beta-Glucosyl Fluoride as Reverse Reaction Donor Substrate and Mechanistic Probe of Inverting Sugar Nucleotide-Dependent Glycosyltransferases

摘要

For a set of flavonoid O- and C-beta-glycosyltransferases, we show that beta-glucosyl fluoride can function as substrate for an enzymatic reaction wherein uridine 5'-diphosphate (UDP) alpha-glucose is synthesized in the presence of UDP. In pH and mutagenesis studies of the C-glycosyltransferase from rice, we show that reaction with the beta-glucosyl fluoride can serve to identify the acid-base catalytic residue of the enzyme (His24). We also show that beta-glucosyl fluoride can rescue activity in an enzyme variant (Ile121Asp) strongly impaired in the canonical reaction wherein flavonoid acceptor is glucosylated from UDP-glucose. Coupling of this variant with the wildtype C-glycosyltransferase in a one-pot reaction enabled efficient 3'-beta-C-glucosylation of phloretin from beta-glucosyl fluoride in the presence of substochiometric amounts of UDP.