Purification, molecular cloning and functional characterization of flavonoid C‐glucosyltransferases from Fagopyrum esculentum M. (buckwheat) cotyledon

Abstract

Summary C‐Glycosides are characterized by their C–C bonds in which the anomeric carbon of the sugar moieties is directly bound to the carbon atom of aglycon. C‐Glycosides are remarkably stable, as their C–C bonds are resistant to glycosidase or acid hydrolysis. A variety of plant species are known to accumulate C‐glycosylflavonoids; however, the genes encoding for enzymes that catalyze C‐glycosylation of flavonoids have been identified only from Oryza sativa (rice) and Zea mays (maize), and have not been identified from dicot plants. In this study, we identified the C‐glucosyltransferase gene from the dicot plant Fagopyrum esculentum M. (buckwheat). We purified two isozymes from buckwheat seedlings that catalyze C‐glucosylation of 2‐hydroxyflavanones, which are expressed specifically in the cotyledon during seed germination. Following purification we isolated the cDNA corresponding to each isozyme [FeCGTa (UGT708C1) and FeCGTb (UGT708C2)]. When expressed in Escherichia coli, both proteins demonstrated C‐glucosylation activity towards 2‐hydroxyflavanones, dihydrochalcone, trihydroxyacetophenones and other related compounds with chemical structures similar to 2′,4′,6′‐trihydroxyacetophenone. Molecular phylogenetic analysis of plant glycosyltransferases shows that flavonoid C‐glycosyltransferases form a different clade with other functionally analyzed plant glycosyltransferases.

Production of <i>C</i>-glucosides of flavonoids and related compounds by <i>Escherichia coli</i> expressing buckwheat <i>C</i>-glucosyltransferase

“…We attempted to produce the C-glucosides of flavonoids and related compounds, using an E. coli strain expressing FeCGTa (Ec-FeCGT). The FeCGTa enzyme catalyzes the C-glucosylation of the open-circular form of 2-hydroxylflavanone and compounds possessing a THAP-like structure, shown in Figure 1 (Nagatomo et al 2014). The substrate dihydrochalcone phloretin (200 µM) was added to the Ec-FeCGT culture, and the medium contents were monitored at appropriate intervals.…”

“…2-Hydroxypinocembrin and 2-hydroxynaringenin were synthesized from chrysin and apigenin, respectively, as previously described (Nagatomo et al 2014). All other chemicals were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), Nacalai Tesque (Kyoto, Japan), and Kanto Chemical Co., Inc. (Tokyo, Japan), unless otherwise specified.…”

“…LC-MS analysis was performed using a Waters UPLC ACQUITY SQD system (Waters) with an electron-spray ionization probe, as described previously (Nagatomo et al 2014).…”

“…AB909375) cDNA subcloned into pET28a(+) (Merck, Darmstadt, Germany) was transformed into E. coli Rosetta ™ 2(DE3) (Merck), as described previously (Nagatomo et al 2014). An aliquot (1 ml) of overnight culture was added to 200 ml of LB medium (Nacalai Tesque) containing kanamycin (50 mg l −1 ) and chloramphenicol (34 mg l −1 ), and this culture was incubated at 37°C with shaking at 150-200 rpm until the OD 600 reached 0.6.…”

“…We recently characterized CGTs from buckwheat (FeCGTa and FeCGTb;Nagatomo et al 2014). These CGTs showed significant activity against flavonoid substrates possessing a basic skeleton of 2′,4′,6′-trihydroxyacetophenone (THAP), such as 2-hydroxyflavanones and dihydroxychalcone.…”