Fig. 1: Mechanistic and structuctural foundations of the proposed hypothesis.

Mechanism of retaining β-glycosidases (a), structural insights on GH3 enzymes (b), and view of the active site of a rBxTW1 model (c). a From up to down, glycosylation and deglycosylation catalyzed by wild-type enzyme; typical thioglycoligase; and GH3 thioglycoligase, where the third acid residue of the latter family is proposed to participate in the reaction mechanism somewhat facilitating the neutralization of the leaving group or accepting a proton from the potential glycosyl acceptor. Nu: nucleophile. b Overlap of five solved structures from the GH3 family and modeled rBxTW1 showing the three conserved acid residues of the active site and a β-d-glucose ligand. E: Catalytic acid/base (Glu); D: Catalytic nucleophile (Asp); E/D: Conserved acid residue (Glu for β-xylosidases and Asp for β-glucosidases). Residue positions are displayed according to the following order: β-glucosidase pdb:2X40³¹ (pale yellow); β-glucosidase pdb:4I3G⁸² (bright orange); β-glucosidase pdb:3ZYZ⁸³ (sand); and β-glucosidase pdb:3AC0⁸⁴ (olive) β-xylosidase pdb:5A7M (blue) and model (SWISS-MODEL tool) of β-xylosidase rBxTW1 (light blue). A glucose ligand and its polar contacts with the corresponding β-glucosidase pdb:3ZYZ are displayed⁸³. The background shows the residues’ environment in rBxTW1. The solved structures are referred by their PBD IDs. c Acid and basic residues in the active site of the rBxTW1 model. The catalytic nucleophile, the catalytic acid/base and the third acid residue are identified as D293, E495, and E91 respectively. The distances between the atoms potentially involved in salt bridges are indicated. Generation of protein images and distance measurements were performed using PyMOL Molecular Graphics System version 1.3.