Expression and characterization of the human intestinal bacterial enzyme which cleaves the c-glycosidic bond in 3″-oxo-puerarin

Abstract

Puerarin (daidzein 8-C-glucoside) is an isoflavone C-glucoside contained in the roots of Pueraria lobata OHWI. We have previously isolated the human intestinal bacterium, strain PUE, which metabolizes puerarin to daidzein, though the enzyme which cleaves C-glycosidic bond has not been clarified. Here, we identified one of the intermediates of enzymatic puerarin C-deglycosylation reaction as 3″-oxo-puerarin (1): C-3 in the glucose moiety connecting to hydroxyl is oxidized to ketone group. 1 was easily isomerized to the mixture of 1, 2″-oxo-puerarin (2a) and cyclic acetal (2b) of 2a in non-enzymatic condition. We identified the putative puerarin-metabolizing operon of strain PUE composed of 8 genes (dgpA–H). Among them, DgpB–C complex was expressed in Escherichia coli, which cleaved the C-glycosidic bond in 1 but not puerarin. These results suggested that the puerarin C-deglycosylation reaction is a two-step enzymatic reaction, including the oxidation reaction at C-3″ in puerarin to give 1, and the subsequent C-deglycosylation of 1 to provide daidzein.