Functional Characterization of Glycosyltransferases from Saussurea involucrata Involved in Rutin Biosynthesis

Saussurea involucrata is a medicinal plant rich in flavonoids, but the glycosyltransferases (GTs) responsible for flavonoid glycosylation in this species remain largely uncharacterized. Here, five GT genes (SiUGT03, SiUGT09, SiUGT60, SiRhaT68-1, and SiRhaT68-2) were identified. Four of the five proteins (SiUGT03, SiUGT09, SiUGT60, and SiRhaT68-1) possessed an intact PSPG-box, and phylogenetic analysis placed SiUGT03 in the 3GT clade and SiRhaT68-1 in the 1,6GT clade. Recombinant proteins were expressed in Escherichia coli as soluble maltose-binding protein (MBP) fusions and purified. In vitro enzymatic assays showed that SiUGT03 catalyzed the 3-O-glucosylation of quercetin to isoquercetin (quercetin-3-O-glucoside), whereas SiRhaT68-1 catalyzed the 1,6-rhamnosylation of isoquercetin to rutin. Among the substrates tested, SiUGT03 was active only toward the flavonols quercetin and kaempferol, while SiRhaT68-1 was active toward isoquercetin and one of the four tested flavone 7-O-glucosides, diosmetin-7-O-glucoside. Kinetic analysis revealed an apparent difference in catalytic efficiency between the two enzymes under the conditions tested (SiUGT03: Km = 36.08 μM, kcat/Km = 0.01983 μM−1·s−1; SiRhaT68-1: Km = 184.70 μM, kcat/Km = 0.0005755 μM−1·s−1); however, since the Km of SiRhaT68-1 was close to the highest substrate concentration tested, this difference should be regarded as preliminary. These results, obtained from separate in vitro assays, demonstrated that the two enzymes catalyze two consecutive terminal glycosylation steps of rutin biosynthesis and represent promising candidates for further exploration of rutin biosynthesis in S. involucrata.