Enzymatic cleavage of the C-glucosidic bond of puerarin by three proteins, Mn(2+), and oxidized form of nicotinamide adenine dinucleotide.

We previously isolated the human intestinal bacterium, strain PUE, which can cleave the C-glucosidic bond of puerarin to yield its aglycone daidzein and glucose. In this study, we partially purified puerarin C-glucosidic bond cleaving enzyme from the cell-free extract of strain PUE and demonstrated that the reaction was catalyzed by at least three proteins, Mn(2+), and oxidized form of nicotinamide adenine dinucleotide (NAD(+)). We completely purified one of the proteins, called protein C, by chromatographic separation in three steps. The molecular mass of protein C was approximately 40 kDa and the amino acid sequence of its N-terminal region shows high homology to those of two putative proteins which belong to Gfo/Idh/MocA family oxidoreductase. Protein C catalyzed hydrogen-deuterium exchange reaction of puerarin to 2"-deuterated puerarin in D(2)O condition, which closely resembles those of glycoside hydrolase family 4 and 109.