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Human glucosylcerebrosidase 2 (GBA2) of the CAZy family GH116 is responsible for the breakdown of glycosphingolipids on the cytoplasmic face of the endoplasmic reticulum and Golgi apparatus. Genetic defects in GBA2 result in spastic paraplegia and cerebellar ataxia, while cross-talk between GBA2 and GBA1 glucosylceramidases may affect Gaucher disease. Here, we report the first three-dimensional structure for any GH116 enzyme, Thermoanaerobacterium xylanolyticum TxGH116 β-glucosidase, alone and in complex with diverse ligands. These structures allow identification of the glucoside binding and active site residues, which are shown to be conserved with GBA2. Mutagenic analysis of TxGH116 and structural modeling of GBA2 provide a detailed structural and functional rationale for pathogenic missense mutations of GBA2.

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Rice BGlu1 b-glucosidase nucleophile mutant E386G is a glycosynthase that can synthesize p-nitrophenyl (pNP)-cellooligosaccharides of up to 11 residues. The X-ray crystal structures of the E386G glycosynthase with and without a-glucosyl fluoride were solved and the a-glucosyl fluoride complex was found to contain an ordered water molecule near the position of the nucleophile of the BGlu1 native structure, which is likely to stabilize the departing fluoride. The structures of E386G glycosynthase in complexes with cellotetraose and cellopentaose confirmed that the side chains of N245, S334, and Y341 interact with glucosyl residues in cellooligosaccharide binding subsites 12, 13, and 14. Mutants in which these residues were replaced in BGlu1 b-glucosidase hydrolyzed cellotetraose and cellopentaose with k cat /K m values similar to those of the wild type enzyme. However, the Y341A, Y341L, and N245V mutants of the E386G glycosynthase synthesize shorter pNPcellooligosaccharides than do the E386G glycosynthase and its S334A mutant, suggesting that Y341 and N245 play important roles in the synthesis of long oligosaccharides. X-ray structural studies revealed that cellotetraose binds to the Y341A mutant of the glycosynthase in a very different, alternative mode not seen in complexes with the E386G glycosynthase, possibly explaining the similar hydrolysis, but poorer synthesis of longer oligosaccharides by Y341 mutants.

Hydrolysis of β-d-mannosides by β-mannosidases typically proceeds via a B 2,5 transition state conformation for the pyranoside ring, while that of β-d-glucosides by β-glucosidases proceeds through a distinct 4 H 3 transition state conformation. However, rice Os7BGlu26 β-glycosidase hydrolyzes 4-nitrophenyl β-d-glucoside and β-d-mannoside with similar efficiencies. The origin of this dual substrate specificity was investigated by kinetic, structural, and computational approaches. The glycosidase inhibitors glucoimidazole and mannoimidazole inhibited Os7BGlu26 with K i values of 2.7 nM and 10.4 μM, respectively. In X-ray crystal structures of complexes with Os7BGlu26, glucoimidazole bound to the active site in a 4 E conformation, while mannoimidazole bound in a B 2,5 conformation, suggesting different transition state conformations. Moreover, calculation of quantum mechanics/molecular mechanics (QM/MM) free energy landscapes showed that 4-nitrophenyl β-d-glucoside adopts a 1 S 3/4 E conformation in the Michaelis complex, while 4-nitrophenyl β-d-mannoside adopts a 1 S 5/B 2,5 conformation. The QM/MM simulations of Os7BGlu26 catalysis of hydrolysis also supported the itineraries of 1 S 3 → 4 E/4 H 3 ⧧ → 4 C 1 for β-d-glucosides and 1 S 5 → B 2,5 ⧧ → O S 2 for β-d-mannosides, thereby revealing that a single glycoside hydrolase can hydrolyze glycosides of different configurations via distinct transition state pyranoside conformations.