Flexibility and Function of Distal Substrate-Binding Tryptophans in the Blue Mussel β-Mannanase <i>Me</i>Man5A and Their Role in Hydrolysis and Transglycosylation
β-Mannanases hydrolyze β-mannans, important components of plant and microalgae cell walls. Retaining β-mannanases can also catalyze transglycosylation, forming new β-mannosidic bonds that are applicable for synthesis. This study focused on the blue mussel (<i>Mytilus edulis</i>) GH5_10 β...
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MDPI AG
2023-09-01
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| author | Simon Birgersson Johan Morrill Olof Stenström Mathias Wiemann Ulrich Weininger Pär Söderhjelm Mikael Akke Henrik Stålbrand |
| author_facet | Simon Birgersson Johan Morrill Olof Stenström Mathias Wiemann Ulrich Weininger Pär Söderhjelm Mikael Akke Henrik Stålbrand |
| author_sort | Simon Birgersson |
| collection | DOAJ |
| description | β-Mannanases hydrolyze β-mannans, important components of plant and microalgae cell walls. Retaining β-mannanases can also catalyze transglycosylation, forming new β-mannosidic bonds that are applicable for synthesis. This study focused on the blue mussel (<i>Mytilus edulis</i>) GH5_10 β-mannanase <i>Me</i>Man5A, which contains two semi-conserved tryptophans (W240 and W281) in the distal subsite +2 of its active site cleft. Variants of <i>Me</i>Man5A were generated by replacing one or both tryptophans with alanines. The substitutions reduced the enzyme’s catalytic efficiency (k<sub>cat</sub>/K<sub>m</sub> using galactomannan) by three-fold (W281A), five-fold (W240A), or 20-fold (W240A/W281A). Productive binding modes were analyzed by <sup>18</sup>O labeling of hydrolysis products and mass spectrometry. Results show that the substitution of both tryptophans was required to shift away from the dominant binding mode of mannopentaose (spanning subsites −3 to +2), suggesting that both tryptophans contribute to glycan binding. NMR spectroscopy and molecular dynamics simulations were conducted to analyze protein flexibility and glycan binding. We suggest that W240 is rigid and contributes to +2 subsite mannosyl specificity, while W281 is flexible, which enables stacking interactions in the +2 subsite by loop movement to facilitate binding. The substitutions significantly reduced or eliminated transglycosylation with saccharides as glycosyl acceptors but had no significant effect on reactions with alcohols. |
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| spelling | doaj.art-6119ba8797c04ddcb3f1db048d4f1ae62023-11-19T09:58:16ZengMDPI AGCatalysts2073-43442023-09-01139128110.3390/catal13091281Flexibility and Function of Distal Substrate-Binding Tryptophans in the Blue Mussel β-Mannanase <i>Me</i>Man5A and Their Role in Hydrolysis and TransglycosylationSimon Birgersson0Johan Morrill1Olof Stenström2Mathias Wiemann3Ulrich Weininger4Pär Söderhjelm5Mikael Akke6Henrik Stålbrand7Biochemistry and Structural Biology, Department of Chemistry, Lund University, P.O. Box 124, S-221 00 Lund, SwedenBiochemistry and Structural Biology, Department of Chemistry, Lund University, P.O. Box 124, S-221 00 Lund, SwedenBiophysical Chemistry, Department of Chemistry, Lund University, P.O. Box 124, S-221 00 Lund, SwedenBiochemistry and Structural Biology, Department of Chemistry, Lund University, P.O. Box 124, S-221 00 Lund, SwedenBiophysical Chemistry, Department of Chemistry, Lund University, P.O. Box 124, S-221 00 Lund, SwedenBiophysical Chemistry, Department of Chemistry, Lund University, P.O. Box 124, S-221 00 Lund, SwedenBiophysical Chemistry, Department of Chemistry, Lund University, P.O. Box 124, S-221 00 Lund, SwedenBiochemistry and Structural Biology, Department of Chemistry, Lund University, P.O. Box 124, S-221 00 Lund, Swedenβ-Mannanases hydrolyze β-mannans, important components of plant and microalgae cell walls. Retaining β-mannanases can also catalyze transglycosylation, forming new β-mannosidic bonds that are applicable for synthesis. This study focused on the blue mussel (<i>Mytilus edulis</i>) GH5_10 β-mannanase <i>Me</i>Man5A, which contains two semi-conserved tryptophans (W240 and W281) in the distal subsite +2 of its active site cleft. Variants of <i>Me</i>Man5A were generated by replacing one or both tryptophans with alanines. The substitutions reduced the enzyme’s catalytic efficiency (k<sub>cat</sub>/K<sub>m</sub> using galactomannan) by three-fold (W281A), five-fold (W240A), or 20-fold (W240A/W281A). Productive binding modes were analyzed by <sup>18</sup>O labeling of hydrolysis products and mass spectrometry. Results show that the substitution of both tryptophans was required to shift away from the dominant binding mode of mannopentaose (spanning subsites −3 to +2), suggesting that both tryptophans contribute to glycan binding. NMR spectroscopy and molecular dynamics simulations were conducted to analyze protein flexibility and glycan binding. We suggest that W240 is rigid and contributes to +2 subsite mannosyl specificity, while W281 is flexible, which enables stacking interactions in the +2 subsite by loop movement to facilitate binding. The substitutions significantly reduced or eliminated transglycosylation with saccharides as glycosyl acceptors but had no significant effect on reactions with alcohols.https://www.mdpi.com/2073-4344/13/9/1281β-mannanaseenzymatic synthesistransglycosylationflexibilitynovel glycosides |
| spellingShingle | Simon Birgersson Johan Morrill Olof Stenström Mathias Wiemann Ulrich Weininger Pär Söderhjelm Mikael Akke Henrik Stålbrand Flexibility and Function of Distal Substrate-Binding Tryptophans in the Blue Mussel β-Mannanase <i>Me</i>Man5A and Their Role in Hydrolysis and Transglycosylation Catalysts β-mannanase enzymatic synthesis transglycosylation flexibility novel glycosides |
| title | Flexibility and Function of Distal Substrate-Binding Tryptophans in the Blue Mussel β-Mannanase <i>Me</i>Man5A and Their Role in Hydrolysis and Transglycosylation |
| title_full | Flexibility and Function of Distal Substrate-Binding Tryptophans in the Blue Mussel β-Mannanase <i>Me</i>Man5A and Their Role in Hydrolysis and Transglycosylation |
| title_fullStr | Flexibility and Function of Distal Substrate-Binding Tryptophans in the Blue Mussel β-Mannanase <i>Me</i>Man5A and Their Role in Hydrolysis and Transglycosylation |
| title_full_unstemmed | Flexibility and Function of Distal Substrate-Binding Tryptophans in the Blue Mussel β-Mannanase <i>Me</i>Man5A and Their Role in Hydrolysis and Transglycosylation |
| title_short | Flexibility and Function of Distal Substrate-Binding Tryptophans in the Blue Mussel β-Mannanase <i>Me</i>Man5A and Their Role in Hydrolysis and Transglycosylation |
| title_sort | flexibility and function of distal substrate binding tryptophans in the blue mussel β mannanase i me i man5a and their role in hydrolysis and transglycosylation |
| topic | β-mannanase enzymatic synthesis transglycosylation flexibility novel glycosides |
| url | https://www.mdpi.com/2073-4344/13/9/1281 |
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