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Flexibility and Function of Distal Substrate-Binding Tryptophans in the Blue Mussel β-Mannanase MeMan5A and Their Role in Hydrolysis and Transglycosylation

Birgersson, Simon LU ; Morrill, Johan LU ; Stenström, Olof LU ; Wiemann, Mathias LU ; Weininger, Ulrich LU ; Söderhjelm, Pär LU ; Akke, Mikael LU orcid and Stålbrand, Henrik LU (2023) In Catalysts 13(9).
Abstract

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 (Mytilus edulis) GH5_10 β-mannanase MeMan5A, which contains two semi-conserved tryptophans (W240 and W281) in the distal subsite +2 of its active site cleft. Variants of MeMan5A were generated by replacing one or both tryptophans with alanines. The substitutions reduced the enzyme’s catalytic efficiency (kcat/Km using galactomannan) by three-fold (W281A), five-fold (W240A), or 20-fold (W240A/W281A). Productive binding modes were analyzed by 18O labeling... (More)

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 (Mytilus edulis) GH5_10 β-mannanase MeMan5A, which contains two semi-conserved tryptophans (W240 and W281) in the distal subsite +2 of its active site cleft. Variants of MeMan5A were generated by replacing one or both tryptophans with alanines. The substitutions reduced the enzyme’s catalytic efficiency (kcat/Km using galactomannan) by three-fold (W281A), five-fold (W240A), or 20-fold (W240A/W281A). Productive binding modes were analyzed by 18O 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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author
; ; ; ; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
enzymatic synthesis, flexibility, novel glycosides, transglycosylation, β-mannanase
in
Catalysts
volume
13
issue
9
article number
1281
publisher
MDPI AG
external identifiers
  • scopus:85172477285
ISSN
2073-4344
DOI
10.3390/catal13091281
language
English
LU publication?
yes
id
231c6bde-139b-4aff-96a4-6c23ea559f9d
date added to LUP
2023-12-18 11:25:14
date last changed
2024-04-02 21:25:54
@article{231c6bde-139b-4aff-96a4-6c23ea559f9d,
  abstract     = {{<p>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 (Mytilus edulis) GH5_10 β-mannanase MeMan5A, which contains two semi-conserved tryptophans (W240 and W281) in the distal subsite +2 of its active site cleft. Variants of MeMan5A 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.</p>}},
  author       = {{Birgersson, Simon and Morrill, Johan and Stenström, Olof and Wiemann, Mathias and Weininger, Ulrich and Söderhjelm, Pär and Akke, Mikael and Stålbrand, Henrik}},
  issn         = {{2073-4344}},
  keywords     = {{enzymatic synthesis; flexibility; novel glycosides; transglycosylation; β-mannanase}},
  language     = {{eng}},
  number       = {{9}},
  publisher    = {{MDPI AG}},
  series       = {{Catalysts}},
  title        = {{Flexibility and Function of Distal Substrate-Binding Tryptophans in the Blue Mussel β-Mannanase MeMan5A and Their Role in Hydrolysis and Transglycosylation}},
  url          = {{http://dx.doi.org/10.3390/catal13091281}},
  doi          = {{10.3390/catal13091281}},
  volume       = {{13}},
  year         = {{2023}},
}