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

Abstract

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, <i>Ri</i>CE2 and <i>Ri</i>CE17, from the Firmicute <i>Roseburia intestinalis</i>, which together deacetylate complex galactoglucomannan (GGM). The three-dimensional (3D) structure of <i>Ri</i>CE17 with a mannopentaose in the active site shows that the CBM35 domain of <i>Ri</i>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 <i>Ri</i>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. <i>Ri</i>CE17 exclusively removes the axially oriented 2-<i>O</i>-acetylations on any mannose residue in an oligosaccharide, including double acetylated mannoses, while the <i>Ri</i>CE2 is active on 3-<i>O-</i>, 4-<i>O-</i>, and 6-<i>O-</i>acetylations. Activity of <i>Ri</i>CE2 is dependent on <i>Ri</i>CE17 removing 2-<i>O</i>-acetylations from double acetylated mannose. Furthermore, transacetylation of oligosaccharides with the 2-<i>O</i>-specific <i>Ri</i>CE17 provided insight into how temperature and pH affects acetyl migration on manno-oligosaccharides.