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

0503890 - MBÚ 2020 RIV CH eng J - Článek v odborném periodiku
Bojarová, Pavla - Kulik, Natalia - Hovorková, Michaela - Slámová, Kristýna - Pelantová, Helena - Křen, Vladimír
The beta-N-Acetylhexosaminidase in the Synthesis of Bioactive Glycans: Protein and Reaction Engineering.
Molecules. Roč. 24, č. 3 (2019), č. článku 599. E-ISSN 1420-3049
Grant CEP: GA MŠMT(CZ) LTC18038
Institucionální podpora: RVO:61388971
Klíčová slova: beta-N-acetylhexosaminidase * galectin-3 * site-directed mutagenesis
Obor OECD: Biochemistry and molecular biology
Impakt faktor: 3.267, rok: 2019
Způsob publikování: Open access
https://www.mdpi.com/1420-3049/24/3/599

N-Acetylhexosamine oligosaccharides terminated with GalNAc act as selective ligands of galectin-3, a biomedically important human lectin. Their synthesis can be accomplished by beta-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. beta-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 beta-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 beta-N-acetylhexosaminidase affinity and selectivity, resulting in a high-yield production of the functionalized GalNAc beta 4GlcNAc disaccharide, a selective ligand of galectin-3.
Trvalý link: http://hdl.handle.net/11104/0295661