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Engineering Bifidobacterium longum Endo-α-N-acetylgalactosaminidase for Neu5Acα2-3Galβ1-3GalNAc reactivity on Fetuin

Hansen, Dennis K. ; Hansen, Anders Lønstrup ; Koivisto, Johanna M. ; Shuoker, Bashar ; Abou Hachem, Maher LU ; Winther, Jakob R. and Willemoës, Martin (2022) In Archives of Biochemistry and Biophysics 725.
Abstract

Endo-α-N-acetylgalactosaminidase from Bifidobacterium longum (EngBF) belongs to the glycoside hydrolase family GH101 and has a strict preference towards the mucin type glycan, Galβ1-3GalNAc, which is O-linked to serine or threonine residues on glycopeptides and -proteins. While other enzymes of the GH101 family exhibit a wider substrate spectrum, no GH101 member has until recently been reported to process the α2-3 sialidated mucin glycan, Neu5Acα2-3Galβ1-3GalNAc. However, work published by others (ACS Chem Biol 2021, 16, 2004–2015) during the preparation of the present manuscript demonstrated that the enzymes from several bacteria are able to hydrolyze this glycan from the fluorophore, methylumbelliferyl. Based on molecular docking... (More)

Endo-α-N-acetylgalactosaminidase from Bifidobacterium longum (EngBF) belongs to the glycoside hydrolase family GH101 and has a strict preference towards the mucin type glycan, Galβ1-3GalNAc, which is O-linked to serine or threonine residues on glycopeptides and -proteins. While other enzymes of the GH101 family exhibit a wider substrate spectrum, no GH101 member has until recently been reported to process the α2-3 sialidated mucin glycan, Neu5Acα2-3Galβ1-3GalNAc. However, work published by others (ACS Chem Biol 2021, 16, 2004–2015) during the preparation of the present manuscript demonstrated that the enzymes from several bacteria are able to hydrolyze this glycan from the fluorophore, methylumbelliferyl. Based on molecular docking using the EngBF homolog, EngSP from Streptococcus pneumoniae, substitution of active site amino acid residues with the potential to allow for accommodation of Neu5Acα2-3Galβ1-3GalNAc were identified. Based on this analysis, the mutant EngBF variants W750A, Q894A, K1199A, E1294A and D1295A were prepared and tested, for activity towards the Neu5Acα2-3Galβ1-3GalNAc O-linked glycan present on bovine fetuin. Among the mutant EngBF variants listed above, only E1294A was shown to release Neu5Acα2-3Galβ1-3GalNAc from fetuin, which subsequently was also demonstrated for the substitutions: E1294 M, E1294H and E1294K. In addition, the kcat/KM of the EngBF variants for cleavage of the Neu5Acα2-3Galβ1-3GalNAc glycan increased between 5 and 70 times from pH 4.5 to pH 6.0.

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author
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organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Enzyme design, GH101, Molecular docking, O-glycanase, Protein engineering, Substrate specificity
in
Archives of Biochemistry and Biophysics
volume
725
article number
109280
publisher
Academic Press
external identifiers
  • scopus:85130511733
  • pmid:35605676
ISSN
0003-9861
DOI
10.1016/j.abb.2022.109280
language
English
LU publication?
yes
additional info
Publisher Copyright: © 2022
id
0f4e3ce3-c73d-4f1e-bb79-98f72d7494bf
date added to LUP
2022-08-18 09:17:24
date last changed
2024-07-10 05:16:03
@article{0f4e3ce3-c73d-4f1e-bb79-98f72d7494bf,
  abstract     = {{<p>Endo-α-N-acetylgalactosaminidase from Bifidobacterium longum (EngBF) belongs to the glycoside hydrolase family GH101 and has a strict preference towards the mucin type glycan, Galβ1-3GalNAc, which is O-linked to serine or threonine residues on glycopeptides and -proteins. While other enzymes of the GH101 family exhibit a wider substrate spectrum, no GH101 member has until recently been reported to process the α2-3 sialidated mucin glycan, Neu5Acα2-3Galβ1-3GalNAc. However, work published by others (ACS Chem Biol 2021, 16, 2004–2015) during the preparation of the present manuscript demonstrated that the enzymes from several bacteria are able to hydrolyze this glycan from the fluorophore, methylumbelliferyl. Based on molecular docking using the EngBF homolog, EngSP from Streptococcus pneumoniae, substitution of active site amino acid residues with the potential to allow for accommodation of Neu5Acα2-3Galβ1-3GalNAc were identified. Based on this analysis, the mutant EngBF variants W750A, Q894A, K1199A, E1294A and D1295A were prepared and tested, for activity towards the Neu5Acα2-3Galβ1-3GalNAc O-linked glycan present on bovine fetuin. Among the mutant EngBF variants listed above, only E1294A was shown to release Neu5Acα2-3Galβ1-3GalNAc from fetuin, which subsequently was also demonstrated for the substitutions: E1294 M, E1294H and E1294K. In addition, the k<sub>cat</sub>/K<sub>M</sub> of the EngBF variants for cleavage of the Neu5Acα2-3Galβ1-3GalNAc glycan increased between 5 and 70 times from pH 4.5 to pH 6.0.</p>}},
  author       = {{Hansen, Dennis K. and Hansen, Anders Lønstrup and Koivisto, Johanna M. and Shuoker, Bashar and Abou Hachem, Maher and Winther, Jakob R. and Willemoës, Martin}},
  issn         = {{0003-9861}},
  keywords     = {{Enzyme design; GH101; Molecular docking; O-glycanase; Protein engineering; Substrate specificity}},
  language     = {{eng}},
  month        = {{08}},
  publisher    = {{Academic Press}},
  series       = {{Archives of Biochemistry and Biophysics}},
  title        = {{Engineering Bifidobacterium longum Endo-α-N-acetylgalactosaminidase for Neu5Acα2-3Galβ1-3GalNAc reactivity on Fetuin}},
  url          = {{http://dx.doi.org/10.1016/j.abb.2022.109280}},
  doi          = {{10.1016/j.abb.2022.109280}},
  volume       = {{725}},
  year         = {{2022}},
}