Acta Cryst. (2013). D69, 2124-2135 [ doi:10.1107/S0907444913020568 ]
Abstract: Rice Os7BGlu26 is a GH1 family glycoside hydrolase with a threefold higher kcat/Km value for 4-nitrophenyl -D-mannoside (4NPMan) compared with 4-nitrophenyl -D-glucoside (4NPGlc). To investigate its selectivity for -D-mannoside and -D-glucoside substrates, the structures of apo Os7BGlu26 at a resolution of 2.20 Å and of Os7BGlu26 with mannose at a resolution of 2.45 Å were elucidated from isomorphous crystals in space group P212121. The (/)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 -D-mannose in the 1S5 skew-boat conformation. Docking of the 1S3, 1S5, 2SO and 3S1 pyranose-ring conformations of 4NPMan and 4NPGlc substrates into the active site of Os7BGlu26 indicated that the lowest energies were in the 1S5 and 1S3 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 -D-mannosidase activity. The mutation of Tyr134 to Trp in Os7BGlu26 resulted in similar kcat/Km values for 4NPMan and 4NPGlc, while mutation of Tyr134 to Phe resulted in a 37-fold higher kcat/Km for 4NPMan than 4NPGlc. Mutation of Cys182 to Thr decreased both the activity and the selectivity for -D-mannoside. It was concluded that interactions with the catalytic acid/base play a significant role in glycon selection.
PDB references: 4jho and 4jie
Keywords: structural analysis; glycoside hydrolases; -mannosidases; rice.
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