Glycoside-metabolizing oxidoreductase D3dgpA from human gut bacterium.

The Gfo/Idh/MocA family enzyme DgpA was known to catalyze the regiospecific oxidation of puerarin to 3"-oxo-puerarin in the presence of 3-oxo-glucose. Here, we discovered that D3dgpA, dgpA cloned from the human gut bacterium Dorea sp. MRG-IFC3, catalyzed the regiospecific oxidation of various C-/O-glycosides, including puerarin, in the presence of methyl ß-D-3-oxo-glucopyranoside. While C-glycosides were converted to 3"- and 2"-oxo-products by D3dgpA, O-glycosides resulted in the formation of aglycones and hexose enediolone from the 3"-oxo-products. From DFT calculations, it was found that isomerization of 3"-oxo-puerarin to 2"-oxo-puerarin required a small activation energy of 9.86 kcal/mol, and the O-glycosidic bond cleavage of 3"-oxo-products was also thermodynamically favored with a small activation energy of 3.49 kcal/mol. In addition, the reaction mechanism of D3dgpA was discussed in comparison to those of Gfo/Idh/MocA and GMC family enzymes. The robust reactivity of D3dgpA was proposed as a new general route for derivatization of glycosides.

Flavonoids Biotransformation by Human Gut Bacterium Dorea sp. MRG-IFC3 Cell-Free Extract.

Human gut bacterium Dorea sp. MRG-IFC3 is unique in that it is capable of metabolizing puerarin, an isoflavone C-glycoside, whereas it shows broad substrate glycosidase activity for the various flavonoid O-glycosides. To address the question on the substrate specificity, as well as biochemical characteristics, cell-free biotransformation of flavonoid glycosides was performed under various conditions. The results showed that there are two different enzyme systems responsible for the metabolism of flavonoid C-glycosides and O-glycosides in the MRG-IFC3 strain. The system responsible for the conversion of puerarin was inducible and comprised of two enzymes. One enzyme oxidizes puerarin to 3"-oxo-puerarin and the other enzyme converts 3"-oxo-puearin to daidzein. The second enzyme was only active toward 3"-oxo-puerarin. The activity of puerarin conversion to daidzein was enhanced in the presence of Mn2+ and NAD+. It was concluded that the puerarin C-deglycosylation by Dorea sp. MRG-IFC3 possibly adopts the same biochemical mechanism as the strain PUE, a species of Dorea longicatena.

C-Glycoside-Metabolizing Human Gut Bacterium, Dorea sp. MRG-IFC3.

Biochemical gut metabolism of dietary bioactive compounds is of great significance in elucidating health-related issues at the molecular level. In this study, a human gut bacterium cleaving C-C glycosidic bond was screened from puerarin conversion to daidzein, and a new, gram-positive C-glycoside-deglycosylating strain, Dorea sp. MRG-IFC3, was isolated from human fecal sample under anaerobic conditions. Though MRG-IFC3 biotransformed isoflavone C-glycoside, it could not metabolize other C-glycosides, such as vitexin, bergenin, and aloin. As evident from the production of the corresponding aglycons from various 7-O-glucosides, MRG-IFC3 strain also showed 7-O-glycoside cleavage activity; however, flavone 3-O-glucoside icariside II was not metabolized. In addition, for mechanism study, C-glycosyl bond cleavage of puerarin by MRG-IFC3 strain was performed in D2O GAM medium. The complete deuterium enrichment on C-8 position of daidzein was confirmed by 1H NMR spectroscopy, and the result clearly proved for the first time that daidzein is produced from puerarin. Two possible reaction intermediates, the quinoids and 8-dehydrodaidzein anion, were proposed for the production of daidzein-8d. These results will provide the basis for the mechanism study of stable C-glycosidic bond cleavage at the molecular level.