Systematic Functional and Computational Analysis of Glucose-Binding Residues in Glycoside Hydrolase Family GH116
Glycoside hydrolases (GH) bind tightly to the sugar moiety at the glycosidic bond being hydrolyzed to stabilize its transition state conformation. We endeavored to assess the importance of glucose-binding residues in GH family 116 (GH116) β-glucosidases, which include human β-glucosylceramidase 2 (G...
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MDPI AG
2022-03-01
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| author | Meng Huang Salila Pengthaisong Ratana Charoenwattanasatien Natechanok Thinkumrob Jitrayut Jitonnom James R. Ketudat Cairns |
| author_facet | Meng Huang Salila Pengthaisong Ratana Charoenwattanasatien Natechanok Thinkumrob Jitrayut Jitonnom James R. Ketudat Cairns |
| author_sort | Meng Huang |
| collection | DOAJ |
| description | Glycoside hydrolases (GH) bind tightly to the sugar moiety at the glycosidic bond being hydrolyzed to stabilize its transition state conformation. We endeavored to assess the importance of glucose-binding residues in GH family 116 (GH116) β-glucosidases, which include human β-glucosylceramidase 2 (GBA2), by mutagenesis followed by kinetic characterization, X-ray crystallography, and ONIOM calculations on <i>Thermoanaerobacterium xylanolyticum Tx</i>GH116, the structural model for GH116 enzymes. Mutations of residues that bind at the glucose C3OH and C4OH caused 27–196-fold increases in <i>K</i><sub>M</sub> for <i>p</i>-nitrophenyl-β-D-glucoside, and significant decreases in the <i>k</i><sub>cat</sub>, up to 5000-fold. At the C6OH binding residues, mutations of E777 decreased the <i>k</i><sub>cat</sub>/<i>K</i><sub>M</sub> by over 60,000-fold, while R786 mutants increased both the <i>K</i><sub>M</sub> (40-fold) and <i>k</i><sub>cat</sub> (2–4-fold). The crystal structures of R786A and R786K suggested a larger entrance to the active site could facilitate their faster rates. ONIOM binding energy calculations identified D452, H507, E777, and R786, along with the catalytic residues E441 and D593, as strong electrostatic contributors to glucose binding with predicted interaction energies > 15 kcal mol<sup>−1</sup>, consistent with the effects of the D452, H507, E777 and R786 mutations on enzyme kinetics. The relative importance of GH116 active site residues in substrate binding and catalysis identified in this work improves the prospects for the design of inhibitors for GBA2 and the engineering of GH116 enzymes for hydrolytic and synthetic applications. |
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| language | English |
| last_indexed | 2024-03-09T20:01:42Z |
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| spelling | doaj.art-304da2e9c3574f829af1cce7c3047a162023-11-24T00:43:11ZengMDPI AGCatalysts2073-43442022-03-0112334310.3390/catal12030343Systematic Functional and Computational Analysis of Glucose-Binding Residues in Glycoside Hydrolase Family GH116Meng Huang0Salila Pengthaisong1Ratana Charoenwattanasatien2Natechanok Thinkumrob3Jitrayut Jitonnom4James R. Ketudat Cairns5School of Chemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, ThailandSchool of Chemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, ThailandCenter for Biomolecular Structure, Function and Application, Suranaree University of Technology, Nakhon Ratchasima 30000, ThailandUnit of Excellence in Computational Molecular Science and Catalysis, and Division of Chemistry, School of Science, University of Phayao, Phayao 56000, ThailandUnit of Excellence in Computational Molecular Science and Catalysis, and Division of Chemistry, School of Science, University of Phayao, Phayao 56000, ThailandSchool of Chemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, ThailandGlycoside hydrolases (GH) bind tightly to the sugar moiety at the glycosidic bond being hydrolyzed to stabilize its transition state conformation. We endeavored to assess the importance of glucose-binding residues in GH family 116 (GH116) β-glucosidases, which include human β-glucosylceramidase 2 (GBA2), by mutagenesis followed by kinetic characterization, X-ray crystallography, and ONIOM calculations on <i>Thermoanaerobacterium xylanolyticum Tx</i>GH116, the structural model for GH116 enzymes. Mutations of residues that bind at the glucose C3OH and C4OH caused 27–196-fold increases in <i>K</i><sub>M</sub> for <i>p</i>-nitrophenyl-β-D-glucoside, and significant decreases in the <i>k</i><sub>cat</sub>, up to 5000-fold. At the C6OH binding residues, mutations of E777 decreased the <i>k</i><sub>cat</sub>/<i>K</i><sub>M</sub> by over 60,000-fold, while R786 mutants increased both the <i>K</i><sub>M</sub> (40-fold) and <i>k</i><sub>cat</sub> (2–4-fold). The crystal structures of R786A and R786K suggested a larger entrance to the active site could facilitate their faster rates. ONIOM binding energy calculations identified D452, H507, E777, and R786, along with the catalytic residues E441 and D593, as strong electrostatic contributors to glucose binding with predicted interaction energies > 15 kcal mol<sup>−1</sup>, consistent with the effects of the D452, H507, E777 and R786 mutations on enzyme kinetics. The relative importance of GH116 active site residues in substrate binding and catalysis identified in this work improves the prospects for the design of inhibitors for GBA2 and the engineering of GH116 enzymes for hydrolytic and synthetic applications.https://www.mdpi.com/2073-4344/12/3/343β-glucosidaseglucose-binding residuesmutagenesisenzyme kineticsX-ray crystallographyONIOM |
| spellingShingle | Meng Huang Salila Pengthaisong Ratana Charoenwattanasatien Natechanok Thinkumrob Jitrayut Jitonnom James R. Ketudat Cairns Systematic Functional and Computational Analysis of Glucose-Binding Residues in Glycoside Hydrolase Family GH116 Catalysts β-glucosidase glucose-binding residues mutagenesis enzyme kinetics X-ray crystallography ONIOM |
| title | Systematic Functional and Computational Analysis of Glucose-Binding Residues in Glycoside Hydrolase Family GH116 |
| title_full | Systematic Functional and Computational Analysis of Glucose-Binding Residues in Glycoside Hydrolase Family GH116 |
| title_fullStr | Systematic Functional and Computational Analysis of Glucose-Binding Residues in Glycoside Hydrolase Family GH116 |
| title_full_unstemmed | Systematic Functional and Computational Analysis of Glucose-Binding Residues in Glycoside Hydrolase Family GH116 |
| title_short | Systematic Functional and Computational Analysis of Glucose-Binding Residues in Glycoside Hydrolase Family GH116 |
| title_sort | systematic functional and computational analysis of glucose binding residues in glycoside hydrolase family gh116 |
| topic | β-glucosidase glucose-binding residues mutagenesis enzyme kinetics X-ray crystallography ONIOM |
| url | https://www.mdpi.com/2073-4344/12/3/343 |
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