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The β-N-Acetylhexosaminidase in the Synthesis of Bioactive Glycans: Protein and Reaction Engineering

N-Acetylhexosamine oligosaccharides terminated with GalNAc act as selective ligands of galectin-3, a biomedically important human lectin. Their synthesis can be accomplished by β-N-acetylhexosaminidases (EC 3.2.1.52). Advantageously, these enzymes tolerate the presence of functional groups in the su...

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Autores principales: Bojarová, Pavla, Kulik, Natalia, Hovorková, Michaela, Slámová, Kristýna, Pelantová, Helena, Křen, Vladimír
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6384963/
https://www.ncbi.nlm.nih.gov/pubmed/30743988
http://dx.doi.org/10.3390/molecules24030599
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author Bojarová, Pavla
Kulik, Natalia
Hovorková, Michaela
Slámová, Kristýna
Pelantová, Helena
Křen, Vladimír
author_facet Bojarová, Pavla
Kulik, Natalia
Hovorková, Michaela
Slámová, Kristýna
Pelantová, Helena
Křen, Vladimír
author_sort Bojarová, Pavla
collection PubMed
description N-Acetylhexosamine oligosaccharides terminated with GalNAc act as selective ligands of galectin-3, a biomedically important human lectin. Their synthesis can be accomplished by β-N-acetylhexosaminidases (EC 3.2.1.52). Advantageously, these enzymes tolerate the presence of functional groups in the substrate molecule, such as the thiourea linker useful for covalent conjugation of glycans to a multivalent carrier, affording glyconjugates. β-N-Acetylhexosaminidases exhibit activity towards both N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) moieties. A point mutation of active-site amino acid Tyr into other amino acid residues, especially Phe, His, and Asn, has previously been shown to strongly suppress the hydrolytic activity of β-N-acetylhexosaminidases, creating enzymatic synthetic engines. In the present work, we demonstrate that Tyr470 is an important mutation hotspot for altering the ratio of GlcNAcase/GalNAcase activity, resulting in mutant enzymes with varying affinity to GlcNAc/GalNAc substrates. The enzyme selectivity may additionally be manipulated by altering the reaction medium upon changing pH or adding selected organic co-solvents. As a result, we are able to fine-tune the β-N-acetylhexosaminidase affinity and selectivity, resulting in a high-yield production of the functionalized GalNAcβ4GlcNAc disaccharide, a selective ligand of galectin-3.
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spelling pubmed-63849632019-02-23 The β-N-Acetylhexosaminidase in the Synthesis of Bioactive Glycans: Protein and Reaction Engineering Bojarová, Pavla Kulik, Natalia Hovorková, Michaela Slámová, Kristýna Pelantová, Helena Křen, Vladimír Molecules Article N-Acetylhexosamine oligosaccharides terminated with GalNAc act as selective ligands of galectin-3, a biomedically important human lectin. Their synthesis can be accomplished by β-N-acetylhexosaminidases (EC 3.2.1.52). Advantageously, these enzymes tolerate the presence of functional groups in the substrate molecule, such as the thiourea linker useful for covalent conjugation of glycans to a multivalent carrier, affording glyconjugates. β-N-Acetylhexosaminidases exhibit activity towards both N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) moieties. A point mutation of active-site amino acid Tyr into other amino acid residues, especially Phe, His, and Asn, has previously been shown to strongly suppress the hydrolytic activity of β-N-acetylhexosaminidases, creating enzymatic synthetic engines. In the present work, we demonstrate that Tyr470 is an important mutation hotspot for altering the ratio of GlcNAcase/GalNAcase activity, resulting in mutant enzymes with varying affinity to GlcNAc/GalNAc substrates. The enzyme selectivity may additionally be manipulated by altering the reaction medium upon changing pH or adding selected organic co-solvents. As a result, we are able to fine-tune the β-N-acetylhexosaminidase affinity and selectivity, resulting in a high-yield production of the functionalized GalNAcβ4GlcNAc disaccharide, a selective ligand of galectin-3. MDPI 2019-02-08 /pmc/articles/PMC6384963/ /pubmed/30743988 http://dx.doi.org/10.3390/molecules24030599 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Bojarová, Pavla
Kulik, Natalia
Hovorková, Michaela
Slámová, Kristýna
Pelantová, Helena
Křen, Vladimír
The β-N-Acetylhexosaminidase in the Synthesis of Bioactive Glycans: Protein and Reaction Engineering
title The β-N-Acetylhexosaminidase in the Synthesis of Bioactive Glycans: Protein and Reaction Engineering
title_full The β-N-Acetylhexosaminidase in the Synthesis of Bioactive Glycans: Protein and Reaction Engineering
title_fullStr The β-N-Acetylhexosaminidase in the Synthesis of Bioactive Glycans: Protein and Reaction Engineering
title_full_unstemmed The β-N-Acetylhexosaminidase in the Synthesis of Bioactive Glycans: Protein and Reaction Engineering
title_short The β-N-Acetylhexosaminidase in the Synthesis of Bioactive Glycans: Protein and Reaction Engineering
title_sort β-n-acetylhexosaminidase in the synthesis of bioactive glycans: protein and reaction engineering
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6384963/
https://www.ncbi.nlm.nih.gov/pubmed/30743988
http://dx.doi.org/10.3390/molecules24030599
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