Acta Crystallographica Section D

Biological Crystallography

Volume 69, Part 10 (October 2013)

research papers

Acta Cryst. (2013). D69, 2124-2135    [ doi:10.1107/S0907444913020568 ]

Structural analysis and insights into the glycon specificity of the rice GH1 Os7BGlu26 [beta]-D-mannosidase

A. Tankrathok, J. Iglesias-Fernández, S. Luang, R. C. Robinson, A. Kimura, C. Rovira, M. Hrmova and J. R. Ketudat Cairns

Abstract: Rice Os7BGlu26 is a GH1 family glycoside hydrolase with a threefold higher kcat/Km value for 4-nitrophenyl [beta]-D-mannoside (4NPMan) compared with 4-nitrophenyl [beta]-D-glucoside (4NPGlc). To investigate its selectivity for [beta]-D-mannoside and [beta]-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 ([beta]/[alpha])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 [beta]-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 [beta]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 [beta]-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; [beta]-mannosidases; rice.

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