise without a correspondingly large number of enzymes having to be present. LaLINS catalyses the transformation of GPP exclusively to®-linalool. The strongly preferred formation of the®-enantiomer corresponds to the configuration of the main constituents of lavender oil,®-linalool and®-linalyl acetate. Therefore the enzyme is likely to be of decisive importance for the aroma profile of Lavandula angustifolia. The third enzyme, LaBERS, is a sesquiterpene synthase and forms predominantly trans-α-bergamotene (74 %) from farnesyl diphosphate (FPP). It is the first trans-α-bergamotene synthase described. With low efficiency it also catalyses the formation of monoterpenes from GPP. Phylogenetically it belongs to class TPS-b of the terpene synthase family, in which almost exclusively monoterpene synthases are classified. It thus represents a further example of a sesquiterpene synthase that presumably emerged from a monoterpene synthase. Although the three terpene synthases are responsible for only a part of the terpenes of the essential oil of Lavandula angustifolia, knowledge of their gene sequences constitutes a basis enabling the targeted genetic modification of lavender plants and the influencing of their aroma profile.

In the essential oil of Lavandula angustifolia the esters of terpene alcohols and short-chain alcohols, above all linalyl acetate, also contribute substantially to the aroma. Since their biosynthesis is catalysed by acyltransferases of the BAHD superfamily, the two sequences LaAT1 and LaAT2 were cloned in full length on the basis of the common amino acid motif DFGWG. Phylogenetically, LaAT1 is related to acyltransferase subgroup V, which requires coenzyme A esters of hydroxycinnamic acids and benzoic acid as donor substrates and transfers the acid residues to the hydroxy or amino functions of quinic acid and shikimic acid as well as anthranilic acid and its derivatives. LaAT2 likewise contains all characteristic sequence elements of the BAHD acyltransferases, but overall shows little similarity to those described so far and could be a member of a new subgroup. LaAT1 and LaAT2 were heterologously expressed in Escherichia coli and Saccharomyces cerevisiae and incubated in a substrate screening with different coenzyme A esters and alcohols. Only LaAT1 was active in enzyme assays and, as the first representative of the BAHD superfamily, catalysed the formation of both amide and ester bonds, which resulted in caffeoyl and coumaroyl shikimate, caffeoyl and coumaroyl tyramine as well as coumaroyl anthranilate. Hitherto the synthesis of the tyramine derivatives in plants has been attributed exclusively to the family of tyramine N-hydroxycinnamoyltransferases, which differ clearly from the BAHD acyltransferases. LaAT1 is thus not involved in aroma biosynthesis, but could assume other important functions in secondary metabolism.