A pair of esterases from a commensal gut bacterium remove acetylations from all positions on complex β-mannans.

β-mannans and xylans are important components of the plant cell wall and they are acetylated to be protected from degradation by glycoside hydrolases. β-mannans are widely present in human and animal diets as fiber from leguminous plants and as thickeners and stabilizers in processed foods. There are many fully characterized acetylxylan esterases (AcXEs); however, the enzymes deacetylating mannans are less understood. Here we present two carbohydrate esterases, CE2 and CE17, from the Firmicute , which together deacetylate complex galactoglucomannan (GGM). The three-dimensional (3D) structure of CE17 with a mannopentaose in the active site shows that the CBM35 domain of CE17 forms a confined complex, where the axially oriented C2-hydroxyl of a mannose residue points toward the Ser41 of the catalytic triad. Cavities on the CE17 surface may accept galactosylations at the C6 positions of mannose adjacent to the mannose residue being deacetylated (subsite -1 and +1). In-depth characterization of the two enzymes using time-resolved NMR, high-performance liquid chromatography (HPLC), and mass spectrometry demonstrates that they work in a complementary manner. CE17 exclusively removes the axially oriented 2--acetylations on any mannose residue in an oligosaccharide, including double acetylated mannoses, while the CE2 is active on 3-, 4-, and 6-acetylations. Activity of CE2 is dependent on CE17 removing 2--acetylations from double acetylated mannose. Furthermore, transacetylation of oligosaccharides with the 2--specific CE17 provided insight into how temperature and pH affects acetyl migration on manno-oligosaccharides.