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, RiCE2 and RiCE17, from the Firmicute Roseburia intestinalis, which together deacetylate complex galactoglucomannan (GGM). The three-dimensional (3D) structure of RiCE17 with a mannopentaose in the active site shows that the CBM35 domain of RiCE17 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 RiCE17 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. RiCE17 exclusively removes the axially oriented 2-O-acetylations on any mannose residue in an oligosaccharide, including double acetylated mannoses, while the RiCE2 is active on 3-O-, 4-O-, and 6-O-acetylations. Activity of RiCE2 is dependent on RiCE17 removing 2-O-acetylations from double acetylated mannose. Furthermore, transacetylation of oligosaccharides with the 2-O-specific RiCE17 provided insight into how temperature and pH affects acetyl migration on manno-oligosaccharides.
Commensal butyrate-producing bacteria belonging to the Firmicutes phylum are abundant in the human gut and are crucial for maintaining health. Currently, insight is lacking into how they target otherwise indigestible dietary fibers and into the trophic interactions they establish with other glycan degraders in the competitive gut environment.
Non-carbohydrate modifications such as acetylations are widespread in food stuffs as well as they play important roles in diverse biological processes. These modifications meet the gut environment and are removed from their carbohydrate substrates by the resident microbiota. Among the most abundant modifications are O-acetylations, contributing to polysaccharides physico-chemical properties such as viscosity and gelling ability, as well as reducing accessibility for glycosyl hydrolases, and thus hindering polysaccharide degradation. Of particular note, O-acetylations increase the overall complexity of a polymer, thus requiring a more advanced degrading machinery for microbes to utilize it. This minireview describes acetylesterases from the gut microbiota that deacetylate various food polysaccharides, either as natural components of food, ingredients, stabilizers of microbial origin, or as part of microbes for food and beverage preparations. These enzymes include members belonging to at least 8 families in the CAZy database, as well as a large number of biochemically characterized esterases that have not been classified yet. Despite different structural folds, most of these acetylesterases have a common acid–base mechanism and belong to the SGNH hydrolase superfamily. We highlight examples of acetylesterases that are highly specific to one substrate and to the position of the acetyl group on the glycosyl residue of the carbohydrate, while other members that have more broad substrate specificity. Current research aimed at unveiling the functions and regioselectivity of acetylesterases will help providing fundamental mechanistic understanding on how dietary components are utilized in the human gut and will aid developing applications of these enzymes to manufacture novel industrial products.
ABSTRACTβ-Mannans are hemicelluloses that are abundant in modern diets as components in seed endosperms and common additives in processed food. Currently, the collective understanding of β-mannan saccharification in the human colon is limited to a few keystone species, which presumably liberate low-molecular-weight mannooligosaccharide fragments that become directly available to the surrounding microbial community. Here we show that a dominant butyrate-producer in the human gut, Faecalibacterium prausnitzii, is able to acquire and degrade various β-mannooligosaccharides (β-MOS), which are derived by the primary mannanolytic activity of neighboring gut microbiota. Detailed biochemical analyses of selected protein components from their two β-mannooligosaccharides (β-MOS) utilization loci (FpMULs) supported a concerted model whereby the imported β-MOS are stepwise disassembled intracellularly by highly adapted enzymes. Coculturing experiments of F. prausnitzii with the primary degrader Bacteroides ovatus on polymeric β-mannan resulted in syntrophic growth and production of butyrate, thus confirming the high efficiency of the FpMULs’ uptake system. Genomic comparison with human F. prausnitzii strains and analyses of 2441 public human metagenomes revealed that FpMULs are highly conserved and distributed worldwide. Together, our results provide a significant advance in the knowledge of β-mannans metabolism and the degree to which its degradation is mediated by cross-feeding interactions between prominent beneficial microbes in the human gut.ImportanceCommensal butyrate-producing bacteria belonging to the Firmicutes phylum are abundant in the human gut and are crucial for maintaining health. Currently, insight is lacking into how they target otherwise indigestible dietary fibers and into the trophic interactions they establish with other glycan degraders in the competitive gut environment. By combining cultivation, genomic and detailed biochemical analyses this work reveals the mechanism enabling F. prausnitzii, as a model clostridial cluster IV Firmicute, to cross-feed and access β-mannan-derived oligosaccharides released in the gut ecosystem by the action of primary degraders. A comprehensive survey of human gut metagenomes shows that FpMULs are ubiquitous in human populations globally, highlighting the importance of microbial metabolism of β-mannans/β-MOS as a common dietary component. Our findings provide a mechanistic understanding of the β-MOS utilization capability by F. prausnitzii that may be exploited to select dietary formulations specifically boosting this beneficial symbiont, thus butyrate production, in the gut.
Abstractβ-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, RiCE2 and RiCEX, from the Firmicute Roseburia intestinalis, which together deacetylate complex galactoglucomannan (GGM). The 3D-structure of RiCEX with a mannopentaose in the active site shows that the CBM35 domain of RiCEX forms a confined complex, where the axially oriented C2-hydroxyl of a mannose residue points towards the Ser41 of the catalytic triad. Cavities on the RiCEX 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, HPLC and mass spectrometry demonstrates that they work in a complementary manner. RiCEX exclusively removes the axially oriented 2-O-acetylations on any mannose residue in an oligosaccharide, including double acetylated mannoses, while the RiCE2 is active on 3-O-, 4-O- and 6-O-acetylations. Activity of RiCE2 is dependent on RiCEX removing 2-O-acetylations from double acetylated mannose. Furthermore, transacetylation of oligosaccharides with the 2-O specific RiCEX provided new insight to how temperature and pH affects acetyl migration on mannooligosaccharides.Significance statementAcetylations are an important feature of hemicellulose, altering the physical properties of the plant cell wall, and limiting enzyme accessibility. Removal of acetyl groups from beta-mannan is a key step towards efficient utilization of mannans as a carbon source for gut microbiota and in biorefineries. We present detailed insight into mannan deacetylation by two highly substrate-specific acetyl-mannan esterases (AcMEs) from a prevalent gut commensal Firmicute, which cooperatively deacetylate complex galactoglucomannan. The 3D structure of RiCEX with mannopentaose in the active site has a unique two-domain architecture including a CBM35 and an SGNH superfamily hydrolytic domain. Discovery of mannan specific esterases improves the understanding of an important step in dietary fiber utilization by gut commensal Firmicutes.
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