Enzymatic β-elimination in natural product O- and C-glycoside deglycosylation

Abstract Biological degradation of natural product glycosides involves, alongside hydrolysis, β-elimination for glycosidic bond cleavage. Here, we discover an O -glycoside β-eliminase (OGE) from Agrobacterium tumefaciens that converts the C3-oxidized O -β- d -glucoside of phloretin (a plant-derived flavonoid) into the aglycone and the 2-hydroxy-3-keto-glycal elimination product. While unrelated in sequence, OGE is structurally homologous to, and shows effectively the same Mn 2+ active site as, the C -glycoside deglycosylating enzyme (CGE) from a human intestinal bacterium implicated in β-elimination of 3-keto C -β- d -glucosides. We show that CGE catalyzes β-elimination of 3-keto O - and C -β- d -glucosides while OGE is specific for the O -glycoside substrate. Substrate comparisons and mutagenesis for CGE uncover positioning of aglycone for protonic assistance by the enzyme as critically important for C -glycoside cleavage. Collectively, our study suggests convergent evolution of active site for β-elimination of 3-keto O -β- d -glucosides. C -Glycoside cleavage is a specialized feature of this active site which is elicited by substrate through finely tuned enzyme-aglycone interactions.