A Bacteroidetes locus dedicated to fungal 1,6-β-glucan degradation: Unique substrate conformation drives specificity of the key endo-1,6-β-glucanase

Abstract

Glycans are major nutrients available to the human gut microbiota. The <i>Bacteroides</i> are generalist glycan degraders, and this function is mediated largely by polysaccharide utilization loci (PULs). The genomes of several <i>Bacteroides</i> species contain a PUL, PUL_{1,6-β-glucan}, that was predicted to target mixed linked plant 1,3;1,4-β-glucans. To test this hypothesis we characterized the proteins encoded by this locus in <i>Bacteroides thetaiotaomicron</i>, a member of the human gut microbiota. We show here that PUL_{1,6-β-glucan} does not orchestrate the degradation of a plant polysaccharide but targets a fungal cell wall glycan, 1,6-β-glucan, which is a growth substrate for the bacterium. The locus is up-regulated by 1,6-β-glucan and encodes two enzymes, a surface endo-1,6-β-glucanase, BT3312, and a periplasmic β-glucosidase that targets primarily 1,6-β-glucans. The non-catalytic proteins encoded by PUL_{1,6-β-glucan} target 1,6-β-glucans and comprise a surface glycan-binding protein and a SusD homologue that delivers glycans to the outer membrane transporter. We identified the central role of the endo-1,6-β-glucanase in 1,6-β-glucan depolymerization by deleting <i>bt3312</i>, which prevented the growth of <i>B. thetaiotaomicron</i> on 1,6-β-glucan. The crystal structure of BT3312 in complex with β-glucosyl-1,6-deoxynojirimycin revealed a TIM barrel catalytic domain that contains a deep substrate-binding cleft tailored to accommodate the hook-like structure adopted by 1,6-β-glucan. Specificity is driven by the complementarity of the enzyme active site cleft and the conformation of the substrate. We also noted that PUL_{1,6-β-glucan} is syntenic to many PULs from other Bacteroidetes, suggesting that utilization of yeast and fungal cell wall 1,6-β-glucans is a widespread adaptation within the human microbiota.