Mechanistic insights into glycoside 3-oxidases involved in C-glycoside metabolism in soil microorganisms

C -glycosides are natural products with important biological activities but are recalcitrant to degradation. Glycoside 3-oxidases (G3Oxs) are recently identified bacterial flavo-oxidases from the glucose-methanol-coline (GMC) superfamily that catalyze the oxidation of C -glycosides with the concomitant reduction of O 2 to H 2 O 2 . This oxidation is followed by C-C acid/base-assisted bond cleavage in two-step C -deglycosylation pathways. Soil and gut microorganisms have different oxidative enzymes, but the details of their catalytic mechanisms are largely unknown. Here, we report that Ps G3Ox oxidizes at 50,000-fold higher specificity ( k cat /K m ) the glucose moiety of mangiferin to 3-keto-mangiferin than free D-glucose to 2-keto-glucose. Analysis of Ps G3Ox X-ray crystal structures and Ps G3Ox in complex with glucose and mangiferin, combined with mutagenesis and molecular dynamics simulations, reveal distinctive features in the topology surrounding the active site that favor catalytically competent conformational states suitable for recognition, stabilization, and oxidation of the glucose moiety of mangiferin. Furthermore, their distinction to pyranose 2-oxidases (P2Oxs) involved in wood decay and recycling is discussed from an evolutionary, structural, and functional viewpoint. Integrated experimental and computational approaches reveal functional and structural details of a key catabolic enzyme that oxidizes recalcitrant C-glycosides, abundant and biologically significant natural molecules, before deglycosylation.