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

A human intestinal bacterium strain related to Dorea species, PUE, can metabolize the isoflavone C-glucoside puerarin (daidzein 8-C-glucoside) to daidzein and glucose. We reported previously that 3μ-oxo-puerarin is an essential reaction intermediate in enzymatic puerarin degradation, and we characterized a bacterial enzyme, the DgpB-DgpC complex, that cleaved the C-glycosidic bond in 3μ-oxopuerarin. However, the exact enzyme catalyzing the oxidation of the C-3μ hydroxyl in puerarin has not been identified. 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 acceptor. In the redox reaction, NAD(H) functioned as the cofactor, which bound tightly but noncovalently to DgpA. Kinetics analysis of DgpA revealed that the reaction proceeded via a ping-pong mechanism. Enzymatic C-deglycosylation of puerarin was achieved by a combination of recombinant DgpA, the DgpB-DgpC complex, and 3-oxo-glucose. In addition, the metabolite derived from the sugar moiety in the 3μ-oxo-puerarin-cleaving reaction catalyzed by the DgpB-DgpC complex was characterized as 1,5-anhydro-D-erythro-hex-1-en-3-ulose, suggesting that the C-glycosidic linkage is cleaved through a μ-elimination-like mechanism.