Structural Biology and Analytical Chemistry Approaches for Characterizing <em>C</em>-Glycoside Metabolic Enzymes in Human Gut Microbiota

The gut microbiota has emerged as a key modulator of host health, particularly because of its capacity to metabolize dietary glycosides. While C-glycosides demonstrate superior chemical stability and resistance to enzymatic cleavage compared to O-glycosides, specialized gut bacteria can selectively metabolize them, improving their bioavailability. Since the identification of microbial C-glycosidase enzymes, the precise reaction mechanisms remain incompletely understood, and standardized methodologies for studying these processes are still not well-established. To address this gap, we introduced an integrated multidisciplinary approach combining structural biology, enzymology, and analytical techniques, including liquid chromatography-tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR), to systematically characterize these enzymatic activities. This comprehensive approach enables precise characterization of C-glycoside metabolism at molecular, enzymatic, and metabolic levels.