Enzymatic Cleavage of the C-Glucosidic Bond of Puerarin by Three Proteins, Mn2+, and Oxidized Form of Nicotinamide Adenine Dinucleotide

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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, Mn²⁺, 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 D₂O condition, which closely resembles those of glycoside hydrolase family 4 and 109.