Engineering Bifidobacterium longum Endo-α-N-acetylgalactosaminidase for Neu5Acα2-3Ga1β1-3Ga1NAc reactivity on Fetuin

Research output: Contribution to journal › Journal article › Research › peer-review

Standard

Engineering Bifidobacterium longum Endo-α-N-acetylgalactosaminidase for Neu5Acα2-3Ga1β1-3Ga1NAc reactivity on Fetuin. / Hansen, Dennis K.; Hansen, Anders Lønstrup; Koivisto, Johanna M.; Shuoker, Bashar; Abou Hachem, Maher; Winther, Jakob R.; Willemoës, Martin.

In: Archives of Biochemistry and Biophysics, Vol. 725, 109280, 2022.

Research output: Contribution to journal › Journal article › Research › peer-review

Harvard

Hansen, DK, Hansen, AL, Koivisto, JM, Shuoker, B, Abou Hachem, M, Winther, JR & Willemoës, M 2022, 'Engineering Bifidobacterium longum Endo-α-N-acetylgalactosaminidase for Neu5Acα2-3Ga1β1-3Ga1NAc reactivity on Fetuin', Archives of Biochemistry and Biophysics, vol. 725, 109280. https://doi.org/10.1016/j.abb.2022.109280

APA

Hansen, D. K., Hansen, A. L., Koivisto, J. M., Shuoker, B., Abou Hachem, M., Winther, J. R., & Willemoës, M. (2022). Engineering Bifidobacterium longum Endo-α-N-acetylgalactosaminidase for Neu5Acα2-3Ga1β1-3Ga1NAc reactivity on Fetuin. Archives of Biochemistry and Biophysics, 725, [109280]. https://doi.org/10.1016/j.abb.2022.109280

Vancouver

Hansen DK, Hansen AL, Koivisto JM, Shuoker B, Abou Hachem M, Winther JR et al. Engineering Bifidobacterium longum Endo-α-N-acetylgalactosaminidase for Neu5Acα2-3Ga1β1-3Ga1NAc reactivity on Fetuin. Archives of Biochemistry and Biophysics. 2022;725. 109280. https://doi.org/10.1016/j.abb.2022.109280

Author

Hansen, Dennis K. ; Hansen, Anders Lønstrup ; Koivisto, Johanna M. ; Shuoker, Bashar ; Abou Hachem, Maher ; Winther, Jakob R. ; Willemoës, Martin. / Engineering Bifidobacterium longum Endo-α-N-acetylgalactosaminidase for Neu5Acα2-3Ga1β1-3Ga1NAc reactivity on Fetuin. In: Archives of Biochemistry and Biophysics. 2022 ; Vol. 725.

Bibtex

@article{ce5a35999e7b44c9b1958fa8522a07f8,
title = "Engineering Bifidobacterium longum Endo-α-N-acetylgalactosaminidase for Neu5Acα2-3Ga1β1-3Ga1NAc reactivity on Fetuin",
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 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.",
keywords = "Enzyme design, GH101, Molecular docking, O-glycanase, Protein engineering, Substrate specificity",
author = "Hansen, {Dennis K.} and Hansen, {Anders L{\o}nstrup} and Koivisto, {Johanna M.} and Bashar Shuoker and {Abou Hachem}, Maher and Winther, {Jakob R.} and Martin Willemo{\"e}s",
note = "Publisher Copyright: {\textcopyright} 2022",
year = "2022",
doi = "10.1016/j.abb.2022.109280",
language = "English",
volume = "725",
journal = "Nitric Oxide: Biology and Chemistry",
issn = "1089-8603",
publisher = "Academic Press",

}

RIS

TY - JOUR

T1 - Engineering Bifidobacterium longum Endo-α-N-acetylgalactosaminidase for Neu5Acα2-3Ga1β1-3Ga1NAc reactivity on Fetuin

AU - Hansen, Dennis K.

AU - Hansen, Anders Lønstrup

AU - Koivisto, Johanna M.

AU - Shuoker, Bashar

AU - Abou Hachem, Maher

AU - Winther, Jakob R.

AU - Willemoës, Martin

N1 - Publisher Copyright: © 2022

PY - 2022

Y1 - 2022

N2 - 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.

AB - 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.

KW - Enzyme design

KW - GH101

KW - Molecular docking

KW - O-glycanase

KW - Protein engineering

KW - Substrate specificity

U2 - 10.1016/j.abb.2022.109280

DO - 10.1016/j.abb.2022.109280

M3 - Journal article

C2 - 35605676

AN - SCOPUS:85130511733

VL - 725

JO - Nitric Oxide: Biology and Chemistry

JF - Nitric Oxide: Biology and Chemistry

SN - 1089-8603

M1 - 109280

ER -

ID: 310388844