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

Anupong Tankrathok, Javier Iglesias-Fernández, Sukanya Luang, Robert C. Robinson, Atsuo Kimura, Carme Rovira, Maria Hrmova, James R. Ketudat Cairns

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)2124-2135
Number of pages12
JournalActa Crystallographica Section D: Biological Crystallography
Volume69
Issue number10
DOIs
Publication statusPublished - Oct 1 2013
Externally publishedYes

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Mannosidases
Mannosides
Glucosides
Mannose
Cats
Ships
Mutation
Catalytic Domain
Acids
4-nitrophenyl
Oryza
Glycoside Hydrolases

Keywords

  • β-mannosidases
  • glycoside hydrolases
  • rice
  • structural analysis

ASJC Scopus subject areas

  • Structural Biology

Cite this

Structural analysis and insights into the glycon specificity of the rice GH1 Os7BGlu26 β-d-mannosidase. / Tankrathok, Anupong; Iglesias-Fernández, Javier; Luang, Sukanya; Robinson, Robert C.; Kimura, Atsuo; Rovira, Carme; Hrmova, Maria; Ketudat Cairns, James R.

In: Acta Crystallographica Section D: Biological Crystallography, Vol. 69, No. 10, 01.10.2013, p. 2124-2135.

Research output: Contribution to journalArticle

Tankrathok, A, Iglesias-Fernández, J, Luang, S, Robinson, RC, Kimura, A, Rovira, C, Hrmova, M & Ketudat Cairns, JR 2013, 'Structural analysis and insights into the glycon specificity of the rice GH1 Os7BGlu26 β-d-mannosidase', Acta Crystallographica Section D: Biological Crystallography, vol. 69, no. 10, pp. 2124-2135. https://doi.org/10.1107/S0907444913020568
Tankrathok, Anupong ; Iglesias-Fernández, Javier ; Luang, Sukanya ; Robinson, Robert C. ; Kimura, Atsuo ; Rovira, Carme ; Hrmova, Maria ; Ketudat Cairns, James R. / Structural analysis and insights into the glycon specificity of the rice GH1 Os7BGlu26 β-d-mannosidase. In: Acta Crystallographica Section D: Biological Crystallography. 2013 ; Vol. 69, No. 10. pp. 2124-2135.
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abstract = "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;{\AA} and of Os7BGlu26 with mannose at a resolution of 2.4514;{\AA} 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.",
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AU - Iglesias-Fernández, Javier

AU - Luang, Sukanya

AU - Robinson, Robert C.

AU - Kimura, Atsuo

AU - Rovira, Carme

AU - Hrmova, Maria

AU - Ketudat Cairns, James R.

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N2 - 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.

AB - 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.

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