Structural analysis and insights into the glycon specificity of the rice GH1 Os7BGlu26 β-d-mannosidase

Rice Os7BGlu26 is a GH1 family glycoside hydrolase with a threefold higher k cat/K m value for 4-nitrophenyl β-d-mannoside (4NPMan) compared with 4-nitrophenyl β-d-glucoside (4NPGlc). To investigate its selectivity for β-d-mannoside and β-d-glucoside substrates, the structures of apo Os7BGlu26 at a resolution of 2.2014;Å and of Os7BGlu26 with mannose at a resolution of 2.4514;Å were elucidated from isomorphous crystals in space group P212121. The (β/)8-barrel structure is similar to other GH1 family structures, but with a narrower active-site cleft. The Os7BGlu26 structure with d-mannose corresponds to a product complex, with β-d-mannose in the ¹ S 5 skew-boat conformation. Docking of the ¹ S 3, ¹ S 5, ² S O and ³ S 1 pyranose-ring conformations of 4NPMan and 4NPGlc substrates into the active site of Os7BGlu26 indicated that the lowest energies were in the ¹ S 5 and ¹ S 3 skew-boat conformations. Comparison of these docked conformers with other rice GH1 structures revealed differences in the residues interacting with the catalytic acid/base between enzymes with and without β-d-mannosidase activity. The mutation of Tyr134 to Trp in Os7BGlu26 resulted in similar k cat/K m values for 4NPMan and 4NPGlc, while mutation of Tyr134 to Phe resulted in a 37-fold higher k cat/K m for 4NPMan than 4NPGlc. Mutation of Cys182 to Thr decreased both the activity and the selectivity for β-d-mannoside. It was concluded that interactions with the catalytic acid/base play a significant role in glycon selection.