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Gut microbiota members from the Bacteroidota phylum play a pivotal role in mammalian health and metabolism. They thrive in this diverse ecosystem due to their notable ability to cope with distinct recalcitrant dietary glycans via polysaccharide utilization loci (PULs). Our study reveals that a PUL from an herbivore gut bacterium belonging to the Bacteroidota phylum, with a gene composition similar to that in the human gut, exhibits extended functionality. While the human gut PUL targets mixed-linkage β-glucans specifically, the herbivore gut PUL also efficiently processes linear and substituted β-1,3-glucans. This gain of function emerges from molecular adaptations in recognition proteins and carbohydrate-active enzymes, including a β-glucosidase specialized for β(1,6)-glucosyl linkages, a typical substitution in β(1,3)-glucans. These findings broaden the existing model for non-cellulosic β-glucans utilization by gut bacteria, revealing an additional layer of functional and evolutionary complexity within the gut microbiota, beyond conventional gene insertions/deletions to intricate biochemical interactions., (© 2024. The Author(s).)

The fundamental and assorted roles of β-1,3-glucans in nature are underpinned on diverse chemistry and molecular structures, demanding sophisticated and intricate enzymatic systems for their processing. In this work, the selectivity and modes of action of a glycoside hydrolase family active on β-1,3-glucans were systematically investigated combining sequence similarity network, phylogeny, X-ray crystallography, enzyme kinetics, mutagenesis and molecular dynamics. This family exhibits a minimalist and versatile (α/β)-barrel scaffold, which can harbor distinguishing exo or endo modes of action, including an ancillary-binding site for the anchoring of triple-helical β-1,3-glucans. The substrate binding occurs via a hydrophobic knuckle complementary to the canonical curved conformation of β-1,3-glucans or through a substrate conformational change imposed by the active-site topology of some fungal enzymes. Together, these findings expand our understanding of the enzymatic arsenal of bacteria and fungi for the breakdown and modification of β-1,3-glucans, which can be exploited for biotechnological applications.