Transglycosidase activity of glycosynthase-type mutants of a fungal GH20 β-N-acetylhexosaminidase.

International Journal of Biological Macromolecules
Jana KapešováKristýna Slámová

Abstract

β-N-Acetylhexosaminidases (CAZy GH20, EC 3.2.1.52) are exo-glycosidases specific for cleaving N-acetylglucosamine and N-acetylgalactosamine moieties of various substrates. The β-N-acetylhexosaminidase from the filamentous fungus Talaromyces flavus (TfHex), a model enzyme in this study, has a broad substrate flexibility and outstanding synthetic ability. We have designed and characterized seven glycosynthase-type variants of TfHex mutated at the catalytic aspartate residue that stabilizes the oxazoline reaction intermediate. Most of the obtained enzyme variants lost the majority of their original hydrolytic activity towards the standard substrate p-nitrophenyl 2-acetamido-2-deoxy-β-D-glucopyranoside (pNP-β-GlcNAc); moreover, the mutants were not active with the proposed glycosynthase donor 2-acetamido-2-deoxy-d-glucopyranosyl-α-fluoride (GlcNAc-α-F) either as would be expected in a glycosynthase. Importantly, the mutant enzymes instead retained a strong transglycosylation activity towards the standard substrate pNP-β-GlcNAc. In summary, five out of seven prepared TfHex variants bearing mutation at the catalytic Asp370 residue acted as efficient transglycosidases, which makes them excellent tools for the synthesis of chitooligosa...Continue Reading

Citations

Apr 4, 2021·International Journal of Molecular Sciences·Mireia Castejón-VilatersanaAntoni Planas
Mar 31, 2021·Biotechnology Advances·Zuzana MészárosKristýna Slámová
Sep 1, 2021·Biotechnology Advances·Zuzana MészárosKristýna Slámová

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