Galactomannans are hemicellulosic dietary fibers which can be fermented in the gut (Fåk et al. 2015). Here we report on the structure-function of enzyme systems for galactomannan digestion among common human gut bacteria, results which contribute to the design of prebiotics.
Gut bacteria may have different synergistic strategies for mannoside and galactomannan digestion. The studied Bifidobacteria express single surface exposed beta-mannanases from either of two glycoside hydrolase (GH) families: GH5 or GH26 (Kulcinskaja et al. 2013, Morrill et al. 2015). Bacteroides ovatus, on the other hand, expresses several GHs from a polysaccharide utilisation locus (PUL), a gene-cluster which is essential for galactomannan utilization (Reddy et al. 2016, Bågenholm et al. 2017). The GHs, two beta-mannanases (BoMan26A, BoMan26B) and an alpha-galactosidase (BoGal36A), act in a sequential manner. The two GH26 beta-mannanases were characterised, including solving the TIM-barrel crystal structures, contributing to a model of the combined function of the enzymes and binding proteins of this galactomannan PUL. BoMan26B is exposed on the cell surface (analysed by immuno-fluorescence) and makes the initial endo-attack on highly branched guar gum galactomannan, explained by the open and extended active site cleft visible in the recent crystal structure. Oligosaccharide products generated by BoMan26B bind (Kd 4 mM by micro-thermophoresis) to a surface-exposed glycan-binding SusD-homolog, predicted to guide saccharide import to the periplasm, where BoMan26A acts in synergy with the periplasmic BoGal36A (Bagenholm et al 2017) and efficiently releases mainly mannobiose from the oligo-saccharides via endo-/exo-hydrolysis, as suggested by time-course product analyses using anion-exchange chromatography (HPAEC) and MALDI mass spectrometry combined with solvent isotope labelling using 18O-water in hydrolysis reactions.
The narrow active site cleft of BoMan26A (Bågenholm et al 2017) and active site loop flexibility, recently studied with 1H, 13C, and 15N NMR, contribute to explain the different mode of attack compared to BoMan26B and selected GH5 beta-mannanases.
Phylogenetic analysis place BoMan26A and BoMan26B in different clades of family GH26, from which we can extrapolate a potentially similar set up with two distinct GH26 beta-mannanases in PUL-encoded systems among several other Bacteroidetes. The genetic data guided the selection of gut bacteria which then were fed on (galacto)-mannosides in co-cultures to address potential synergistic cross-feeding between species. For this, glycan conversion and up-take were analysed with HPAEC.
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