Enantioselectivities of Uridine Diphosphate-Glucose:Monoterpenol

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Chapter 5

Enantioselectivities of Uridine Diphosphate-Glucose:Monoterpenol Glucosyltransferases from Grapevine (Vitis vinifera L.) Friedericke Bönisch,1 Johanna Frotscher,2 Sarah Stanitzek,3 Ernst Rühl,3 Oliver Bitz,3 Wilfried Schwab,1 and Matthias Wüst*,2 1Biotechnology

of Natural Products, Technische Universität München, Liesel-Beckmann-Strasse 1, 85354 Freising, Germany 2Geisenheim University, Department of Grapebreeding, Von-Lade-Strasse 1, 65366 Geisenheim, Germany 3Bioanalytics, Institute of Nutritional and Food Sciences, University of Bonn, Endenicher Allee 11-13, D-53115 Bonn, Germany *E-mail: [email protected].

Aroma-active substances, like monoterpene alcohols, often occur as non-aromatic, glycosidically bound forms and accumulate during grape ripening. Although these compounds are a precious source of aroma, little is known about the enzymes catalyzing this glycosylation. Recently, heterologous expression and biochemical assays of candidate genes extracted from the V. vinifera (Vv) genome database has led to the identification of several UDP-glucose:monoterpenol β-D-glucosyltransferases (UDP-Glc GTs). Here, kinetic resolution of racemic monoterpenols is demonstrated for several of these GTs. Thus, the enantioselective “aroma-hiding” biochemistry of these enzymes is shown for the first time in grapevine.

© 2015 American Chemical Society Engel and Takeoka; Importance of Chirality to Flavor Compounds ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Terpenoids represent one of the major classes of natural products and are used in many applications, from health care and pharmaceutical uses to color, flavor and fragrance compounds in food and cosmetics. Biosynthesis in grapevine occurs through either the mevalonic acid or the 1-deoxy-D-xylulose 5-phosphate pathway (1–3). In plants, terpenoids are further conjugated with sugars which are linked to the active groups OH and/or COOH. In grapes (Vitis vinifera) and wines, where the importance of monoterpenes to varietal flavor is widely recognized, a major fraction of these compounds is present as non-volatile, aroma-inactive terpene glycosides (Figure 1) (4, 5).

Figure 1. Structures of selected free monoterpenols and their glycosylated conjugates found in grapes and wines.

Although this water-soluble fraction is a precious source of aroma, little is known about the genes and their encoded enzymes catalyzing the glycosylation of terpenols in grapes (6). A functional analysis of Arabidopsis thaliana glycosyltransferase (GT) genes has yielded 27 sequences whose encoded proteins glucosylate a diversity of terpenes (7). In a previous project, we identified 67 homologous, putative GT sequences in the Vitis vinifera genome database and performed spatial and temporal expression analysis of nine selected potential VvGT genes in different grapes varieties (8). Comparison with the levels of terpene glycosides in different tissues and heterologous expression followed by biochemical characterization yielded the first three monoterpenyl GTs from V. vinifera (9). Here, we demonstrate that these GTs are able to discriminate between the enantiomers of the chiral monoterpenols linalool and citronellol. 78 Engel and Takeoka; Importance of Chirality to Flavor Compounds ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

Experimental Plant Material and Chemicals


V. vinifera grapevines of different cultivars (Gewürztraminer, Muscat, White Riesling) were grown in the Geisenheim University vineyard at Geisenheim, Germany, during vintages 2011 and 2012. Grape berries were collected as previously described between 4 and 18 weeks after bloom (8, 9). Citronellyl ß-D-glucoside was synthesized according to the Koenigs-Knorr-procedure (10). Linaloyl ß-D-glucoside was synthesized from linalool, as less reactive tertiary alcohol, according to a modified Koenigs-Knorr-procedure, using another catalyst (11). Spectral data of the synthesized compounds were in all cases in good agreement with previously published data. Transcription Analysis, Heterologous Protein Expression, and Assays RNA was extracted from different grape tissues and transcription analysis of seven putative GT genes was performed by GeXP profiling (12). Furthermore, DNA was extracted and comparative sequencing was performed as previously described (9). Recombinant protein was expressed as GST fusion proteins in E. coli BL21 (DE3) pLysS. After a purification step by GST bind resin (8), activity assays were conducted. A reaction mixture containing buffer solution, UDP-Glucose, racemic substrate and enzyme was applied, and activity measurements were carried out via GC/MS and LC-MS/MS measurements. To determine the enantioselectivities of the GTs the enantiomeric ratios of glucosidically bound citronellol and linalool were determined by enantioselective GC-MS. Following the incubation, residual citronellol and linalool were completely removed by extraction with dichloromethane. Citronellyl ß-D-glucoside which remained in the aqueous phase was hydrolyzed by HCl (2 mL, 0.1 M, pH 1) for one hour at 100 °C to release citronellol (13). In case of linalyl ß-D-glucoside, an enzymatic hydrolysis (AR 2000, citric acid buffer pH 4, 24 h) was applied due to the instability of linalool in acid solutions (14). Hydrolysis of a synthetic 1:1 mixture of (R)- and (S)-linalyl ß-D-glucoside revealed that AR 2000 does not discriminate between the two diastereomeric glucosides. After hydrolysis, citronellol and linalool were analyzed by SPME-GC/MS as previously described (9). GC-MS and LC-MS/MS Analysis A Varian Saturn 3900 GC/Varian Saturn 2100T-MS ion-trap was used for the analysis of free monoterpenes. EI (electron impact ionization)-MS spectra were recorded from m/z 40 to 300 (ionization energy 70 eV). The column was a DiAcß (heptakis-(2,3-di-O-acetyl-6-O-tert-butyldimethylsilyl)-ß-cyclodextrin), 26 m x 0.32 mm i.d. with a 0.1 µm film. For HPLC-MS/MS analysis of monoterpenyl-ß-D-glucosides, an HP 1050 HPLC system coupled to an API 2000 (Applied Biosystems, AB Sciex, Framingham, USA) triple-quadrupole-MS was used. The RP-column was eluted with a linear gradient of water/acetonitrile containing 0.2% ammonia. The column temperature was maintained at 40 79 Engel and Takeoka; Importance of Chirality to Flavor Compounds ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

°C. The mass spectrometer was operated in ESI-MRM negative ion mode. Nitrogen was used as curtain (setting 20), nebulizing and collision gas (setting 1; collision energy was -20 eV). Monoterpenyl ß-D-glucosides were identified by the following characteristic MRM transitions: LinGlc: m/z 315→161(Glu), 315→113(Glu); CitrGlc: m/z 317→101(Glu), 317→161(Glu).


Results and Discussion Previous research has shown that monoterpenol ß-glucosides in Vitis vinifera are formed by the action of glycosyltransferases (GTs) that catalyze the transfer of glucose from uridine diphosphate glucose (UDP-Glc) to the acceptor molecule (Figure 2) (8, 9). These GTs recognize small-molecule scaffolds and belong to the so-called class 1 family that are characterized by a conserved plant secondary product GT motif (PSPG motif) (7). Beside the glucosylation of a rather broad monoterpenol spectrum, these GTs show also considerable activities toward aliphatic alcohols and benzoic compounds and thus add to the chemical diversity of the grapevine metabolome. Because numerous monoterpenols in V. vinifera are chiral (e.g., citronellol, linalool, hotrienol and alpha-terpineol; Figure 1) the enantioselectivity of these GTs is of interest. The relationship between the absolute configuration and the odor quality of chiral aroma compounds is well known and an enantiodiscriminating effect of GTs might thus modulate the aroma quality of the final wine (15). To determine the enantioselectivities of GTs that were shown to catalyze the glucosylation of chiral monoterpenols, racemic mixtures of citronellol and linalool were incubated with three different GTs, namely VvGT7, VvGT14a, and VvGT15a. The identities of the generated ß-glucosides were confirmed by the synthesis of reference compounds and analysis by LC-MS/MS. Because the diastereomeric glucosides of the respective monoterpenol enantiomers could not be separated by LC, the generated glucosides were hydrolyzed and the enantiomeric ratios of the liberated aglycons were determined by enantioselective GC/MS using a modified cyclodextrin as chiral selector. Figure 3A/B shows the results of the enantioselective analysis of citronellol, liberated by acid-catalyzed hydrolysis from its glucoside, that was generated from racemic citronellol and VvGT7 (linalool was not glucosylated by VvGT7). VvGT7 clearly preferred (R)- over (S)-citronellol even in long term assays. However, VvGT14a and VvGT15a preferentially glucosylated (S)-citronellol in short-term assays and enantioselectivity was not observed in long-term studies. This kinetic effect is characteristic for studies on the kinetic resolution of racemates. In this context it is noteworthy that free and glycosylated citronellol in grape berries is usually (S)-configured with an enantiomeric excess greater than 90% (16). Thus, it is obvious that VvGT7 is probably not involved in the production of (S)-citronellyl-ß-D-glucoside. VvGT14a also glucosylated linalool and preferred (R)-linalool even in long term assays (Figure 3D). In grape berries free linalool is usually present as S-configured enantiomer with enantiomeric purities up to 99.8 % in Muscat varieties (17, 18). However, in the glycosidically bound fraction of linalool in Morio Muscat and Muscat Ottonel 80 Engel and Takeoka; Importance of Chirality to Flavor Compounds ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


the (R)-enantiomer is enriched (up to 12.7%) (17). This previously observed enantiodiscrimination can be explained by the preference of VvGT14a for the (R)-enantiomer as described above.

Figure 2. Sequential biosynthesis of glycosides by glycosyltransferases. GT1 is a glucosyltransferase while GT2 is a postulated apiosyltransferase which has not yet been characterized in V. vinifera.

Figure 3. Separation of racemic mixtures of citronellol (A) and linalool (C) by enantioselective GC. When racemic citronellol is incubated with VvGT7 and the generated glucoside is hydrolyzed only (R)-citronellol is detected (B). The analogous reaction sequence using VvGT14a and racemic linalool yields (R)-enriched linalool (D). x are impurities. 81 Engel and Takeoka; Importance of Chirality to Flavor Compounds ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

Conclusions UDP-glucose:monoterpenol-GTs from V. vinifera are able to discriminate between the enantiomers of linalool and citronellol. Thus, the enantiomeric ratios of free and glycosylated chiral monoterpenols might differ considerably. The potential aroma of the bound fraction of volatiles is therefore not only modulated by the substrate specificities of the involved GTs but also by their enantioselectivities.


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83 Engel and Takeoka; Importance of Chirality to Flavor Compounds ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

Enantioselectivities of Uridine Diphosphate-Glucose:Monoterpenol

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