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 kcat/Km 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 re...
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| Main Authors: | , , , , , , , |
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| Other Authors: | |
| Format: | Article |
| Language: | English |
| Published: |
2016
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| Subjects: | |
| Online Access: | https://hdl.handle.net/10356/82124 http://hdl.handle.net/10220/41104 |
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| Institution: | Nanyang Technological University |
| Language: | English |
| Summary: | Rice Os7BGlu26 is a GH1 family glycoside hydrolase with a threefold higher kcat/Km 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 2.20 Å and of Os7BGlu26 with mannose at a resolution of 2.45 Å were elucidated from isomorphous crystals in space group P212121. The (β/α)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 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 β-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 β-D-mannoside. It was concluded that interactions with the catalytic acid/base play a significant role in glycon selection. |
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