Functional characterization of terpene glycosyltransferases from Vitis vinifera
Zusammenfassung der Projektergebnisse
Monoterpenols serve various biological functions and accumulate in grapes (Vitis vinifera) where a major fraction occurs as non-volatile glycosides. We have screened the V. vinifera genome for sequences with similarity to terpene uridine diphosphate glycosyltransferases (UGT) from Arabidopsis thaliana. Spatial and temporal expression levels of potential VvGT (Vitis vinifera glycosyltransferase) genes were determined in five different grape varieties. A ripening related expression pattern was shown for four candidates. Transcript accumulation correlated with the production of monoterpenyl ß-D-glucosides in grape exocarp during ripening and was low in vegetative tissue. Heterologous expression and biochemical assays of candidate genes led to the identification of UDP-glucose:monoterpenol β-D-glucosyltransferases (VvGT7, 14 and 15). The VvGT7 gene was expressed in various tissues in accordance with monoterpenyl glucoside accumulation in V. vinifera cultivars. Twelve allelic VvGT7 genes were isolated from five cultivars and their encoded proteins biochemically analyzed. They varied in substrate preference and catalytic activity. Three amino acids, which corresponded to none of the determinants previously identified for other plant GTs were found to be important for enzymatic catalysis. Site specific mutagenesis along with the analysis of allelic proteins also revealed amino acids that impact catalytic activity and substrate tolerance. Targeted functional screening of recombinant UGTs for their biological substrates was also performed by activity based metabolite profiling (ABMP) employing for the first time a physiologic library of aglycones build from glycosides isolated from grapes. This approach led to the identification of UDP-glucose:monoterpenol β-D-glucosyltransferases VvGT14 and VvGT15. Whereas VvGT14a glucosylated geraniol, R,S-citronellol, and nerol with similar efficiency, the three allelic forms VvGT15a-c preferred geraniol over nerol. Kinetic resolution of R,S-citronellol and R,S-linalool was shown for VvGT15a and VvGT14a, respectively. ABMP revealed geraniol as major biological substrate but also disclosed that these UGTs may add to the production of further glycoconjugate in planta. ABMP of aglycone libraries provides a versatile tool to uncover novel biologically relevant substrates of small molecule glycosyltransferases that often show broad sugar acceptor promiscuity.
Projektbezogene Publikationen (Auswahl)
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A UDP-glucose:monoterpenol glucosyltransferase adds to the chemical diversity of the grapevine metabolome (Vitis vinifera L). Plant Physiology 165 (2014), 561-581
F. Bönisch, J. Frotscher, S. Stanitzek, E. Rühl, M. Wüst, O. Bitz, W. Schwab
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Activity based profiling of a physiologic aglycone library reveals sugar acceptor promiscuity of family 1 UDP-glucosyltransferases from Vitis vinifera. Plant Physiology (2014)
F. Bönisch, J. Frotscher, S. Stanitzek, E. Rühl, M. Wüst, O. Bitz, W. Schwab
