Teune_2025_Nat.Commun_16_7034

Carbohydrate esterases modify polysaccharides by removing different ester moieties thereby affecting their physicochemical properties and their accessibility by glycoside hydrolases. We determined the full-length structures of two members (Fl8CE20_II and PpCE20_II) from the carbohydrate esterase family 20 (CE20) by X-ray crystallography that feature an ancillary domain, inserted into the catalytic SGNH-hydrolase domain. Detailed structural analysis identifies a so far undescribed catalytic triad architecture which lacks the typical aspartate for polarization of the histidine but instead reveals a precisely coordinated water molecule mediating contact between the His and Asp. This coordinated water in the Ser-His-(H(2)O-Asp/Asn) motif, as further confirmed by mutational studies and by determination of kinetic constants, is crucial for catalytic activity. We therefore term this active site architecture a water-mediated catalytic triad.