Fig. 6: Structures of four of the O-glycan active GH16 family members characterised in this study.
a Crystal structures of BACCAC_02680E143Q, BF4060, BACCAC_03717, and Amuc_0724. The loops extending from the active site that are proposed to be involved in substrate specificity in GH16 enzymes are termed ‘fingers’ and are colour coded. b Subsites −1 to −3 of BACCAC_02680E143Q and BF4060 have the product of TriLacNAc cleavage bound (Galβ1,4GlcNAcβ1,3 Gal; shown in symbol form next to the structures with the sugar in each subsite labelled). The residues interacting directly with sugar are shown as sticks. The aromatic residues shared with β-glucanase GH16 family members that drive specificity for a β1,3 between the −1 and −2 sugars are shown (W129, W138 and W131, W140; See Supplementary Fig. 20 for active sites of BACCAC_03717, and Amuc_0724). c A surface representation of the regions surrounding the −1 subsite showing the selection for the axial O4 of Gal in the three Bacteroides enzymes, while Amuc_0724 has a more open ‘tunnel’ like space that appears to also allow accommodation of the equatorial O4 of Glc. The product from BACCAC_02680E143Q was overlaid in the BACCAC_03717, and Amuc_0724 structures. Colours represent the different ‘fingers’. d A view of the predicted +1 subsite of BF4060 and BACCAC_03717 overlaid with the glucose from the +1 subsite of a laminarinase from Phaenerochaete chrysosporium. The +1 subsites are much more closed for BF4060 and BACCAC_02680E143Q compared to BACCAC_03717, and Amuc_0724. e, An overview of the monosaccharides occupying the different subsites in GH16 family members with different activities. Linkages also shown. It should be noted that the sulfation will be variable along the O-glycan chain and there will also be fucose decorations. This situation is similar to porphyran, where the polysaccharide can have a variable composition, but the subsite occupancy shown here reflects the observations of structures of enzyme-glycan complexes currently available for porphyranases.
