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.
|Number of pages||12|
|Journal||Acta Crystallographica Section D: Biological Crystallography|
|Publication status||Published - Oct 2013|
- glycoside hydrolases
- structural analysis
ASJC Scopus subject areas
- Structural Biology