Deglycosylation of isoflavone C–glucoside puerarin by combination of two recombinant bacterial enzymes and 3–oxo–glucose

Abstract

AbstractC–Glucosides are resistant to glycoside hydrolase activity because the anomeric carbon of glucose is directly connected to aglycone via carbon-carbon bonding. A human intestinal bacterium strain PUE related to Dorea species can metabolize the isoflavone C–glucoside puerarin (daidzein 8–C–glucoside) to daidzein and glucose by more than three bacterial enzymes which have not been well-characterized. We previously reported that 3”–oxo–puerarin is an essential reaction intermediate in enzymatic puerarin degradation and characterized a bacterial enzyme of DgpB–C complex which cleaved the C–glycosidic bond in 3”–oxo–puerarin. However, the exact enzyme catalyzing the oxidation of C–3” hydroxyl in puerarin has not been identified, and the other metabolite corresponding to the precursor of D–glucose, derived from the sugar moiety in 3”–oxo–puerarin in the cleaving reaction catalyzed by the DgpB–C complex, remains unknown.In this study, we demonstrated that recombinant DgpA, a Gfo/Idh/MocA family oxidoreductase, catalyzed puerarin oxidation in the presence of 3–oxo–glucose as the hydride accepter. In addition, enzymatic C–deglycosylation of puerarin was achieved by a combination of recombinant DgpA, DgpB–C complex, and 3–oxo–glucose. Furthermore, the metabolite derived from the sugar moiety in 3”–oxo–puerarin cleaving reaction catalyzed by DgpB–C complex was characterized as 1,5–anhydro–D–erythro –hex–1–en–3–ulose, suggesting that the C–glycosidic linkage is cleaved through a β–elimination like mechanism.ImportanceOne important role of the gut microbiota is to metabolize dietary nutrients and supplements such as flavonoid glycosides. Ingested glycosides are metabolized by intestinal bacteria to more absorbable aglycones and further degradation products which show beneficial effects in humans. Although numerous glycoside hydrolases that catalyze O–deglycosylation have been reported, enzymes responsible for C–deglycosylation are still limited. In this study, we characterized enzymes involved in C–deglycosylation of puerarin from a human intestinal bacterium PUE. To our knowledge, this is the first report of the expression, purification and characterization of an oxidoreductase involved in C–glucoside degradation. This study provides new insights for the elucidation of mechanisms of enzymatic C–deglycosylation.