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Bacteria in the human gut including Ruminococcus bromii and Eubacterium rectale encode starch-active enzymes that dictate how these bacteria interact with starch to initiate a metabolic cascade that leads to increased butyrate. Here, we determined the structures of two predicted secreted glycoside hydrolase 13 subfamily 36 (GH13_36) enzymes: ErAmy13B complexed with maltotetraose from E. rectale and RbAmy5 from R. bromii. The structures show a limited binding pocket extending from –2 through +2 subsites with limited possibilities for substrate interaction beyond this, which contributes to the propensity for members of this family to produce maltose as their main product. The enzyme structures reveal subtle differences in the +1/+2 subsites that may restrict the recognition of larger starch polymers by ErAmy13B. Our bioinformatic analysis of the biochemically characterized members of the GH13_36 subfamily, which includes the cell-surface GH13 SusG from Bacteroides thetaiotaomicron, suggests that these maltogenic amylases (EC 3.2.1.133) are usually localized to the outside of the cell, display a range of substrate preferences, and most likely contribute to maltose liberation at the cell surface during growth on starch. A broader comparison between GH13_36 and other maltogenic amylase subfamilies explain how the activity profiles of these enzymes are influenced by their structures.

Resistant starch is a prebiotic with breakdown by gut bacteria requiring the action of specialized amylases and starch-binding proteins. The human gut symbiont Ruminococcus bromii expresses granular starch-binding protein Sas6 (Starch Adherence System member 6) that consists of two starch-specific carbohydrate binding modules from family 26 (RbCBM26) and family 74 (RbCBM74). Here we present the crystal structures of Sas6 and RbCBM74 with a double helical dimer of maltodecaose bound along an extended surface groove. Binding data combined with native mass spectrometry suggest that RbCBM26 binds short maltooligosaccharides while RbCBM74 can bind single and double helical α-glucans. Our results support a model by which RbCBM74 and RbCBM26 bind neighboring α-glucan chains at the granule surface. CBM74s are conserved among starch granule-degrading bacteria and our work provides molecular insight into how this structure is accommodated by select gut species.

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