To obtain glycosyltransferases that are useful for structural and functional modification of bioactive compounds by glycosylation, an array of cDNAs encoding plant secondary product glycosyltransferases (PSPGs) were cloned from Chinese wolfberry (Lycium barbarum L.) fruits. The cloning strategy was based on high sequence conservation of the C-terminal region of PSPGs. Nine of these cDNAs were heterologously expressed in Escherichia coli, resulting in the production of catalytically active proteins, thereby allowing for potential specific glycosylation of a wide range of natural products. We found that one of these PSPGs, UGT73A10, uniquely displayed regiospecific glucosyl transfer activity toward flavan-3-ols (e.g., (+)-catechin and epigallocatechin gallate), which occur only rarely in nature as glucosides. Thus, the biochemistry of this enzyme was characterized in detail. The enzyme was then used as a biocatalyst of the regiospecific glucosylation of (+)-catechin. UGT73A10 was highly specific for the glycosyl donor, UDP-glucose, and showed broad acceptor specificity with the highest preference for naringenin (kcat/Km value, 0.097 s-1 μM-1). UGT73A10 was phylogenetically related to the flavonoid 7-O-glucosyltransferases and, in fact, glucosylated the 7-position of naringenin. UGT73A10-catalyzed glucosyl transfer to (+)-catechin was obtained in high yield (83%). Surprisingly, spectroscopic analyses showed that the transfer product was 4′-O-β-d-glucopyranoside, and not the 7-O-β-d-glucoside, of (+)-catechin. Stability studies showed that the transfer product was more stable than (+)-catechin under alkaline conditions and at elevated temperatures.
ASJC Scopus subject areas
- Process Chemistry and Technology