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Acceptor Specificity of β-N-Acetylhexosaminidase from Talaromyces flavus: A Rational Explanation

Fungal β-N-acetylhexosaminidases, though hydrolytic enzymes in vivo, are useful tools in the preparation of oligosaccharides of biological interest. The β-N-acetylhexosaminidase from Talaromyces flavus is remarkable in terms of its synthetic potential, broad substrate specificity, and tolerance to s...

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Autores principales: Garcia-Oliva, Cecilia, Hoyos, Pilar, Petrásková, Lucie, Kulik, Natalia, Pelantová, Helena, Cabanillas, Alfredo H., Rumbero, Ángel, Křen, Vladimír, Hernáiz, María J., Bojarová, Pavla
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6940953/
https://www.ncbi.nlm.nih.gov/pubmed/31817903
http://dx.doi.org/10.3390/ijms20246181
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author Garcia-Oliva, Cecilia
Hoyos, Pilar
Petrásková, Lucie
Kulik, Natalia
Pelantová, Helena
Cabanillas, Alfredo H.
Rumbero, Ángel
Křen, Vladimír
Hernáiz, María J.
Bojarová, Pavla
author_facet Garcia-Oliva, Cecilia
Hoyos, Pilar
Petrásková, Lucie
Kulik, Natalia
Pelantová, Helena
Cabanillas, Alfredo H.
Rumbero, Ángel
Křen, Vladimír
Hernáiz, María J.
Bojarová, Pavla
author_sort Garcia-Oliva, Cecilia
collection PubMed
description Fungal β-N-acetylhexosaminidases, though hydrolytic enzymes in vivo, are useful tools in the preparation of oligosaccharides of biological interest. The β-N-acetylhexosaminidase from Talaromyces flavus is remarkable in terms of its synthetic potential, broad substrate specificity, and tolerance to substrate modifications. It can be heterologously produced in Pichia pastoris in a high yield. The mutation of the Tyr470 residue to histidine greatly enhances its transglycosylation capability. The aim of this work was to identify the structural requirements of this model β-N-acetylhexosaminidase for its transglycosylation acceptors and formulate a structure–activity relationship study. Enzymatic reactions were performed using an activated glycosyl donor, 4-nitrophenyl N-acetyl-β-d-glucosaminide or 4-nitrophenyl N-acetyl-β-d-galactosaminide, and a panel of glycosyl acceptors of varying structural features (N-acetylglucosamine, glucose, N-acetylgalactosamine, galactose, N-acetylmuramic acid, and glucuronic acid). The transglycosylation products were isolated and structurally characterized. The C-2 N-acetamido group in the acceptor molecule was found to be essential for recognition by the enzyme. The presence of the C-2 hydroxyl moiety strongly hindered the normal course of transglycosylation, yielding unique non-reducing disaccharides in a low yield. Moreover, whereas the gluco-configuration at C-4 steered the glycosylation into the β(1-4) position, the galacto-acceptor afforded a β(1-6) glycosidic linkage. The Y470H mutant enzyme was tested with acceptors based on β-glycosides of uronic acid and N-acetylmuramic acid. With the latter acceptor, we were able to isolate and characterize one glycosylation product in a low yield. To our knowledge, this is the first example of enzymatic glycosylation of an N-acetylmuramic acid derivative. In order to explain these findings and predict enzyme behavior, a modeling study was accomplished that correlated with the acquired experimental data.
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spelling pubmed-69409532020-01-09 Acceptor Specificity of β-N-Acetylhexosaminidase from Talaromyces flavus: A Rational Explanation Garcia-Oliva, Cecilia Hoyos, Pilar Petrásková, Lucie Kulik, Natalia Pelantová, Helena Cabanillas, Alfredo H. Rumbero, Ángel Křen, Vladimír Hernáiz, María J. Bojarová, Pavla Int J Mol Sci Article Fungal β-N-acetylhexosaminidases, though hydrolytic enzymes in vivo, are useful tools in the preparation of oligosaccharides of biological interest. The β-N-acetylhexosaminidase from Talaromyces flavus is remarkable in terms of its synthetic potential, broad substrate specificity, and tolerance to substrate modifications. It can be heterologously produced in Pichia pastoris in a high yield. The mutation of the Tyr470 residue to histidine greatly enhances its transglycosylation capability. The aim of this work was to identify the structural requirements of this model β-N-acetylhexosaminidase for its transglycosylation acceptors and formulate a structure–activity relationship study. Enzymatic reactions were performed using an activated glycosyl donor, 4-nitrophenyl N-acetyl-β-d-glucosaminide or 4-nitrophenyl N-acetyl-β-d-galactosaminide, and a panel of glycosyl acceptors of varying structural features (N-acetylglucosamine, glucose, N-acetylgalactosamine, galactose, N-acetylmuramic acid, and glucuronic acid). The transglycosylation products were isolated and structurally characterized. The C-2 N-acetamido group in the acceptor molecule was found to be essential for recognition by the enzyme. The presence of the C-2 hydroxyl moiety strongly hindered the normal course of transglycosylation, yielding unique non-reducing disaccharides in a low yield. Moreover, whereas the gluco-configuration at C-4 steered the glycosylation into the β(1-4) position, the galacto-acceptor afforded a β(1-6) glycosidic linkage. The Y470H mutant enzyme was tested with acceptors based on β-glycosides of uronic acid and N-acetylmuramic acid. With the latter acceptor, we were able to isolate and characterize one glycosylation product in a low yield. To our knowledge, this is the first example of enzymatic glycosylation of an N-acetylmuramic acid derivative. In order to explain these findings and predict enzyme behavior, a modeling study was accomplished that correlated with the acquired experimental data. MDPI 2019-12-07 /pmc/articles/PMC6940953/ /pubmed/31817903 http://dx.doi.org/10.3390/ijms20246181 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
Garcia-Oliva, Cecilia
Hoyos, Pilar
Petrásková, Lucie
Kulik, Natalia
Pelantová, Helena
Cabanillas, Alfredo H.
Rumbero, Ángel
Křen, Vladimír
Hernáiz, María J.
Bojarová, Pavla
Acceptor Specificity of β-N-Acetylhexosaminidase from Talaromyces flavus: A Rational Explanation
title Acceptor Specificity of β-N-Acetylhexosaminidase from Talaromyces flavus: A Rational Explanation
title_full Acceptor Specificity of β-N-Acetylhexosaminidase from Talaromyces flavus: A Rational Explanation
title_fullStr Acceptor Specificity of β-N-Acetylhexosaminidase from Talaromyces flavus: A Rational Explanation
title_full_unstemmed Acceptor Specificity of β-N-Acetylhexosaminidase from Talaromyces flavus: A Rational Explanation
title_short Acceptor Specificity of β-N-Acetylhexosaminidase from Talaromyces flavus: A Rational Explanation
title_sort acceptor specificity of β-n-acetylhexosaminidase from talaromyces flavus: a rational explanation
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6940953/
https://www.ncbi.nlm.nih.gov/pubmed/31817903
http://dx.doi.org/10.3390/ijms20246181
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