Fig. 1: Exo-hydrolytic enzymes of the GH3 family with an entrapped Glc molecule and a ligand-free form, and their phylogeny.

a Hordeum vulgare HvExoI with entrapped Glc (PDB 3WLH; atomic colours), Bacteroides ovatus β-glucosidase with perfused Glc (PDB 5JP0; green cpk), and a ligand-free form of Pseudoalteromonas sp. exo-(1,4;1,3)-β-glucanase (PDB 3UT0; yellow cpk). Distances ached of HvExoI residues to Glc in the −1 subsite (black dashes) are within 3.3 Å. HvExoI and structurally equivalent residues in 3UT0 and 5JP0 superpose with respective RMSD values of 0.7 Å and 1.2 Å. b Surface morphology of the active site pocket of HvExoI coloured by an electrostatic potential: white, neutral; red, −5 kT·e⁻¹. Separations (dashed lines) between Glc and key interacting residues are within 3.3 Å. The positions of the −1 subsites in panels a and b are indicated. c Unrooted phylogenetic tree of representative GH3 family members (accession numbers listed in Supplementary Table 1) revealed the presence of four major clades with five activities [β-d-glucan glucohydrolases (EC 3.2.1.-), β-d-glucosidases (EC 3.2.1.21), β-N-acetylhexosaminidases (EC 3.2.1.52), β-d-xylosidases (EC 3.2.1.37), and α-l-arabinofuranosidases (EC 3.2.1.55)]. In β-d-glucan glucohydrolases, three subclades were identified with residue variability in the aromatic clamp (residue numbering corresponding to HvExoI; PDB 1EX1). The percentage of replicate trees with taxa clustering together in the bootstrap test (1000 replicates) are shown at node bipartitions.