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Engineering Bifidob...
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Hansen, Dennis K.University of Copenhagen
(author)
Engineering Bifidobacterium longum Endo-α-N-acetylgalactosaminidase for Neu5Acα2-3Galβ1-3GalNAc reactivity on Fetuin
- Article/chapterEnglish2022
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Numbers
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LIBRIS-ID:oai:lup.lub.lu.se:0f4e3ce3-c73d-4f1e-bb79-98f72d7494bf
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https://lup.lub.lu.se/record/0f4e3ce3-c73d-4f1e-bb79-98f72d7494bfURI
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https://doi.org/10.1016/j.abb.2022.109280DOI
Supplementary language notes
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Language:English
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Summary in:English
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Subject category:art swepub-publicationtype
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Subject category:ref swepub-contenttype
Notes
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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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Hansen, Anders LønstrupUniversity of Copenhagen
(author)
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Koivisto, Johanna M.University of Copenhagen
(author)
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Shuoker, BasharTechnical University of Denmark
(author)
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Abou Hachem, MaherLund University,Lunds universitet,Bioteknik,Centrum för tillämpade biovetenskaper,Kemiska institutionen,Institutioner vid LTH,Lunds Tekniska Högskola,Biotechnology,Center for Applied Life Sciences,Department of Chemistry,Departments at LTH,Faculty of Engineering, LTH,Technical University of Denmark(Swepub:lu)biot-mah
(author)
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Winther, Jakob R.University of Copenhagen
(author)
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Willemoës, MartinUniversity of Copenhagen
(author)
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University of CopenhagenTechnical University of Denmark
(creator_code:org_t)
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In:Archives of Biochemistry and Biophysics: Elsevier BV7250003-9861
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