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Molecules 2019, 24(3), 599; https://doi.org/10.3390/molecules24030599

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

1
Laboratory of Biotransformation, Institute of Microbiology, Czech Academy of Sciences, Vídeňská 1083, CZ-14220 Praha 4, Czech Republic
2
Center for Nanobiology and Structural Biology, Institute of Microbiology, Czech Academy of Sciences, Zámek 136, CZ-37333 Nové Hrady, Czech Republic
3
Laboratory of Molecular Structure Characterization, Institute of Microbiology, Czech Academy of Sciences, Vídeňská 1083, CZ-14220 Praha 4, Czech Republic
*
Author to whom correspondence should be addressed.
Academic Editor: Lothar Elling
Received: 13 January 2019 / Revised: 4 February 2019 / Accepted: 6 February 2019 / Published: 8 February 2019
(This article belongs to the Special Issue Synthesis and Biological Applications of Glycoconjugates Ⅱ)
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Abstract

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. View Full-Text
Keywords: β-N-acetylhexosaminidase; galectin-3; molecular modeling; site-directed mutagenesis; solvent; substrate specificity; transglycosidase β-N-acetylhexosaminidase; galectin-3; molecular modeling; site-directed mutagenesis; solvent; substrate specificity; transglycosidase
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Bojarová, P.; Kulik, N.; Hovorková, M.; Slámová, K.; Pelantová, H.; Křen, V. The β-N-Acetylhexosaminidase in the Synthesis of Bioactive Glycans: Protein and Reaction Engineering. Molecules 2019, 24, 599.

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