Molecular and biochemical characterization of the UDP-glucose: Anthocyanin 5-O-glucosyltransferase from Vitis amurensis

UDP-glucose: anthocyanin 5-O-glucosyltransferase from Vitis amurensis grapes (Va5GT), which catalyzes the glucosylation of anthocyanidin monoglucosides at the 5-O-position, is crucial for the biosynthesis of anthocyanidin bis-glucosides. [Display omitted] •A gene encoding Va5GT glucosyltransferase from V. amurensis was cloned and the recombinant protein was expressed in E. coli.•The enzymatic properties (substrate specificity and enzyme kinetics) of Va5GT were established.•The previously speculated subcellular location of 5GTs in the cytoplasm was confirmed.•Transcriptional expression of Va5GT in related V. amurensis cultivars was analyzed. Generally, red Vitis vinifera grapes only contain monoglucosidic anthocyanins, whereas most non-vinifera red grapes of the Vitis genus have both monoglucosidic and bis-glucosidic anthocyanins, the latter of which are believed to be more hydrophilic and more stable. Although previous studies have established the biosynthetic mechanism for formation of monoglucosidic anthocyanins, less attention has been paid to that of bis-glucosidic anthocyanins. In the present research, the full-length cDNA of UDP-glucose: anthocyanin 5-O-glucosyltransferase from Vitis amurensis Rupr. cv. ‘Zuoshanyi’ grape (Va5GT) was cloned. After acquisition and purification of recombinant Va5GT, its enzymatic parameters were systematically analyzed in vitro. Recombinant Va5GT used malvidin-3-O-glucoside as its optimum glycosidic acceptor when UDP-glucose was used as the glycosidic donor. Va5GT-GFP was found to be located in the cytoplasm by analyzing its subcellular localization with a laser-scanning confocal fluorescence microscope, and this result was coincident with its metabolic function of modifying anthocyanins in grape cells. Furthermore, the relationship between the transcriptional expression of Va5GT and the accumulation of anthocyanidin bis-glucosides during berry development suggested that Va5GT is a key enzyme in the biosynthesis of bis-glucosidic anthocyanins in V. amurensis grape berries.