The Catalytic Acid–Base in GH109 Resides in a Conserved GGHGG Loop and Allows for Comparable α‑Retaining and β‑Inverting Activity in an <i>N</i>‑Acetylgalactosaminidase from <i>Akkermansia muciniphila</i>

Enzymes active on glycosidic bonds are defined according to the stereochemistry of both substrates and products of the reactions they catalyze. The CAZy classification further assigns these enzymes into sequence-based families sharing a common stereochemistry for substrates (either α- or β-) and products (i.e., inverting or retaining mechanism). Here we describe the N-acetylgalactosaminidases AmGH109A and AmGH109B (i.e., GH109: glycoside hydrolase family 109) from the human gut symbiont Akkermansia muciniphila. Notably, AmGH109A displays α-retaining and β-inverting N-acetylgalactosaminidase activities with comparable efficiencies on natural disaccharides. This dual specificity could provide an advantage in targeting a broader range of host-derived glycans. We rationalize this discovery through bioinformatics, structural, mutational, and computational studies, unveiling a histidine residing in a conserved GGHGG motif as the elusive catalytic acid–base of the GH109 family.