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

Autor: Hansen DK; The Linderstrøm-Lang Centre for Protein Science, Section for Biomolecular Sciences, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200, Copenhagen, Denmark., Hansen AL; The Linderstrøm-Lang Centre for Protein Science, Section for Biomolecular Sciences, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200, Copenhagen, Denmark., Koivisto JM; The Linderstrøm-Lang Centre for Protein Science, Section for Biomolecular Sciences, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200, Copenhagen, Denmark., Shuoker B; Section for Protein Chemistry and Enzyme Technology, Department of Biotechnology and Biomedicine, Technical University of Denmark, Building 224, DK-2800, Kgs. Lyngby, Denmark; Biotechnology, Department of Chemistry (KILU), Lund University, Post Office Box 124, 221 00, Lund, Sweden., Abou Hachem M; Section for Protein Chemistry and Enzyme Technology, Department of Biotechnology and Biomedicine, Technical University of Denmark, Building 224, DK-2800, Kgs. Lyngby, Denmark., Winther JR; The Linderstrøm-Lang Centre for Protein Science, Section for Biomolecular Sciences, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200, Copenhagen, Denmark., Willemoës M; The Linderstrøm-Lang Centre for Protein Science, Section for Biomolecular Sciences, Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200, Copenhagen, Denmark. Electronic address: willemoes@bio.ku.dk.
Jazyk: angličtina
Zdroj: Archives of biochemistry and biophysics [Arch Biochem Biophys] 2022 Aug 15; Vol. 725, pp. 109280. Date of Electronic Publication: 2022 May 21.
DOI: 10.1016/j.abb.2022.109280
Abstrakt: 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 cat /K M 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.
(Copyright © 2022. Published by Elsevier Inc.)
Databáze: MEDLINE
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